Nucleocytoplasmic Shuttling of the Golgi Phosphatidylinositol 4-Kinase Pik1 Is Regulated by 14-3-3 Proteins and Coordinates Golgi Function with Cell Growth

PubMed Central

Demmel, Lars; Beck, Mike; Klose, Christian; Schlaitz, Anne-Lore; Gloor, Yvonne; Hsu, Peggy P.; Havlis, Jan; Shevchenko, Andrej; Krause, Eberhard; Kalaidzidis, Yannis

2008-01-01

The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p–14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling. PMID:18172025

Chlamydia trachomatis Intercepts Golgi-Derived Sphingolipids through a Rab14-Mediated Transport Required for Bacterial Development and Replication

PubMed Central

Capmany, Anahí; Damiani, María Teresa

2010-01-01

Chlamydia trachomatis are obligate intracellular bacteria that survive and replicate in a bacterial-modified phagosome called inclusion. As other intracellular parasites, these bacteria subvert the phagocytic pathway to avoid degradation in phagolysosomes and exploit trafficking pathways to acquire both energy and nutrients essential for their survival. Rabs are host proteins that control intracellular vesicular trafficking. Rab14, a Golgi-related Rab, controls Golgi to endosomes transport. Since Chlamydia establish a close relationship with the Golgi apparatus, the recruitment and participation of Rab14 on inclusion development and bacteria growth were analyzed. Time course analysis revealed that Rab14 associated with inclusions by 10 h post infection and was maintained throughout the entire developmental cycle. The recruitment was bacterial protein synthesis-dependent but independent of microtubules and Golgi integrity. Overexpression of Rab14 dominant negative mutants delayed inclusion enlargement, and impaired bacteria replication as determined by IFU. Silencing of Rab14 by siRNA also decreased bacteria multiplication and infectivity. By electron microscopy, aberrant bacteria were observed in cells overexpressing the cytosolic negative Rab14 mutant. Our results showed that Rab14 facilitates the delivery of sphingolipids required for bacterial development and replication from the Golgi to chlamydial inclusions. Novel anti-chlamydial therapies could be developed based on the knowledge of how bacteria subvert host vesicular transport events through Rabs manipulation. PMID:21124879

Chlamydia trachomatis intercepts Golgi-derived sphingolipids through a Rab14-mediated transport required for bacterial development and replication.

PubMed

Capmany, Anahí; Damiani, María Teresa

2010-11-22

Chlamydia trachomatis are obligate intracellular bacteria that survive and replicate in a bacterial-modified phagosome called inclusion. As other intracellular parasites, these bacteria subvert the phagocytic pathway to avoid degradation in phagolysosomes and exploit trafficking pathways to acquire both energy and nutrients essential for their survival. Rabs are host proteins that control intracellular vesicular trafficking. Rab14, a Golgi-related Rab, controls Golgi to endosomes transport. Since Chlamydia establish a close relationship with the Golgi apparatus, the recruitment and participation of Rab14 on inclusion development and bacteria growth were analyzed. Time course analysis revealed that Rab14 associated with inclusions by 10 h post infection and was maintained throughout the entire developmental cycle. The recruitment was bacterial protein synthesis-dependent but independent of microtubules and Golgi integrity. Overexpression of Rab14 dominant negative mutants delayed inclusion enlargement, and impaired bacteria replication as determined by IFU. Silencing of Rab14 by siRNA also decreased bacteria multiplication and infectivity. By electron microscopy, aberrant bacteria were observed in cells overexpressing the cytosolic negative Rab14 mutant. Our results showed that Rab14 facilitates the delivery of sphingolipids required for bacterial development and replication from the Golgi to chlamydial inclusions. Novel anti-chlamydial therapies could be developed based on the knowledge of how bacteria subvert host vesicular transport events through Rabs manipulation.

Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis.

PubMed

Zhang, Yi; Nikolovski, Nino; Sorieul, Mathias; Vellosillo, Tamara; McFarlane, Heather E; Dupree, Ray; Kesten, Christopher; Schneider, René; Driemeier, Carlos; Lathe, Rahul; Lampugnani, Edwin; Yu, Xiaolan; Ivakov, Alexander; Doblin, Monika S; Mortimer, Jenny C; Brown, Steven P; Persson, Staffan; Dupree, Paul

2016-06-09

As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus.

Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools*

PubMed Central

Goto, Asako; Charman, Mark; Ridgway, Neale D.

2016-01-01

Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50–70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology domain probe. The cellular mass of phosphatidylinositol monophosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) was unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of sphingomyelin synthesis, suggesting that 25OH must be exchanged for PI-4P to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P specifically recruits pleckstrin homology domain-containing proteins involved in lipid transfer and metabolism, such as CERT. PMID:26601944

Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools.

PubMed

Goto, Asako; Charman, Mark; Ridgway, Neale D

2016-01-15

Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50-70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology domain probe. The cellular mass of phosphatidylinositol monophosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) was unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of sphingomyelin synthesis, suggesting that 25OH must be exchanged for PI-4P to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P specifically recruits pleckstrin homology domain-containing proteins involved in lipid transfer and metabolism, such as CERT. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Saccharomyces cerevisiae proteinase A excretion and wine making.

PubMed

Song, Lulu; Chen, Yefu; Du, Yongjing; Wang, Xibin; Guo, Xuewu; Dong, Jian; Xiao, Dongguang

2017-11-09

Proteinase A (PrA), the major protease in Saccharomyces cerevisiae, plays an essential role in zymogen activation, sporulation, and other physiological processes in vivo. The extracellular secretion of PrA often occurs during alcoholic fermentation, especially in the later stages when the yeast cells are under stress conditions, and affects the quality and safety of fermented products. Thus, the mechanism underlying PrA excretion must be explored to improve the quality and safety of fermented products. This paper briefly introduces the structure and physiological function of PrA. Two transport routes of PrA, namely, the Golgi-to-vacuole pathway and the constitutive Golgi-to-plasma membrane pathway, are also discussed. Moreover, the research history and developments on the mechanism of extracellular PrA secretion are described. In addition, it is briefly discussed that calcium homeostasis plays an important role in the secretory pathway of proteins, implying that the regulation of PrA delivery to the plasma membrane requires the involvement of calcium ion. Finally, this review focuses on the effects of PrA excretion on wine making (including Chinese rice wine, grape wine, and beer brewage) and presents strategies to control PrA excretion.

The ceramide-enriched trans-Golgi compartments reorganize together with other parts of the Golgi apparatus in response to ATP-depletion.

PubMed

Meisslitzer-Ruppitsch, Claudia; Röhrl, Clemens; Ranftler, Carmen; Neumüller, Josef; Vetterlein, Monika; Ellinger, Adolf; Pavelka, Margit

2011-02-01

In this study, the ceramide-enriched trans-Golgi compartments representing sites of synthesis of sphingomyelin and higher organized lipids were visualized in control and ATP-depleted hepatoma and endothelial cells using internalization of BODIPY-ceramide and the diaminobenzidine photooxidation method for combined light-electron microscopical exploration. Metabolic stress induced by lowering the cellular ATP-levels leads to reorganizations of the Golgi apparatus and the appearance of tubulo-glomerular bodies and networks. The results obtained with three different protocols, in which BODIPY-ceramide either was applied prior to, concomitantly with, or after ATP-depletion, revealed that the ceramide-enriched compartments reorganize together with other parts of the Golgi apparatus under these conditions. They were found closely associated with and integrated in the tubulo-glomerular bodies formed in response to ATP-depletion. This is in line with the changes of the staining patterns obtained with the Helix pomatia lectin and the GM130 and TGN46 immuno-reactions occurring in response to ATP-depletion and is confirmed by 3D electron tomography. The 3D reconstructions underlined the glomerular character of the reorganized Golgi apparatus and demonstrated continuities of ceramide positive and negative parts. Most interestingly, BODIPY-ceramide becomes concentrated in compartments of the tubulo-glomerular Golgi bodies, even though the reorganization took place before BODIPY-ceramide administration. This indicates maintained functionalities although the regular Golgi stack organization is abolished; the results provide novel insights into Golgi structure-function relationships, which might be relevant for cells affected by metabolic stress.

A Two-Tier Golgi-Based Control of Organelle Size Underpins the Functional Plasticity of Endothelial Cells

PubMed Central

Ferraro, Francesco; Kriston-Vizi, Janos; Metcalf, Daniel J.; Martin-Martin, Belen; Freeman, Jamie; Burden, Jemima J.; Westmoreland, David; Dyer, Clare E.; Knight, Alex E.; Ketteler, Robin; Cutler, Daniel F.

2014-01-01

Summary Weibel-Palade bodies (WPBs), endothelial-specific secretory granules that are central to primary hemostasis and inflammation, occur in dimensions ranging between 0.5 and 5 μm. How their size is determined and whether it has a functional relevance are at present unknown. Here, we provide evidence for a dual role of the Golgi apparatus in controlling the size of these secretory carriers. At the ministack level, cisternae constrain the size of nanostructures (“quanta”) of von Willebrand factor (vWF), the main WPB cargo. The ribbon architecture of the Golgi then allows copackaging of a variable number of vWF quanta within the continuous lumen of the trans-Golgi network, thereby generating organelles of different sizes. Reducing the WPB size abates endothelial cell hemostatic function by drastically diminishing platelet recruitment, but, strikingly, the inflammatory response (the endothelial capacity to engage leukocytes) is unaltered. Size can thus confer functional plasticity to an organelle by differentially affecting its activities. PMID:24794632

Inhibition of intra-Golgi transport in vitro by mitotic kinase.

PubMed

Stuart, R A; Mackay, D; Adamczewski, J; Warren, G

1993-02-25

It has previously been shown that exocytic and endocytic membrane traffic are inhibited in mitotic mammalian cells. Here we have used a cell-free intra-Golgi transport assay supplemented with heterologous cytosols to mimic this effect in vitro. Cytosols with high histone kinase activity, made either from mitotic cells or by cyclin A treatment of interphase cells, inhibited intra-Golgi transport by up to 75%. Inhibition of transport was reversed by the kinase inhibitor staurosporine or by reduction in ATP levels leading to inactivation of histone kinase. The data indicate that cell cycle control of intra-Golgi transport is due to a reversible modification of cytosol, and this assay system may be used to study the molecular mechanism of mitotic transport inhibition in mammalian cells.

Discrete Determinants in ArfGAP2/3 Conferring Golgi Localization and Regulation by the COPI Coat

PubMed Central

Kliouchnikov, Lena; Bigay, Joëlle; Mesmin, Bruno; Parnis, Anna; Rawet, Moran; Goldfeder, Noga; Antonny, Bruno

2009-01-01

From yeast to mammals, two types of GTPase-activating proteins, ArfGAP1 and ArfGAP2/3, control guanosine triphosphate (GTP) hydrolysis on the small G protein ADP-ribosylation factor (Arf) 1 at the Golgi apparatus. Although functionally interchangeable, they display little similarity outside the catalytic GTPase-activating protein (GAP) domain, suggesting differential regulation. ArfGAP1 is controlled by membrane curvature through its amphipathic lipid packing sensor motifs, whereas Golgi targeting of ArfGAP2 depends on coatomer, the building block of the COPI coat. Using a reporter fusion approach and in vitro assays, we identified several functional elements in ArfGAP2/3. We show that the Golgi localization of ArfGAP3 depends on both a central basic stretch and a carboxy-amphipathic motif. The basic stretch interacts directly with coatomer, which we found essential for the catalytic activity of ArfGAP3 on Arf1-GTP, whereas the carboxy-amphipathic motif interacts directly with lipid membranes but has minor role in the regulation of ArfGAP3 activity. Our findings indicate that the two types of ArfGAP proteins that reside at the Golgi use a different combination of protein–protein and protein–lipid interactions to promote GTP hydrolysis in Arf1-GTP. PMID:19109418

Effects of brefeldin A on the endomembrane system and germ tube formation of the tetraspore of Gelidium floridanum (Rhodophyta, Florideophyceae).

PubMed

Simioni, Carmen; Rover, Ticiane; Schmidt, Éder C; de L Felix, Marthiellen R; Polo, Luz Karime; Santos, Rodrigo Dos; Costa, Giulia Burle; Kreusch, Marianne; Pereira, Debora T; Ouriques, Luciane C; Bouzon, Zenilda L

2014-06-01

Gelidium floridanum W.R. Taylor tetraspores are units of dispersal and are responsible for substrate attachment. This study aimed to examine evidence of direct interaction between germ tube formation and Golgi activity during tetraspore germination of G. floridanum. After release, the tetraspores were incubated with brefeldin A (BFA) in concentrations of 4 and 8 μM over a 6 h period. The controls and treatments were analyzed with light, fluorescence (FM4-64 dye) and transmission electron microscopy. In the control samples, the Golgi bodies were responsible for germ tube formation. In contrast, BFA-treated samples were observed to inhibit spore adhesion and germ tube formation. These tetraspores also showed an increase in volume (≥30 μm width). BFA treatment also resulted in the disassembly of Golgi cisternae and the formation of vesiculated areas of the cytoplasm, blocking the secretion of protein and amorphous matrix polysaccharides. When stained with FM4-64, the control samples showed fluorescence in the apical region of the germ tube, but the treated samples showed an intense fluorescence throughout the cytoplasm. From these results, we can conclude that the germ tube is formed by the incorporation of vesicles derived from Golgi. Thus, vesicle secretion and Golgi organization are basic processes and essential in adhesion and tube formation. By blocking the secretion of protein and amorphous matrix polysaccharides, BFA treatment precluded tetraspore germination. © 2014 Phycological Society of America.

Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis

PubMed Central

Zhang, Yi; Nikolovski, Nino; Sorieul, Mathias; Vellosillo, Tamara; McFarlane, Heather E.; Dupree, Ray; Kesten, Christopher; Schneider, René; Driemeier, Carlos; Lathe, Rahul; Lampugnani, Edwin; Yu, Xiaolan; Ivakov, Alexander; Doblin, Monika S.; Mortimer, Jenny C.; Brown, Steven P.; Persson, Staffan; Dupree, Paul

2016-01-01

As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus. PMID:27277162

Intracellular accumulation and oligosaccharide processing of alkaline phosphatase under disassembly of the Golgi complex caused by brefeldin A.

PubMed

Takami, N; Oda, K; Fujiwara, T; Ikehara, Y

1990-12-27

Electron microscopic observations showed that the fungal metabolite brefeldin A caused disassembly of the Golgi complex in human choriocarcinoma cells and accumulation of alkaline phosphatase (ALP) in the endoplasmic reticulum (ER) and nuclear envelope, where ALP was not apparently detectable in control cells. Pulse/chase experiments with [35S]methionine demonstrated that in the control cells, ALP synthesized as a 63-kDa precursor form was rapidly converted to a 66-kDa form, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type, which was expressed on the cell surface after 30 min of chase. In contrast, in the brefeldin-A-treated cells the precursor was gradually converted to a 65-kDa form, slightly smaller than the control mature form, which was not expressed on the cell surface even after a prolonged time of chase. Kinetics of the ALP processing in the brefeldin-A-treated cells demonstrated that the precursor was initially converted to an intermediate form, partially sensitive to endo-beta-N-acetylglucosaminidase H (endo H), then to an endo-H-resistant 65-kDa form. In addition, this form was found to be sensitive to neuraminidase digestion, though its sialylation was not so complete as that of the control mature form. Taken together, these results suggest that under disassembly of the Golgi complex caused by brefeldin A, oligosaccharide-processing enzymes including sialyltransferase, an enzyme in the trans Golgi cisterna(e) and/or the trans Golgi network, might be redistributed into the ER and involved in processing of the oligosaccharides of ALP accumulating there.

Demonstration that endoplasmic reticulum-associated degradation of glycoproteins can occur downstream of processing by endomannosidase.

PubMed

Kukushkin, Nikolay V; Alonzi, Dominic S; Dwek, Raymond A; Butters, Terry D

2011-08-15

During quality control in the ER (endoplasmic reticulum), nascent glycoproteins are deglucosylated by ER glucosidases I and II. In the post-ER compartments, glycoprotein endo-α-mannosidase provides an alternative route for deglucosylation. Previous evidence suggests that endomannosidase non-selectively deglucosylates glycoproteins that escape quality control in the ER, facilitating secretion of aberrantly folded as well as normal glycoproteins. In the present study, we employed FOS (free oligosaccharides) released from degrading glycoproteins as biomarkers of ERAD (ER-associated degradation), allowing us to gain a global rather than single protein-centred view of ERAD. Glucosidase inhibition was used to discriminate between glucosidase- and endomannosidase-mediated ERAD pathways. Endomannosidase expression was manipulated in CHO (Chinese-hamster ovary)-K1 cells, naturally lacking a functional version of the enzyme, and HEK (human embryonic kidney)-293T cells. Endomannosidase was shown to decrease the levels of total FOS, suggesting decreased rates of ERAD. However, following pharmacological inhibition of ER glucosidases I and II, endomannosidase expression resulted in a partial switch between glucosylated FOS, released from ER-confined glycoproteins, to deglucosylated FOS, released from endomannosidase-processed glycoproteins transported from the Golgi/ERGIC (ER/Golgi intermediate compartment) to the ER. Using this approach, we have identified a previously unknown pathway of glycoprotein flow, undetectable by the commonly employed methods, in which secretory cargo is targeted back to the ER after being processed by endomannosidase. © The Authors Journal compilation © 2011 Biochemical Society

A reversible Renilla luciferase protein complementation assay for rapid identification of protein–protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus

PubMed Central

Lund, Christian H.; Bromley, Jennifer R.; Stenbæk, Anne; Rasmussen, Randi E.; Scheller, Henrik V.; Sakuragi, Yumiko

2015-01-01

A growing body of evidence suggests that protein–protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. We tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. Our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta. PMID:25326916

A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus

DOE PAGES

Lund, C. H.; Bromley, J. R.; Stenbaek, A.; ...

2014-10-18

A growing body of evidence suggests that protein–protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. Wemore » tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. In conclusion, our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta.« less

Architecture of the Mammalian Golgi

PubMed Central

Klumperman, Judith

2011-01-01

Since its first visualization in 1898, the Golgi has been a topic of intense morphological research. A typical mammalian Golgi consists of a pile of stapled cisternae, the Golgi stack, which is a key station for modification of newly synthesized proteins and lipids. Distinct stacks are interconnected by tubules to form the Golgi ribbon. At the entrance site of the Golgi, the cis-Golgi, vesicular tubular clusters (VTCs) form the intermediate between the endoplasmic reticulum and the Golgi stack. At the exit site of the Golgi, the trans-Golgi, the trans-Golgi network (TGN) is the major site of sorting proteins to distinct cellular locations. Golgi functioning can only be understood in light of its complex architecture, as was revealed by a range of distinct electron microscopy (EM) approaches. In this article, a general concept of mammalian Golgi architecture, including VTCs and the TGN, is described. PMID:21502307

Galacturonomannan and Golgi-derived membrane linked to growth and shaping of biogenic calcite

NASA Technical Reports Server (NTRS)

Marsh, M. E.; Ridall, A. L.; Azadi, P.; Duke, P. J.

2002-01-01

The coccolithophores are valuable models for the design and synthesis of composite materials, because the cellular machinery controlling the nucleation, growth, and patterning of their calcitic scales (coccoliths) can be examined genetically. The coccoliths are formed within the Golgi complex and are the major CaCO(3) component in limestone sediments-particularly those of the Cretaceous period. In this study, we describe mutants lacking a sulfated galacturonomannan and show that this polysaccharide in conjunction with the Golgi-derived membrane is directly linked to the growth and shaping of coccolith calcite but not to the initial orientated nucleation of the mineral phase.

Flying saucer1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed mucilage.

PubMed

Voiniciuc, Catalin; Dean, Gillian H; Griffiths, Jonathan S; Kirchsteiger, Kerstin; Hwang, Yeen Ting; Gillett, Alan; Dow, Graham; Western, Tamara L; Estelle, Mark; Haughn, George W

2013-03-01

Pectins are complex polysaccharides that form the gel matrix of the primary cell wall and are abundant in the middle lamella that holds plant cells together. Their degree of methylesterification (DM) impacts wall strength and cell adhesion since unesterified pectin regions can cross-link via Ca(2+) ions to form stronger gels. Here, we characterize flying saucer1 (fly1), a novel Arabidopsis thaliana seed coat mutant, which displays primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence. These defects appear to result from a lower DM in mucilage and are enhanced by the addition of Ca(2+) or completely rescued using alkaline Ca(2+) chelators. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. FLY1 is orthologous to TRANSMEMBRANE UBIQUITIN LIGASE1, a Golgi-localized E3 ligase involved in the quality control of membrane proteins in yeast. However, FLY1-yellow fluorescent protein (YFP) fusions are localized in punctae that are predominantly distinct from the Golgi and the trans-Golgi network/early endosome in the seed coat epidermis. Wortmannin treatment, which induces the fusion of late endosomes in plants, resulted in enlarged FLY1-YFP bodies. We propose that FLY1 regulates the DM of pectin in mucilage, potentially by recycling pectin methylesterase enzymes in the endomembrane system of seed coat epidermal cells.

FLYING SAUCER1 Is a Transmembrane RING E3 Ubiquitin Ligase That Regulates the Degree of Pectin Methylesterification in Arabidopsis Seed Mucilage[W

PubMed Central

Voiniciuc, Cătălin; Dean, Gillian H.; Griffiths, Jonathan S.; Kirchsteiger, Kerstin; Hwang, Yeen Ting; Gillett, Alan; Dow, Graham; Western, Tamara L.; Estelle, Mark; Haughn, George W.

2013-01-01

Pectins are complex polysaccharides that form the gel matrix of the primary cell wall and are abundant in the middle lamella that holds plant cells together. Their degree of methylesterification (DM) impacts wall strength and cell adhesion since unesterified pectin regions can cross-link via Ca2+ ions to form stronger gels. Here, we characterize flying saucer1 (fly1), a novel Arabidopsis thaliana seed coat mutant, which displays primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence. These defects appear to result from a lower DM in mucilage and are enhanced by the addition of Ca2+ or completely rescued using alkaline Ca2+ chelators. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. FLY1 is orthologous to TRANSMEMBRANE UBIQUITIN LIGASE1, a Golgi-localized E3 ligase involved in the quality control of membrane proteins in yeast. However, FLY1–yellow fluorescent protein (YFP) fusions are localized in punctae that are predominantly distinct from the Golgi and the trans-Golgi network/early endosome in the seed coat epidermis. Wortmannin treatment, which induces the fusion of late endosomes in plants, resulted in enlarged FLY1-YFP bodies. We propose that FLY1 regulates the DM of pectin in mucilage, potentially by recycling pectin methylesterase enzymes in the endomembrane system of seed coat epidermal cells. PMID:23482858

HID-1 controls formation of large dense core vesicles by influencing cargo sorting and trans-Golgi network acidification

PubMed Central

Hummer, Blake H.; de Leeuw, Noah F.; Burns, Christian; Chen, Lan; Joens, Matthew S.; Hosford, Bethany; Fitzpatrick, James A. J.; Asensio, Cedric S.

2017-01-01

Large dense core vesicles (LDCVs) mediate the regulated release of neuropeptides and peptide hormones. They form at the trans-Golgi network (TGN), where their soluble content aggregates to form a dense core, but the mechanisms controlling biogenesis are still not completely understood. Recent studies have implicated the peripheral membrane protein HID-1 in neuropeptide sorting and insulin secretion. Using CRISPR/Cas9, we generated HID-1 KO rat neuroendocrine cells, and we show that the absence of HID-1 results in specific defects in peptide hormone and monoamine storage and regulated secretion. Loss of HID-1 causes a reduction in the number of LDCVs and affects their morphology and biochemical properties, due to impaired cargo sorting and dense core formation. HID-1 KO cells also exhibit defects in TGN acidification together with mislocalization of the Golgi-enriched vacuolar H+-ATPase subunit isoform a2. We propose that HID-1 influences early steps in LDCV formation by controlling dense core formation at the TGN. PMID:29074564

Trans-Membrane Area Asymmetry Controls the Shape of Cellular Organelles

PubMed Central

Beznoussenko, Galina V.; Pilyugin, Sergei S.; Geerts, Willie J. C.; Kozlov, Michael M.; Burger, Koert N. J.; Luini, Alberto; Derganc, Jure; Mironov, Alexander A.

2015-01-01

Membrane organelles often have complicated shapes and differ in their volume, surface area and membrane curvature. The ratio between the surface area of the cytosolic and luminal leaflets (trans-membrane area asymmetry (TAA)) determines the membrane curvature within different sites of the organelle. Thus, the shape of the organelle could be critically dependent on TAA. Here, using mathematical modeling and stereological measurements of TAA during fast transformation of organelle shapes, we present evidence that suggests that when organelle volume and surface area are constant, TAA can regulate transformation of the shape of the Golgi apparatus, endosomal multivesicular bodies, and microvilli of brush borders of kidney epithelial cells. Extraction of membrane curvature by small spheres, such as COPI-dependent vesicles within the Golgi (extraction of positive curvature), or by intraluminal vesicles within endosomes (extraction of negative curvature) controls the shape of these organelles. For instance, Golgi tubulation is critically dependent on the fusion of COPI vesicles with Golgi cisternae, and vice versa, for the extraction of membrane curvature into 50–60 nm vesicles, to induce transformation of Golgi tubules into cisternae. Also, formation of intraluminal ultra-small vesicles after fusion of endosomes allows equilibration of their TAA, volume and surface area. Finally, when microvilli of the brush border are broken into vesicles and microvilli fragments, TAA of these membranes remains the same as TAA of the microvilli. Thus, TAA has a significant role in transformation of organelle shape when other factors remain constant. PMID:25761238

Trafficking-deficient hERG K+ channels linked to long QT syndrome are regulated by a microtubule-dependent quality control compartment in the ER

PubMed Central

Smith, Jennifer L.; McBride, Christie M.; Nataraj, Parvathi S.; Bartos, Daniel C.; January, Craig T.

2011-01-01

The human ether-a-go-go related gene (hERG) encodes the voltage-gated K+ channel that underlies the rapidly activating delayed-rectifier current in cardiac myocytes. hERG is synthesized in the endoplasmic reticulum (ER) as an “immature” N-linked glycoprotein and is terminally glycosylated in the Golgi apparatus. Most hERG missense mutations linked to long QT syndrome type 2 (LQT2) reduce the terminal glycosylation and functional expression. We tested the hypothesis that a distinct pre-Golgi compartment negatively regulates the trafficking of some LQT2 mutations to the Golgi apparatus. We found that treating cells in nocodazole, a microtubule depolymerizing agent, altered the subcellular localization, functional expression, and glycosylation of the LQT2 mutation G601S-hERG differently from wild-type hERG (WT-hERG). G601S-hERG quickly redistributed to peripheral compartments that partially colocalized with KDEL (Lys-Asp-Glu-Leu) chaperones but not calnexin, Sec31, or the ER golgi intermediate compartment (ERGIC). Treating cells in E-4031, a drug that increases the functional expression of G601S-hERG, prevented the accumulation of G601S-hERG to the peripheral compartments and increased G601S-hERG colocalization with the ERGIC. Coexpressing the temperature-sensitive mutant G protein from vesicular stomatitis virus, a mutant N-linked glycoprotein that is retained in the ER, showed it was not restricted to the same peripheral compartments as G601S-hERG at nonpermissive temperatures. We conclude that the trafficking of G601S-hERG is negatively regulated by a microtubule-dependent compartment within the ER. Identifying mechanisms that prevent the sorting or promote the release of LQT2 channels from this compartment may represent a novel therapeutic strategy for LQT2. PMID:21490315

In the grass species Brachypodium distachyon, the production of mixed-linkage (1,3;1,4)-β-glucan (MLG) occurs in the Golgi apparatus.

PubMed

Kim, Sang-Jin; Zemelis-Durfee, Starla; Jensen, Jacob Krüger; Wilkerson, Curtis G; Keegstra, Kenneth; Brandizzi, Federica

2018-03-01

Mixed-linkage (1,3;1,4)-β-glucan (MLG) is a glucose polymer with beneficial effects on human health and high potential for the agricultural industry. MLG is present predominantly in the cell wall of grasses and is synthesized by cellulose synthase-like F or H families of proteins, with CSLF6 being the best-characterized MLG synthase. Although the function of this enzyme in MLG production has been established, the site of MLG synthesis in the cell is debated. It has been proposed that MLG is synthesized at the plasma membrane, as occurs for cellulose and callose; in contrast, it has also been proposed that MLG is synthesized in the Golgi apparatus, as occurs for other matrix polysaccharides of the cell wall. Testing these conflicting possibilities is fundamentally important in the general understanding of the biosynthesis of the plant cell wall. Using immuno-localization analyses with MLG-specific antibody in Brachypodium and in barley, we found MLG present in the Golgi, in post-Golgi structures and in the cell wall. Accordingly, analyses of a functional fluorescent protein fusion of CSLF6 stably expressed in Brachypodium demonstrated that the enzyme is localized in the Golgi. We also established that overproduction of MLG causes developmental and growth defects in Brachypodium as also occur in barley. Our results indicated that MLG production occurs in the Golgi similarly to other cell wall matrix polysaccharides, and supports the broadly applicable model in grasses that tight mechanisms control optimal MLG accumulation in the cell wall during development and growth. This work addresses the fundamental question of where mixed linkage (1,3;1,4)-β-glucan (MLG) is synthesized in plant cells. By analyzing the subcellular localization of MLG and MLG synthase in an endogenous system, we demonstrated that MLG synthesis occurs at the Golgi in Brachypodium and barley. A growth inhibition due to overproduced MLG in Brachypodium supports the general applicability of the model that a tight control of the cell wall polysaccharides accumulation is needed to maintain growth homeostasis during development. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Concerted effort of centrosomal and Golgi-derived microtubules is required for proper Golgi complex assembly but not for maintenance

PubMed Central

Vinogradova, Tatiana; Paul, Raja; Grimaldi, Ashley D.; Loncarek, Jadranka; Miller, Paul M.; Yampolsky, Dmitry; Magidson, Valentin; Khodjakov, Alexey; Mogilner, Alex; Kaverina, Irina

2012-01-01

Assembly of an integral Golgi complex is driven by microtubule (MT)-dependent transport. Conversely, the Golgi itself functions as an unconventional MT-organizing center (MTOC). This raises the question of whether Golgi assembly requires centrosomal MTs or can be self-organized, relying on its own MTOC activity. The computational model presented here predicts that each MT population is capable of gathering Golgi stacks but not of establishing Golgi complex integrity or polarity. In contrast, the concerted effort of two MT populations would assemble an integral, polarized Golgi complex. Indeed, while laser ablation of the centrosome did not alter already-formed Golgi complexes, acentrosomal cells fail to reassemble an integral complex upon nocodazole washout. Moreover, polarity of post-Golgi trafficking was compromised under these conditions, leading to strong deficiency in polarized cell migration. Our data indicate that centrosomal MTs complement Golgi self-organization for proper Golgi assembly and motile-cell polarization. PMID:22262454

A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus.

PubMed

Lund, Christian H; Bromley, Jennifer R; Stenbæk, Anne; Rasmussen, Randi E; Scheller, Henrik V; Sakuragi, Yumiko

2015-01-01

A growing body of evidence suggests that protein-protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. We tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. Our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

FAM21 directs SNX27–retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus

PubMed Central

Lee, Seongju; Chang, Jaerak; Blackstone, Craig

2016-01-01

The endosomal network maintains cellular homeostasis by sorting, recycling and degrading endocytosed cargoes. Retromer organizes the endosomal sorting pathway in conjunction with various sorting nexin (SNX) proteins. The SNX27–retromer complex has recently been identified as a major endosomal hub that regulates endosome-to-plasma membrane recycling by preventing lysosomal entry of cargoes. Here, we show that SNX27 directly interacts with FAM21, which also binds retromer, within the Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complex. This interaction is required for the precise localization of SNX27 at an endosomal subdomain as well as for recycling of SNX27-retromer cargoes. Furthermore, FAM21 prevents cargo transport to the Golgi apparatus by controlling levels of phosphatidylinositol 4-phosphate, which facilitates cargo dissociation at the Golgi. Together, our results demonstrate that the SNX27–retromer–WASH complex directs cargoes to the plasma membrane by blocking their transport to lysosomes and the Golgi. PMID:26956659

LARGE, an intellectual disability-associated protein, regulates AMPA-type glutamate receptor trafficking and memory.

PubMed

Seo, Bo Am; Cho, Taesup; Lee, Daniel Z; Lee, Joong-Jae; Lee, Boyoung; Kim, Seong-Wook; Shin, Hee-Sup; Kang, Myoung-Goo

2018-06-18

Mutations in the human LARGE gene result in severe intellectual disability and muscular dystrophy. How LARGE mutation leads to intellectual disability, however, is unclear. In our proteomic study, LARGE was found to be a component of the AMPA-type glutamate receptor (AMPA-R) protein complex, a main player for learning and memory in the brain. Here, our functional study of LARGE showed that LARGE at the Golgi apparatus (Golgi) negatively controlled AMPA-R trafficking from the Golgi to the plasma membrane, leading to down-regulated surface and synaptic AMPA-R targeting. In LARGE knockdown mice, long-term potentiation (LTP) was occluded by synaptic AMPA-R overloading, resulting in impaired contextual fear memory. These findings indicate that the fine-tuning of AMPA-R trafficking by LARGE at the Golgi is critical for hippocampus-dependent memory in the brain. Our study thus provides insights into the pathophysiology underlying cognitive deficits in brain disorders associated with intellectual disability.

Quantitative analysis of intra-Golgi transport shows intercisternal exchange for all cargo

PubMed Central

Dmitrieff, Serge; Rao, Madan; Sens, Pierre

2013-01-01

The mechanisms controlling the transport of proteins through the Golgi stack of mammalian and plant cells is the subject of intense debate, with two models, cisternal progression and intercisternal exchange, emerging as major contenders. A variety of transport experiments have claimed support for each of these models. We reevaluate these experiments using a single quantitative coarse-grained framework of intra-Golgi transport that accounts for both transport models and their many variants. Our analysis makes a definitive case for the existence of intercisternal exchange both for small membrane proteins and large protein complexes––this implies that membrane structures larger than the typical protein-coated vesicles must be involved in transport. Notwithstanding, we find that current observations on protein transport cannot rule out cisternal progression as contributing significantly to the transport process. To discriminate between the different models of intra-Golgi transport, we suggest experiments and an analysis based on our extended theoretical framework that compare the dynamics of transiting and resident proteins. PMID:24019488

FAM21 directs SNX27-retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus.

PubMed

Lee, Seongju; Chang, Jaerak; Blackstone, Craig

2016-03-09

The endosomal network maintains cellular homeostasis by sorting, recycling and degrading endocytosed cargoes. Retromer organizes the endosomal sorting pathway in conjunction with various sorting nexin (SNX) proteins. The SNX27-retromer complex has recently been identified as a major endosomal hub that regulates endosome-to-plasma membrane recycling by preventing lysosomal entry of cargoes. Here, we show that SNX27 directly interacts with FAM21, which also binds retromer, within the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex. This interaction is required for the precise localization of SNX27 at an endosomal subdomain as well as for recycling of SNX27-retromer cargoes. Furthermore, FAM21 prevents cargo transport to the Golgi apparatus by controlling levels of phosphatidylinositol 4-phosphate, which facilitates cargo dissociation at the Golgi. Together, our results demonstrate that the SNX27-retromer-WASH complex directs cargoes to the plasma membrane by blocking their transport to lysosomes and the Golgi.

Content of endoplasmic reticulum and Golgi complex membranes positively correlates with the proliferative status of brain cells.

PubMed

Silvestre, David C; Maccioni, Hugo J F; Caputto, Beatriz L

2009-03-01

Although the molecular and cellular basis of particular events that lead to the biogenesis of membranes in eukaryotic cells has been described in detail, understanding of the intrinsic complexity of the pleiotropic response by which a cell adjusts the overall activity of its endomembrane system to accomplish these requirements is limited. Here we carried out an immunocytochemical and biochemical examination of the content and quality of the endoplasmic reticulum (ER) and Golgi apparatus membranes in two in vivo situations characterized by a phase of active cell proliferation followed by a phase of declination in proliferation (rat brain tissue at early and late developmental stages) or by permanent active proliferation (gliomas and their most malignant manifestation, glioblastomas multiforme). It was found that, in highly proliferative phases of brain development (early embryo brain cells), the content of ER and Golgi apparatus membranes, measured as total lipid phosphorous content, is higher than in adult brain cells. In addition, the concentration of protein markers of ER and Golgi is also higher in early embryo brain cells and in human glioblastoma multiforme cells than in adult rat brain or in nonpathological human brain cells. Results suggest that the amount of endomembranes and the concentration of constituent functional proteins diminish as cells decline in their proliferative activity.

The cerebellar Golgi cell and spatiotemporal organization of granular layer activity

PubMed Central

D'Angelo, Egidio; Solinas, Sergio; Mapelli, Jonathan; Gandolfi, Daniela; Mapelli, Lisa; Prestori, Francesca

2013-01-01

The cerebellar granular layer has been suggested to perform a complex spatiotemporal reconfiguration of incoming mossy fiber signals. Central to this role is the inhibitory action exerted by Golgi cells over granule cells: Golgi cells inhibit granule cells through both feedforward and feedback inhibitory loops and generate a broad lateral inhibition that extends beyond the afferent synaptic field. This characteristic connectivity has recently been investigated in great detail and been correlated with specific functional properties of these neurons. These include theta-frequency pacemaking, network entrainment into coherent oscillations and phase resetting. Important advances have also been made in terms of determining the membrane and synaptic properties of the neuron, and clarifying the mechanisms of activation by input bursts. Moreover, voltage sensitive dye imaging and multi-electrode array (MEA) recordings, combined with mathematical simulations based on realistic computational models, have improved our understanding of the impact of Golgi cell activity on granular layer circuit computations. These investigations have highlighted the critical role of Golgi cells in: generating dense clusters of granule cell activity organized in center-surround structures, implementing combinatorial operations on multiple mossy fiber inputs, regulating transmission gain, and cut-off frequency, controlling spike timing and burst transmission, and determining the sign, intensity and duration of long-term synaptic plasticity at the mossy fiber-granule cell relay. This review considers recent advances in the field, highlighting the functional implications of Golgi cells for granular layer network computation and indicating new challenges for cerebellar research. PMID:23730271

Protein quality control in the early secretory pathway

PubMed Central

Anelli, Tiziana; Sitia, Roberto

2008-01-01

Eukaryotic cells are able to discriminate between native and non-native polypeptides, selectively transporting the former to their final destinations. Secretory proteins are scrutinized at the endoplasmic reticulum (ER)–Golgi interface. Recent findings reveal novel features of the underlying molecular mechanisms, with several chaperone networks cooperating in assisting the maturation of complex proteins and being selectively induced to match changing synthetic demands. ‘Public' and ‘private' chaperones, some of which enriched in specializes subregions, operate for most or selected substrates, respectively. Moreover, sequential checkpoints are distributed along the early secretory pathway, allowing efficiency and fidelity in protein secretion. PMID:18216874

ZFPL1, a novel ring finger protein required for cis-Golgi integrity and efficient ER-to-Golgi transport.

PubMed

Chiu, Chi-Fang; Ghanekar, Yashoda; Frost, Laura; Diao, Aipo; Morrison, Daniel; McKenzie, Eddie; Lowe, Martin

2008-04-09

The Golgi apparatus occupies a central position within the secretory pathway, but the molecular mechanisms responsible for its assembly and organization remain poorly understood. We report here the identification of zinc finger protein-like 1 (ZFPL1) as a novel structural component of the Golgi apparatus. ZFPL1 is a conserved and widely expressed integral membrane protein with two predicted zinc fingers at the N-terminus, the second of which is a likely ring domain. ZFPL1 directly interacts with the cis-Golgi matrix protein GM130. Depletion of ZFPL1 results in the accumulation of cis-Golgi matrix proteins in the intermediate compartment (IC) and the tubulation of cis-Golgi and IC membranes. Loss of ZFPL1 function also impairs cis-Golgi assembly following brefeldin A washout and slows the rate of cargo trafficking into the Golgi apparatus. Effects upon Golgi matrix protein localization and cis-Golgi structure can be rescued by wild-type ZFPL1 but not mutants defective in GM130 binding. Together, these data suggest that ZFPL1 has an important function in maintaining the integrity of the cis-Golgi and that it does so through interactions with GM130.

cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells

PubMed Central

Ito, Yoko; Uemura, Tomohiro; Shoda, Keiko; Fujimoto, Masaru; Ueda, Takashi; Nakano, Akihiko

2012-01-01

The Golgi apparatus forms stacks of cisternae in many eukaryotic cells. However, little is known about how such a stacked structure is formed and maintained. To address this question, plant cells provide a system suitable for live-imaging approaches because individual Golgi stacks are well separated in the cytoplasm. We established tobacco BY-2 cell lines expressing multiple Golgi markers tagged by different fluorescent proteins and observed their responses to brefeldin A (BFA) treatment and BFA removal. BFA treatment disrupted cis, medial, and trans cisternae but caused distinct relocalization patterns depending on the proteins examined. Medial- and trans-Golgi proteins, as well as one cis-Golgi protein, were absorbed into the endoplasmic reticulum (ER), but two other cis-Golgi proteins formed small punctate structures. After BFA removal, these puncta coalesced first, and then the Golgi stacks regenerated from them in the cis-to-trans order. We suggest that these structures have a property similar to the ER-Golgi intermediate compartment and function as the scaffold of Golgi regeneration. PMID:22740633

cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells.

PubMed

Ito, Yoko; Uemura, Tomohiro; Shoda, Keiko; Fujimoto, Masaru; Ueda, Takashi; Nakano, Akihiko

2012-08-01

The Golgi apparatus forms stacks of cisternae in many eukaryotic cells. However, little is known about how such a stacked structure is formed and maintained. To address this question, plant cells provide a system suitable for live-imaging approaches because individual Golgi stacks are well separated in the cytoplasm. We established tobacco BY-2 cell lines expressing multiple Golgi markers tagged by different fluorescent proteins and observed their responses to brefeldin A (BFA) treatment and BFA removal. BFA treatment disrupted cis, medial, and trans cisternae but caused distinct relocalization patterns depending on the proteins examined. Medial- and trans-Golgi proteins, as well as one cis-Golgi protein, were absorbed into the endoplasmic reticulum (ER), but two other cis-Golgi proteins formed small punctate structures. After BFA removal, these puncta coalesced first, and then the Golgi stacks regenerated from them in the cis-to-trans order. We suggest that these structures have a property similar to the ER-Golgi intermediate compartment and function as the scaffold of Golgi regeneration.

Role of N-linked polymannose oligosaccharides in targeting glycoproteins for endoplasmic reticulum-associated degradation.

PubMed

Spiro, R G

2004-05-01

Misfolded or incompletely assembled multisubunit glycoproteins undergo endoplasmic reticulum-associated degradation (ERAD) regulated in large measure by their N-linked polymannose oligosaccharides. In this quality control system lectin interaction with Glc(3)Man(9)GlcNAc(2) glycans after trimming with endoplasmic reticulum (ER) alpha-glucosidases and alpha-mannosidases sorts out persistently unfolded glycoproteins for N-deglycosylation and proteolytic degradation. Monoglucosylated (Glc(1)Man(9)GlcNAc(2)) glycoproteins take part in the calnexin/calreticulin glucosylation-deglucosylation cycle, while the Man(8)GlcNAc(2) isomer B product of ER mannosidase I interacts with EDEM. Proteasomal degradation requires retrotranslocation into the cytosol through a Sec61 channel and deglycosylation by peptide: N-glycosidase (PNGase); in alternate models both PNGase and proteasomes may be either free in the cytosol or ER membrane-imbedded/attached. Numerous proteins appear to undergo nonproteasomal degradation in which deglycosylation and proteolysis take place in the ER lumen. The released free oligosaccharides (OS) are transported to the cytosol as OS-GlcNAc(2) along with similar components produced by the hydrolytic action of the oligosaccharyltransferase, where they together with OS from the proteasomal pathway are trimmed to Man(5)GlcNAc(1) by the action of cytosolic endo-beta- N-acetylglucosaminidase and alpha-mannosidase before entering the lysosomes. Some misfolded glycoproteins can recycle between the ER, intermediate and Golgi compartments, where they are further processed before ERAD. Moreover, properly folded glycoproteins with mannose-trimmed glycans can be deglucosylated in the Golgi by endomannosidase, thereby releasing calreticulin and permitting formation of complex OS. A number of regulatory controls have been described, including the glucosidase-glucosyltransferase shuttle, which controls the level of Glc(3)Man(9)GlcNAc(2)-P-P-Dol, and the unfolded protein response, which enhances synthesis of components of the quality control system.

Expression, sorting, and segregation of Golgi proteins during germ cell differentiation in the testis

PubMed Central

Au, Catherine E.; Hermo, Louis; Byrne, Elliot; Smirle, Jeffrey; Fazel, Ali; Simon, Paul H. G.; Kearney, Robert E.; Cameron, Pamela H.; Smith, Charles E.; Vali, Hojatollah; Fernandez-Rodriguez, Julia; Ma, Kewei; Nilsson, Tommy; Bergeron, John J. M.

2015-01-01

The molecular basis of changes in structure, cellular location, and function of the Golgi apparatus during male germ cell differentiation is unknown. To deduce cognate Golgi proteins, we isolated germ cell Golgi fractions, and 1318 proteins were characterized, with 20 localized in situ. The most abundant protein, GL54D of unknown function, is characterized as a germ cell–specific Golgi-localized type II integral membrane glycoprotein. TM9SF3, also of unknown function, was revealed to be a universal Golgi marker for both somatic and germ cells. During acrosome formation, several Golgi proteins (GBF1, GPP34, GRASP55) localize to both the acrosome and Golgi, while GL54D, TM9SF3, and the Golgi trafficking protein TMED7/p27 are segregated from the acrosome. After acrosome formation, GL54D, TM9SF3, TMED4/p25, and TMED7/p27 continue to mark Golgi identity as it migrates away from the acrosome, while the others (GBF1, GPP34, GRASP55) remain in the acrosome and are progressively lost in later steps of differentiation. Cytoplasmic HSP70.2 and the endoplasmic reticulum luminal protein-folding enzyme PDILT are also Golgi recruited but only during acrosome formation. This resource identifies abundant Golgi proteins that are expressed differentially during mitosis, meiosis, and postacrosome Golgi migration, including the last step of differentiation. PMID:25808494

Golgi Glycosylation

PubMed Central

Stanley, Pamela

2011-01-01

Glycosylation is a very common modification of protein and lipid, and most glycosylation reactions occur in the Golgi. Although the transfer of initial sugar(s) to glycoproteins or glycolipids occurs in the ER or on the ER membrane, the subsequent addition of the many different sugars that make up a mature glycan is accomplished in the Golgi. Golgi membranes are studded with glycosyltransferases, glycosidases, and nucleotide sugar transporters arrayed in a generally ordered manner from the cis-Golgi to the trans-Golgi network (TGN), such that each activity is able to act on specific substrate(s) generated earlier in the pathway. The spectrum of glycosyltransferases and other activities that effect glycosylation may vary with cell type, and thus the final complement of glycans on glycoconjugates is variable. In addition, glycan synthesis is affected by Golgi pH, the integrity of Golgi peripheral membrane proteins, growth factor signaling, Golgi membrane dynamics, and cellular stress. Knowledge of Golgi glycosylation has fostered the development of assays to identify mechanisms of intracellular vesicular trafficking and facilitated glycosylation engineering of recombinant glycoproteins. PMID:21441588

How Do Rab Proteins Determine Golgi Structure?

PubMed Central

Liu, Shijie; Storrie, Brian

2015-01-01

Rab proteins, small GTPases, are key regulators of mammalian Golgi apparatus organization. Based on the effect of Rab activation state, Rab proteins fall into two functional classes. In Class1, inactivation induces Golgi ribbon fragmentation and/or redistribution of Golgi enzymes to the ER, while overexpression of wild type or activation has little, if any, effect on Golgi ribbon organization. In Class 2, the reverse is true. We give emphasis to Rab6, the most abundant Golgi-associated Rab protein. Rab6 depletion in HeLa cells causes an increase in Golgi cisternal number, longer, more continuous cisternae, and a pronounced accumulation of vesicles; the effect of Rab6 on Golgi ribbon organization is probably through regulation of vesicle transport. In effector studies, motor proteins and their regulators are found to be key Rab6 effectors. A related Rab, Rab41, affects Golgi ribbon organization in a contrasting manner. The balance between minus- and plus-end directed motor recruitment may well be the major Rab-dependent factor in Golgi ribbon organization. PMID:25708460

Golgi-associated Rab14, a new regulator for Chlamydia trachomatis infection outcome.

PubMed

Capmany, Anahí; Leiva, Natalia; Damiani, María Teresa

2011-09-01

Chlamydia trachomatis is the causing agent of the most frequent bacterial sexually-transmitted diseases worldwide and is an underlying cause of chronic pelvic inflammatory diseases and cervical cancer. It is an obligate intracellular bacterium that establishes a close relationship with the Golgi complex and parasites the biosynthetic machinery of host cells. In a recent study, we have demonstrated that Rab14, a newly-described Golgi-associated Rab, is involved in the delivery of sphingolipids to the growing bacteria-containing vacuole. The interference with Rab14-controlled trafficking pathways delays chlamydial inclusion enlargement, decreases bacterial lipid uptake, negatively impact on bacterial differentiation, and reduces bacterial progeny and infectivity. C. trachomatis manipulation of host trafficking pathways for the acquisition of endogenously-biosynthesized nutrients arises as one of the characteristics of this highly evolved pathogen. The development of therapeutic strategies targeted to interfere with bacterium-host cell interaction is a new challenge for pharmacological approaches to control chlamydial infections.

Golgi-associated Rab14, a new regulator for Chlamydia trachomatis infection outcome

PubMed Central

Capmany, Anahí; Leiva, Natalia

2011-01-01

Chlamydia trachomatis is the causing agent of the most frequent bacterial sexually-transmitted diseases worldwide and is an underlying cause of chronic pelvic inflammatory diseases and cervical cancer. It is an obligate intracellular bacterium that establishes a close relationship with the Golgi complex and parasites the biosynthetic machinery of host cells. In a recent study, we have demonstrated that Rab14, a newly-described Golgi-associated Rab, is involved in the delivery of sphingolipids to the growing bacteria-containing vacuole. The interference with Rab14-controlled trafficking pathways delays chlamydial inclusion enlargement, decreases bacterial lipid uptake, negatively impact on bacterial differentiation, and reduces bacterial progeny and infectivity. C. trachomatis manipulation of host trafficking pathways for the acquisition of endogenously-biosynthesized nutrients arises as one of the characteristics of this highly evolved pathogen. The development of therapeutic strategies targeted to interfere with bacterium-host cell interaction is a new challenge for pharmacological approaches to control chlamydial infections. PMID:22046472

A role for Sar1 and ARF1 GTPases during Golgi biogenesis in the protozoan parasite Trypanosoma brucei

PubMed Central

Yavuz, Sevil; Warren, Graham

2017-01-01

A single Golgi stack is duplicated and partitioned into two daughter cells during the cell cycle of the protozoan parasite Trypanosoma brucei. The source of components required to generate the new Golgi and the mechanism by which it forms are poorly understood. Using photoactivatable GFP, we show that the existing Golgi supplies components directly to the newly forming Golgi in both intact and semipermeabilized cells. The movement of a putative glycosyltransferase, GntB, requires the Sar1 and ARF1 GTPases in intact cells. In addition, we show that transfer of GntB from the existing Golgi to the new Golgi can be recapitulated in semipermeabilized cells and is sensitive to the GTP analogue GTPγS. We suggest that the existing Golgi is a key source of components required to form the new Golgi and that this process is regulated by small GTPases. PMID:28495798

COPI selectively drives maturation of the early Golgi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Papanikou, Effrosyni; Day, Kasey J.; Austin, II, Jotham

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generatemore » partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Lastly, our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins.« less

COPI selectively drives maturation of the early Golgi

DOE PAGES

Papanikou, Effrosyni; Day, Kasey J.; Austin, II, Jotham; ...

2015-12-28

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generatemore » partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Lastly, our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins.« less

Golgi Localized Barley MTP8 Proteins Facilitate Mn Transport

PubMed Central

Pedas, Pai; Schiller Stokholm, Michaela; Hegelund, Josefine Nymark; Ladegård, Anne Hald; Schjoerring, Jan Kofod; Husted, Søren

2014-01-01

Many metabolic processes in plants are regulated by manganese (Mn) but limited information is available on the molecular mechanisms controlling cellular Mn homeostasis. In this study, a yeast assay was used to isolate and characterize two genes, MTP8.1 and MTP8.2, which encode membrane-bound proteins belonging to the cation diffusion facilitator (CDF) family in the cereal species barley (Hordeum vulgare). Transient expression in onion epidermal cells showed that MTP8.1 and MTP8.2 proteins fused to the green fluorescent protein (GFP) are localized to Golgi. When heterologously expressed in yeast, MTP8.1 and MTP8.2 were found to be Mn transporters catalysing Mn efflux in a similar manner as the Golgi localized endogenous yeast protein Pmr1p. The level of MTP8.1 transcripts in barley roots increased with external Mn supply ranging from deficiency to toxicity, while MTP8.2 transcripts decreased under the same conditions, indicating non-overlapping functions for the two genes. In barley leaves, the expression of both MTP8 genes declined in response to toxic Mn additions to the roots suggesting a role in ensuring proper delivery of Mn to Golgi. Based on the above we suggest that barley MTP8 proteins are involved in Mn loading to the Golgi apparatus and play a role in Mn homeostasis by delivering Mn to Mn-dependent enzymes and/or by facilitating Mn efflux via secretory vesicles. This study highlights the importance of MTP transporters in Mn homeostasis and is the first report of Golgi localized Mn2+ transport proteins in a monocot plant species. PMID:25486417

Partitioning of the Golgi Apparatus during Mitosis in Living HeLa Cells

PubMed Central

Shima, David T.; Haldar, Kasturi; Pepperkok, Rainer; Watson, Rose; Warren, Graham

1997-01-01

The Golgi apparatus of HeLa cells was fluorescently tagged with a green fluorescent protein (GFP), localized by attachment to the NH2-terminal retention signal of N-acetylglucosaminyltransferase I (NAGT I). The location was confirmed by immunogold and immunofluorescence microscopy using a variety of Golgi markers. The behavior of the fluorescent Golgi marker was observed in fixed and living mitotic cells using confocal microscopy. By metaphase, cells contained a constant number of Golgi fragments dispersed throughout the cytoplasm. Conventional and cryoimmunoelectron microscopy showed that the NAGT I–GFP chimera (NAGFP)-positive fragments were tubulo-vesicular mitotic Golgi clusters. Mitotic conversion of Golgi stacks into mitotic clusters had surprisingly little effect on the polarity of Golgi membrane markers at the level of fluorescence microscopy. In living cells, there was little self-directed movement of the clusters in the period from metaphase to early telophase. In late telophase, the Golgi ribbon began to be reformed by a dynamic process of congregation and tubulation of the newly inherited Golgi fragments. The accuracy of partitioning the NAGFP-tagged Golgi was found to exceed that expected for a stochastic partitioning process. The results provide direct evidence for mitotic clusters as the unit of partitioning and suggest that precise regulation of the number, position, and compartmentation of mitotic membranes is a critical feature for the ordered inheritance of the Golgi apparatus. PMID:9182657

Downregulation of the small GTPase SAR1A: a key event underlying alcohol-induced Golgi fragmentation in hepatocytes

PubMed Central

Petrosyan, Armen; Cheng, Pi-Wan; Clemens, Dahn L.; Casey, Carol A.

2015-01-01

The hepatic asialoglycoprotein receptor (ASGP-R) is posttranslationally modified in the Golgi en route to the plasma membrane, where it mediates clearance of desialylated serum glycoproteins. It is known that content of plasma membrane-associated ASGP-R is decreased after ethanol exposure, although the mechanisms remain elusive. Previously, we found that formation of compact Golgi requires dimerization of the largest Golgi matrix protein giantin. We hypothesize that ethanol-impaired giantin function may be related to altered trafficking of ASGP-R. Here we report that in HepG2 cells expressing alcohol dehydrogenase and hepatocytes of ethanol-fed rats, ethanol metabolism results in Golgi disorganization. This process is initiated by dysfunction of SAR1A GTPase followed by altered COPII vesicle formation and impaired Golgi delivery of the protein disulfide isomerase A3 (PDIA3), an enzyme that catalyzes giantin dimerization. Additionally, we show that SAR1A gene silencing in hepatocytes mimics the effect of ethanol: dedimerization of giantin, arresting PDIA3 in the endoplasmic reticulum (ER) and large-scale alterations in Golgi architecture. Ethanol-induced Golgi fission has no effect on ER-to-Golgi transportation of ASGP-R, however, it results in its deposition in cis-medial-, but not trans-Golgi. Thus, alcohol-induced deficiency in COPII vesicle formation predetermines Golgi fragmentation which, in turn, compromises the Golgi-to-plasma membrane transportation of ASGP-R. PMID:26607390

Sac1--Vps74 structure reveals a mechanism to terminate phosphoinositide signaling in the Golgi apparatus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cai, Yiying; Deng, Yongqiang; Horenkamp, Florian

2014-08-25

Sac1 is a phosphoinositide phosphatase of the endoplasmic reticulum and Golgi apparatus that controls organelle membrane composition principally via regulation of phosphatidylinositol 4-phosphate signaling. We present a characterization of the structure of the N-terminal portion of yeast Sac1, containing the conserved Sac1 homology domain, in complex with Vps74, a phosphatidylinositol 4-kinase effector and the orthologue of human GOLPH3. The interface involves the N-terminal subdomain of the Sac1 homology domain, within which mutations in the related Sac3/Fig4 phosphatase have been linked to Charcot–Marie–Tooth disorder CMT4J and amyotrophic lateral sclerosis. Disruption of the Sac1–Vps74 interface results in a broader distribution of phosphatidylinositolmore » 4-phosphate within the Golgi apparatus and failure to maintain residence of a medial Golgi mannosyltransferase. The analysis prompts a revision of the membrane-docking mechanism for GOLPH3 family proteins and reveals how an effector of phosphoinositide signaling serves a dual function in signal termination.« less

Golgi as an MTOC: making microtubules for its own good

PubMed Central

Zhu, Xiaodong; Kaverina, Irina

2013-01-01

In cells, microtubules (MTs) are nucleated at MT-organizing centers (MTOCs). The centrosome-based MTOCs organize radial MT arrays which are often not optimal for polarized trafficking. A recently discovered subset of non-centrosomal MTs nucleated at the Golgi has proven to be indispensable for the Golgi organization, post-Golgi trafficking and cell polarity. Here, we summarize the history of this discovery, known molecular prerequisites of MT nucleation at the Golgi and unique functions of Golgi-derived MTs. PMID:23821162

COPI selectively drives maturation of the early Golgi

PubMed Central

Papanikou, Effrosyni; Day, Kasey J; Austin, Jotham; Glick, Benjamin S

2015-01-01

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generate partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins. DOI: http://dx.doi.org/10.7554/eLife.13232.001 PMID:26709839

Mena–GRASP65 interaction couples actin polymerization to Golgi ribbon linking

PubMed Central

Tang, Danming; Zhang, Xiaoyan; Huang, Shijiao; Yuan, Hebao; Li, Jie; Wang, Yanzhuang

2016-01-01

In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking. PMID:26538023

Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum.

PubMed

Hallée, Stéphanie; Thériault, Catherine; Gagnon, Dominic; Kehrer, Jessica; Frischknecht, Friedrich; Mair, Gunnar R; Richard, Dave

2018-03-26

Compared with other eukaryotic cell types, malaria parasites appear to possess a more rudimentary Golgi apparatus being composed of dispersed, unstacked cis and trans-cisternae. Despite playing a central role in the secretory pathway of the parasite, few Plasmodium Golgi resident proteins have been characterised. We had previously identified a new Golgi resident protein of unknown function, which we had named Golgi Protein 1, and now show that it forms a complex with a previously uncharacterised transmembrane protein (Golgi Protein 2, GP2). The Golgi Protein complex localises to the cis-Golgi throughout the erythrocytic cycle and potentially also during the mosquito stages. Analysis of parasite strains where GP1 expression is conditionally repressed and/or the GP2 gene is inactivated reveals that though the Golgi protein complex is not essential at any stage of the parasite life cycle, it is important for optimal asexual development in the blood stages. © 2018 John Wiley & Sons Ltd.

Non-canonical features of the Golgi apparatus in bipolar epithelial neural stem cells

PubMed Central

Taverna, Elena; Mora-Bermúdez, Felipe; Strzyz, Paulina J.; Florio, Marta; Icha, Jaroslav; Haffner, Christiane; Norden, Caren; Wilsch-Bräuninger, Michaela; Huttner, Wieland B.

2016-01-01

Apical radial glia (aRG), the stem cells in developing neocortex, are unique bipolar epithelial cells, extending an apical process to the ventricle and a basal process to the basal lamina. Here, we report novel features of the Golgi apparatus, a central organelle for cell polarity, in mouse aRGs. The Golgi was confined to the apical process but not associated with apical centrosome(s). In contrast, in aRG-derived, delaminating basal progenitors that lose apical polarity, the Golgi became pericentrosomal. The aRG Golgi underwent evolutionarily conserved, accordion-like compression and extension concomitant with cell cycle-dependent nuclear migration. Importantly, in line with endoplasmic reticulum but not Golgi being present in the aRG basal process, its plasma membrane contained glycans lacking Golgi processing, consistent with direct ER-to-cell surface membrane traffic. Our study reveals hitherto unknown complexity of neural stem cell polarity, differential Golgi contribution to their specific architecture, and fundamental Golgi re-organization upon cell fate change. PMID:26879757

Golgi Membrane Dynamics Viewed Through a Lens of Lipids

PubMed Central

Bankaitis, Vytas A.; Garcia-Mata, Rafael; Mousley, Carl J.

2012-01-01

Summary The striking morphology of the Golgi complex has fascinated cell biologists since its discovery over 100 years ago. Yet, despite intense efforts to understand how membrane flow relates to Golgi form and function, this organelle continues to baffle cell biologists and biochemists alike. Fundamental questions regarding Golgi function, while hotly debated, remain unresolved. While Golgi function is historically described from a protein-centric point of view, we now appreciate that conceptual frameworks for how lipid metabolism is integrated with Golgi biogenesis and function are essential for a mechanistic understanding of this fascinating organelle. It is from a lipid-centric perspective that we discuss the larger question of Golgi dynamics and membrane trafficking. We review the growing body of evidence for how lipid metabolism is integrally written into the engineering of the Golgi system, and highlight questions for future study. PMID:22625862

Onco-Golgi: Is Fragmentation a Gate to Cancer Progression?

PubMed Central

Petrosyan, Armen

2015-01-01

The Golgi apparatus-complex is a highly dynamic organelle which is considered the “heart” of intracellular transportation. Since its discovery by Camillo Golgi in 1873, who described it as the “black reaction,” and despite the enormous volume of publications about Golgi, this apparatus remains one of the most enigmatic of the cytoplasmic organelles. A typical mammalian Golgi consists of a parallel series of flattened, disk-shaped cisternae which align into stacks. The tremendous volume of Golgi-related incoming and outgoing traffic is mediated by different motor proteins, including members of the dynein, kinesin, and myosin families. Yet in spite of the strenuous work it performs, Golgi contrives to maintain its monolithic morphology and orchestration of matrix and residential proteins. However, in response to stress, alcohol, and treatment with many pharmacological drugs over time, Golgi undergoes a kind of disorganization which ranges from mild enlargement to critical scattering. While fragmentation of the Golgi was confirmed in cancer by electron microscopy almost fifty years ago, it is only in recent years that we have begun to understand the significance of Golgi fragmentation in the biology of tumors. Below author would like to focus on how Golgi fragmentation opens the doors for cascades of fatal pathways which may facilitate cancer progression and metastasis. Among the issues addressed will be the most important cancer-specific hallmarks of Golgi fragmentation, including aberrant glycosylation, abnormal expression of the Ras GTPases, dysregulation of kinases, and hyperactivity of myosin motor proteins. PMID:27064441

Multidimensional fractionation is a requirement for quantitation of Golgi-resident glycosylation enzymes from cultured human cells.

PubMed

Lin, Chi-Hung; Chik, Jenny H L; Packer, Nicolle H; Molloy, Mark P

2015-02-06

Glycosylation results from the concerted action of glycosylation enzymes in the secretory pathway. In general, gene expression serves as the primary control mechanism, but post-translational fine-tuning of glycosylation enzyme functions is often necessary for efficient synthesis of specific glycan epitopes. While the field of glycomics has rapidly advanced, there lacks routine proteomic methods to measure expression of specific glycosylation enzymes needed to fill the gap between mRNA expression and the glycomic profile in a "reverse genomics" workflow. Toward developing this workflow we enriched Golgi membranes from two human colon cancer cell lines by sucrose density centrifugation and further mass-based fractionation by SDS-PAGE. We then applied mass spectrometry to demonstrate a doubling in the number of Golgi resident proteins identified, compared to the unenriched, low speed centrifuged supernatant of lysed cells. A total of 35 Golgi-resident glycosylation enzymes, of which 23 were glycosyltransferases, were identified making this the largest protein database so far of Golgi resident glycosylation enzymes experimentally identified in cultured human cells. We developed targeted mass spectrometry assays for specific quantitation of many of these glycosylation enzymes. Our results show that alterations in abundance of glycosylation enzymes at the protein level were generally consistent with the resultant glycomic profiles, but not necessarily with the corresponding glycosyltransferase mRNA expression as exemplified by the case of O-glycan core 1 T synthase.

Fluvastatin delays propagation of viral infection in isolated rat FDB myofibers but does not affect exocytic membrane trafficking.

PubMed

Nevalainen, Mika; Metsikkö, Kalervo

2015-11-01

We have utilized the enveloped viral model to study the effect of fluvastatin on membrane trafficking in isolated rat myofibers. Our immunofluorescence studies constantly showed that infections in myofibers, which were treated with fluvastatin prior and during the infection with either vesicular stomatitis virus (VSV) or influenza A virus, propagated more slowly than in control myofibers without drug treatment. Experiments with a virus expressing Dad1 tagged with green fluorescent protein (GFP-Dad1) showed that fluvastatin did not affect its distribution within the ER/SR network and immunofluorescence staining for GM130 did not show any marked effect on the structure of the Golgi components. Furthermore, fluvastatin did not inhibit trafficking of the chimeric transport marker VSV temperature sensitive G protein (tsG-GFP) from the ER to the Golgi. We next subjected VSV infected myofibers for pulse-chase labeling experiments and found that fluvastatin did not slow down the ER-to-Golgi trafficking or Golgi to plasma membrane trafficking of the viral glycoprotein. These studies show that fluvastatin inhibited the propagation of viral infection in skeletal myofibers but no adverse effect on the exocytic trafficking could be demonstrated. These results suggest that other effects of statins rather than inhibition of ER-to-Golgi trafficking might be behind the myotoxic effects of the statins. © 2015 International Federation for Cell Biology.

Isoform-specific tethering links the Golgi ribbon to maintain compartmentalization

PubMed Central

Jarvela, Timothy; Linstedt, Adam D.

2014-01-01

Homotypic membrane tethering by the Golgi reassembly and stacking proteins (GRASPs) is required for the lateral linkage of mammalian Golgi ministacks into a ribbon-like membrane network. Although GRASP65 and GRASP55 are specifically localized to cis and medial/trans cisternae, respectively, it is unknown whether each GRASP mediates cisternae-specific tethering and whether such specificity is necessary for Golgi compartmentalization. Here each GRASP was tagged with KillerRed (KR), expressed in HeLa cells, and inhibited by 1-min exposure to light. Significantly, inactivation of either GRASP unlinked the Golgi ribbon, and the immediate effect of GRASP65-KR inactivation was a loss of cis- rather than trans-Golgi integrity, whereas inactivation of GRASP55-KR first affected the trans- and not the cis-Golgi. Thus each GRASP appears to play a direct and cisternae-specific role in linking ministacks into a continuous membrane network. To test the consequence of loss of cisternae-specific tethering, we generated Golgi membranes with a single GRASP on all cisternae. Remarkably, the membranes exhibited the full connectivity of wild-type Golgi ribbons but were decompartmentalized and defective in glycan processing. Thus the GRASP isoforms specifically link analogous cisternae to ensure Golgi compartmentalization and proper processing. PMID:24227884

Kinesin is the motor for microtubule-mediated Golgi-to-ER membrane traffic [published errata appear in J Cell Biol 1995 Mar;128(5):following 988 and 1995 May;129(3):893

PubMed Central

1995-01-01

The distribution and dynamics of both the ER and Golgi complex in animal cells are known to be dependent on microtubules; in many cell types the ER extends toward the plus ends of microtubules at the cell periphery and the Golgi clusters at the minus ends of microtubules near the centrosome. In this study we provide evidence that the microtubule motor, kinesin, is present on membranes cycling between the ER and Golgi and powers peripherally directed movements of membrane within this system. Immunolocalization of kinesin at both the light and electron microscopy levels in NRK cells using the H1 monoclonal antibody to kinesin heavy chain, revealed kinesin to be associated with all membranes of the ER/Golgi system. At steady-state at 37 degrees C, however, kinesin was most concentrated on peripherally distributed, pre- Golgi structures containing beta COP and vesicular stomatitis virus glycoprotein newly released from the ER. Upon temperature reduction or nocodazole treatment, kinesin's distribution shifted onto the Golgi, while with brefeldin A (BFA)-treatment, kinesin could be found in both Golgi-derived tubules and in the ER. This suggested that kinesin associates with membranes that constitutively cycle between the ER and Golgi. Kinesin's role on these membranes was examined by microinjecting kinesin antibody. Golgi-to-ER but not ER-to-Golgi membrane transport was found to be inhibited by the microinjected anti-kinesin, suggesting kinesin powers the microtubule plus end-directed recycling of membrane to the ER, and remains inactive on pre-Golgi intermediates that move toward the Golgi complex. PMID:7844144

Golgi apparatus partitioning during cell division.

PubMed

Rabouille, Catherine; Jokitalo, Eija

2003-01-01

This review discusses the mitotic segregation of the Golgi apparatus. The results from classical biochemical and morphological studies have suggested that in mammalian cells this organelle remains distinct during mitosis, although highly fragmented through the formation of mitotic Golgi clusters of small tubules and vesicles. Shedding of free Golgi-derived vesicles would consume Golgi clusters and disperse this organelle throughout the cytoplasm. Vesicles could be partitioned in a stochastic and passive way between the two daughter cells and act as a template for the reassembly of this key organelle. This model has recently been modified by results obtained using GFP- or HRP-tagged Golgi resident enzymes, live cell imaging and electron microscopy. Results obtained with these techniques show that the mitotic Golgi clusters are stable entities throughout mitosis that partition in a microtubule spindle-dependent fashion. Furthermore, a newer model proposes that at the onset of mitosis, the Golgi apparatus completely loses its identity and is reabsorbed into the endoplasmic reticulum. This suggests that the partitioning of the Golgi apparatus is entirely dependent on the partitioning of the endoplasmic reticulum. We critically discuss both models and summarize what is known about the molecular mechanisms underlying the Golgi disassembly and reassembly during and after mitosis. We will also review how the study of the Golgi apparatus during mitosis in other organisms can answer current questions and perhaps reveal novel mechanisms.

Expression of a dominant allele of human ARF1 inhibits membrane traffic in vivo

PubMed Central

1994-01-01

ADP-ribosylation factor (ARF) proteins and inhibitory peptides derived from ARFs have demonstrated activities in a number of in vitro assays that measure ER-to-Golgi and intra-Golgi transport and endosome fusion. To better understand the roles of ARF proteins in vivo, stable cell lines were obtained from normal rat kidney (NRK) cells transfected with either wild-type or a dominant activating allele ([Q71L]) of the human ARF1 gene under the control of the interferon-inducible mouse Mx1 promoter. Upon addition of interferon, expression of ARF1 proteins increased with a half-time of 7-8 h, as determined by immunoblot analysis. Induction of mutant ARF1, but not wild-type ARF1, led to an inhibition of protein secretion with kinetics similar to that observed for induction of protein expression. Examination of the Golgi apparatus and the ER by indirect immunofluorescence or transmission electron microscopy revealed that expression of low levels of mutant ARF1 protein correlated with a dramatic increase in vesiculation of the Golgi apparatus and expansion of the ER lumen, while expression of substantially higher levels of wild-type ARF1 had no discernible effect. Endocytosis was also inhibited by expression of mutant ARF1, but not by the wild-type protein. Finally, the expression of [Q71L]ARF1, but not wild-type ARF1, antagonized the actions of brefeldin A, as determined by the delayed loss of ARF and beta-COP from Golgi membranes and disruption of the Golgi apparatus. General models for the actions of ARF1 in membrane traffic events are discussed. PMID:8294513

Membrane adhesion dictates Golgi stacking and cisternal morphology.

PubMed

Lee, Intaek; Tiwari, Neeraj; Dunlop, Myun Hwa; Graham, Morven; Liu, Xinran; Rothman, James E

2014-02-04

Two classes of proteins that bind to each other and to Golgi membranes have been implicated in the adhesion of Golgi cisternae to each other to form their characteristic stacks: Golgi reassembly and stacking proteins 55 and 65 (GRASP55 and GRASP65) and Golgin of 45 kDa and Golgi matrix protein of 130 kDa. We report here that efficient stacking occurs in the absence of GRASP65/55 when either Golgin is overexpressed, as judged by quantitative electron microscopy. The Golgi stacks in these GRASP-deficient HeLa cells were normal both in morphology and in anterograde cargo transport. This suggests the simple hypothesis that the total amount of adhesive energy gluing cisternae dictates Golgi cisternal stacking, irrespective of which molecules mediate the adhesive process. In support of this hypothesis, we show that adding artificial adhesive energy between cisternae and mitochondria by dimerizing rapamycin-binding domain and FK506-binding protein domains that are attached to cisternal adhesive proteins allows mitochondria to invade the stack and even replace Golgi cisternae within a few hours. These results indicate that although Golgi stacking is a highly complicated process involving a large number of adhesive and regulatory proteins, the overriding principle of a Golgi stack assembly is likely to be quite simple. From this simplified perspective, we propose a model, based on cisternal adhesion and cisternal maturation as the two core principles, illustrating how the most ancient form of Golgi stacking might have occurred using only weak cisternal adhesive processes because of the differential between the rate of influx and outflux of membrane transport through the Golgi.

Membrane adhesion dictates Golgi stacking and cisternal morphology

PubMed Central

Lee, Intaek; Tiwari, Neeraj; Dunlop, Myun Hwa; Graham, Morven; Liu, Xinran; Rothman, James E.

2014-01-01

Two classes of proteins that bind to each other and to Golgi membranes have been implicated in the adhesion of Golgi cisternae to each other to form their characteristic stacks: Golgi reassembly and stacking proteins 55 and 65 (GRASP55 and GRASP65) and Golgin of 45 kDa and Golgi matrix protein of 130 kDa. We report here that efficient stacking occurs in the absence of GRASP65/55 when either Golgin is overexpressed, as judged by quantitative electron microscopy. The Golgi stacks in these GRASP-deficient HeLa cells were normal both in morphology and in anterograde cargo transport. This suggests the simple hypothesis that the total amount of adhesive energy gluing cisternae dictates Golgi cisternal stacking, irrespective of which molecules mediate the adhesive process. In support of this hypothesis, we show that adding artificial adhesive energy between cisternae and mitochondria by dimerizing rapamycin-binding domain and FK506-binding protein domains that are attached to cisternal adhesive proteins allows mitochondria to invade the stack and even replace Golgi cisternae within a few hours. These results indicate that although Golgi stacking is a highly complicated process involving a large number of adhesive and regulatory proteins, the overriding principle of a Golgi stack assembly is likely to be quite simple. From this simplified perspective, we propose a model, based on cisternal adhesion and cisternal maturation as the two core principles, illustrating how the most ancient form of Golgi stacking might have occurred using only weak cisternal adhesive processes because of the differential between the rate of influx and outflux of membrane transport through the Golgi. PMID:24449908

Division of the intermediate compartment at the onset of mitosis provides a mechanism for Golgi inheritance.

PubMed

Marie, Michaël; Dale, Hege A; Kouprina, Nina; Saraste, Jaakko

2012-11-15

As mammalian cells prepare for mitosis, the Golgi ribbon is first unlinked into its constituent stacks and then transformed into spindle-associated, pleiomorphic membrane clusters in a process that remains enigmatic. Also, it remains unclear whether Golgi inheritance involves the incorporation of Golgi enzymes into a pool of coat protein I (COPI) vesicles, or their COPI-independent transfer to the endoplasmic reticulum (ER). Based on the observation that the intermediate compartment (IC) at the ER-Golgi boundary is connected to the centrosome, we examined its mitotic fate and possible role in Golgi breakdown. The use of multiple imaging techniques and markers revealed that the IC elements persist during the M phase, maintain their compositional and structural properties and remain associated with the mitotic spindle, forming circular arrays at the spindle poles. At G2/M transition, the movement of the pericentrosomal domain of the IC (pcIC) to the cell centre and its expansion coincide with the unlinking of the Golgi ribbon. At prophase, coupled to centrosome separation, the pcIC divides together with recycling endosomes, providing novel landmarks for mitotic entry. We provide evidence that the permanent IC elements function as way stations during the COPI-dependent dispersal of Golgi components at prometa- and metaphase, indicating that they correspond to the previously described Golgi clusters. In addition, they continue to communicate with the vesicular 'Golgi haze' and thus are likely to provide templates for Golgi reassembly. These results implicate the IC in mitotic Golgi inheritance, resulting in a model that integrates key features of the two previously proposed pathways.

Image-based drug screen identifies HDAC inhibitors as novel Golgi disruptors synergizing with JQ1

PubMed Central

Gendarme, Mathieu; Baumann, Jan; Ignashkova, Tatiana I.; Lindemann, Ralph K.; Reiling, Jan H.

2017-01-01

The Golgi apparatus is increasingly recognized as a major hub for cellular signaling and is involved in numerous pathologies, including neurodegenerative diseases and cancer. The study of Golgi stress-induced signaling pathways relies on the selectivity of the available tool compounds of which currently only a few are known. To discover novel Golgi-fragmenting agents, transcriptomic profiles of cells treated with brefeldin A, golgicide A, or monensin were generated and compared with a database of gene expression profiles from cells treated with other bioactive small molecules. In parallel, a phenotypic screen was performed for compounds that alter normal Golgi structure. Histone deacetylase (HDAC) inhibitors and DNA-damaging agents were identified as novel Golgi disruptors. Further analysis identified HDAC1/HDAC9 as well as BRD8 and DNA-PK as important regulators of Golgi breakdown mediated by HDAC inhibition. We provide evidence that combinatorial HDACi/(+)-JQ1 treatment spurs synergistic Golgi dispersal in several cancer cell lines, pinpointing a possible link between drug-induced toxicity and Golgi morphology alterations. PMID:29074567

Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy.

PubMed Central

Preuss, D; Mulholland, J; Franzusoff, A; Segev, N; Botstein, D

1992-01-01

The membrane compartments responsible for Golgi functions in wild-type Saccharomyces cerevisiae were identified and characterized by immunoelectron microscopy. Using improved fixation methods, Golgi compartments were identified by labeling with antibodies specific for alpha 1-6 mannose linkages, the Sec7 protein, or the Ypt1 protein. The compartments labeled by each of these antibodies appear as disk-like structures that are apparently surrounded by small vesicles. Yeast Golgi typically are seen as single, isolated cisternae, generally not arranged into parallel stacks. The location of the Golgi structures was monitored by immunoelectron microscopy through the yeast cell cycle. Several Golgi compartments, apparently randomly distributed, were always observed in mother cells. During the initiation of new daughter cells, additional Golgi structures cluster just below the site of bud emergence. These Golgi enter daughter cells at an early stage, raising the possibility that much of the bud's growth might be due to secretory vesicles formed as well as consumed entirely within the daughter. During cytokinesis, the Golgi compartments are concentrated near the site of cell wall synthesis. Clustering of Golgi both at the site of bud formation and at the cell septum suggests that these organelles might be directed toward sites of rapid cell surface growth. Images PMID:1381247

The yeast Golgi apparatus: insights and mysteries

PubMed Central

Papanikou, Effrosyni; Glick, Benjamin S.

2009-01-01

The Golgi apparatus is known to modify and sort newly synthesized secretory proteins. However, fundamental mysteries remain about the structure, operation, and dynamics of this organelle. Important insights have emerged from studying the Golgi in yeasts. For example, yeasts have provided direct evidence for Golgi cisternal maturation, a mechanism that is likely to be broadly conserved. Here, we highlight features of the yeast Golgi as well as challenges that lie ahead. PMID:19879270

Protein Kinase A Activity Is Necessary for Fission and Fusion of Golgi to Endoplasmic Reticulum Retrograde Tubules

PubMed Central

Tenorio, María J.; Luchsinger, Charlotte; Mardones, Gonzalo A.

2015-01-01

It is becoming increasingly accepted that together with vesicles, tubules play a major role in the transfer of cargo between different cellular compartments. In contrast to our understanding of the molecular mechanisms of vesicular transport, little is known about tubular transport. How signal transduction molecules regulate these two modes of membrane transport processes is also poorly understood. In this study we investigated whether protein kinase A (PKA) activity regulates the retrograde, tubular transport of Golgi matrix proteins from the Golgi to the endoplasmic reticulum (ER). We found that Golgi-to-ER retrograde transport of the Golgi matrix proteins giantin, GM130, GRASP55, GRASP65, and p115 was impaired in the presence of PKA inhibitors. In addition, we unexpectedly found accumulation of tubules containing both Golgi matrix proteins and resident Golgi transmembrane proteins. These tubules were still attached to the Golgi and were highly dynamic. Our data suggest that both fission and fusion of retrograde tubules are mechanisms regulated by PKA activity. PMID:26258546

The Hsp90 chaperone complex regulates GDI-dependent Rab recycling.

PubMed

Chen, Christine Y; Balch, William E

2006-08-01

Rab GTPase regulated hubs provide a framework for an integrated coding system, the membrome network, that controls the dynamics of the specialized exocytic and endocytic membrane architectures found in eukaryotic cells. Herein, we report that Rab recycling in the early exocytic pathways involves the heat-shock protein (Hsp)90 chaperone system. We find that Hsp90 forms a complex with guanine nucleotide dissociation inhibitor (GDI) to direct recycling of the client substrate Rab1 required for endoplasmic reticulum (ER)-to-Golgi transport. ER-to-Golgi traffic is inhibited by the Hsp90-specific inhibitors geldanamycin (GA), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), and radicicol. Hsp90 activity is required to form a functional GDI complex to retrieve Rab1 from the membrane. Moreover, we find that Hsp90 is essential for Rab1-dependent Golgi assembly. The observation that the highly divergent Rab GTPases Rab1 involved in ER-to-Golgi transport and Rab3A involved in synaptic vesicle fusion require Hsp90 for retrieval from membranes lead us to now propose that the Hsp90 chaperone system may function as a general regulator for Rab GTPase recycling in exocytic and endocytic trafficking pathways involved in cell signaling and proliferation.

Control of cerebellar granule cell output by sensory-evoked Golgi cell inhibition

PubMed Central

Duguid, Ian; Branco, Tiago; Chadderton, Paul; Arlt, Charlotte; Powell, Kate; Häusser, Michael

2015-01-01

Classical feed-forward inhibition involves an excitation–inhibition sequence that enhances the temporal precision of neuronal responses by narrowing the window for synaptic integration. In the input layer of the cerebellum, feed-forward inhibition is thought to preserve the temporal fidelity of granule cell spikes during mossy fiber stimulation. Although this classical feed-forward inhibitory circuit has been demonstrated in vitro, the extent to which inhibition shapes granule cell sensory responses in vivo remains unresolved. Here we combined whole-cell patch-clamp recordings in vivo and dynamic clamp recordings in vitro to directly assess the impact of Golgi cell inhibition on sensory information transmission in the granule cell layer of the cerebellum. We show that the majority of granule cells in Crus II of the cerebrocerebellum receive sensory-evoked phasic and spillover inhibition prior to mossy fiber excitation. This preceding inhibition reduces granule cell excitability and sensory-evoked spike precision, but enhances sensory response reproducibility across the granule cell population. Our findings suggest that neighboring granule cells and Golgi cells can receive segregated and functionally distinct mossy fiber inputs, enabling Golgi cells to regulate the size and reproducibility of sensory responses. PMID:26432880

Are Rab Proteins the Link Between Golgi Organization and Membrane Trafficking?

PubMed Central

Liu, Shijie; Storrie, Brian

2014-01-01

The fundamental separation of Golgi function between subcompartments termed cisternae is conserved across all eukaryotes. Likewise, Rab proteins, small GTPases of the Ras superfamily, are putative common coordinators of Golgi organization and protein transport. However, despite sequence conservation, e.g., Rab6 and Ypt6 are conserved proteins between humans and yeast, the fundamental organization of the organelle can vary profoundly. In the yeast Sacchromyces cerevisiae, the Golgi cisternae are physically separated from one another while, in mammalian cells, the cisternae are stacked one upon the other. Moreover, in mammalian cells many Golgi stacks are typically linked together to generate a ribbon structure. Do evolutionarily conserved Rab proteins regulate secretory membrane trafficking and diverse Golgi organization in a common manner? In mammalian cells, some Golgi associated Rab proteins function in coordination of protein transport and maintenance of Golgi organization. These include Rab6, Rab33B, Rab1, Rab2, Rab18 and Rab43. In yeast, these include Ypt1, Ypt32 and Ypt6. Here, based on evidence from both yeast and mammalian cells, we speculate on the essential role of Rab proteins in Golgi organization and protein transport. PMID:22581368

Fluorescence recovery after photobleaching measured by confocal microscopy as a tool for the analysis of vesicular lipid transport and plasma membrane mobility

NASA Astrophysics Data System (ADS)

Schmitz, Gerd; Goetz, Alexandra; Orso, Evelyn; Rothe, Gregor

1998-04-01

The vesicular transport of lipids from the endoplasmic reticulum via the Golgi apparatus affects the composition of the plasma membrane. The purpose of our study was to develop an in vitro test system for characterization of vesicular lipid transport kinetics by using confocal microscopy and fluorescence recovery after photobleaching (FRAP). Fibroblasts from two patients homozygous for the hypercatabolic HDL deficiency syndrome Tangier disease and 4 control subjects were pulsed with the C6-NBD-ceramide for 30 minutes. Chase incubation at room temperature resulted in the metabolic accumulation of fluorescent C6-NBD-sphingolyelin and C6-NBD-glycosylceramides in the medial- and trans-Golgi region. Cells were analyzed with an inverted Leica TCS microscope. Calibration was performed through the analysis of diffusion of 50 nm microparticles embedded in media of different viscosity. An acousto optical tunable filter (AOTF) was used for the selective bleaching of the medial- and trans- Golgi region followed by analysis of the fluorescence recovery for 4 minutes. Post-bleach fluorescence recovery through the trans-Golgi-oriented transport of NBD-sphingomyelin was calculated from 2-dimensional scans. Tangier fibroblasts displayed a retarded recovery of fluorescence in the trans- Golgi region. This suggests that the vesicular transport of sphingomyelin and cholesterol is disturbed in Tangier disease confirming data from our laboratory generated with radiometabolites on whole cells. Our data suggest that FRAP analysis allows a sensitive kinetic and spatially resolved analysis of disturbances of vesicular lipid transport.

Molecular Characterization of Human MUC16 (CA125) in Breast Cancer

DTIC Science & Technology

2015-04-01

and O-linked glycosylation takes place as it progresses through the cis-medial-trans Golgi apparatus . In addition, the juxta-membrane ectodomain...swapping experiment. Further, the cleavage of MUC16 was found to take place in the Golgi /post- Golgi compartments and is dependent on the acidic pH in the...therapeutic interventions based on MUC16. 15. SUBJECT TERMS Mucin 16 (MUC16), MUC16-Cter, Golgi /post- Golgi , nuclear localization 16. SECURITY

LIMK1 regulates Golgi dynamics, traffic of Golgi-derived vesicles, and process extension in primary cultured neurons.

PubMed

Rosso, Silvana; Bollati, Flavia; Bisbal, Mariano; Peretti, Diego; Sumi, Tomoyuki; Nakamura, Toshikazu; Quiroga, Santiago; Ferreira, Adriana; Cáceres, Alfredo

2004-07-01

In this study, we examined the subcellular distribution and functions of LIMK1 in developing neurons. Confocal microscopy, subcellular fractionation, and expression of several epitope-tagged LIMK1 constructs revealed that LIMK1 is enriched in the Golgi apparatus and growth cones, with the LIM domain required for Golgi localization and the PDZ domain for its presence at neuritic tips. Overexpression of wild-type LIMK1 suppresses the formation of trans-Golgi derived tubules, and prevents cytochalasin D-induced Golgi fragmentation, whereas that of a kinase-defective mutant has the opposite effect. Transfection of wild-type LIMK1 accelerates axon formation and enhances the accumulation of Par3/Par6, insulin-like growth factor (IGF)1 receptors, and neural cell adhesion molecule (NCAM) at growth cones, while inhibiting the Golgi export of synaptophysin-containing vesicles. These effects were dependent on the Golgi localization of LIMK1, paralleled by an increase in cofilin phosphorylation and phalloidin staining in the region of the Golgi apparatus, and prevented by coexpression of constitutive active cofilin. The long-term overexpression of LIMK1 produces growth cone collapse and axon retraction, an effect that is dependent on its growth cone localization. Together, our results suggest an important role for LIMK1 in axon formation that is related with its ability to regulate Golgi dynamics, membrane traffic, and actin cytoskeletal organization.

Characterization of a cis-Golgi matrix protein, GM130

PubMed Central

1995-01-01

Antisera raised to a detergent- and salt-resistant matrix fraction from rat liver Golgi stacks were used to screen an expression library from rat liver cDNA. A full-length clone was obtained encoding a protein of 130 kD (termed GM130), the COOH-terminal domain of which was highly homologous to a Golgi human auto-antigen, golgin-95 (Fritzler et al., 1993). Biochemical data showed that GM130 is a peripheral cytoplasmic protein that is tightly bound to Golgi membranes and part of a larger oligomeric complex. Predictions from the protein sequence suggest that GM130 is an extended rod-like protein with coiled-coil domains. Immunofluorescence microscopy showed partial overlap with medial- and trans-Golgi markers but almost complete overlap with the cis-Golgi network (CGN) marker, syntaxin5. Immunoelectron microscopy confirmed this location showing that most of the GM130 was located in the CGN and in one or two cisternae on the cis-side of the Golgi stack. GM130 was not re-distributed to the ER in the presence of brefeldin A but maintained its overlap with syntaxin5 and a partial overlap with the ER- Golgi intermediate compartment marker, p53. Together these results suggest that GM130 is part of a cis-Golgi matrix and has a role in maintaining cis-Golgi structure. PMID:8557739

Identification and characterization of GSRP-56, a novel Golgi-localized spectrin repeat-containing protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kobayashi, Yuko; Katanosaka, Yuki; Iwata, Yuko

2006-10-01

Spectrin repeat (SR)-containing proteins are important for regulation of integrity of biomembranes, not only the plasma membrane but also those of intracellular organelles, such as the Golgi, nucleus, endo/lysosomes, and synaptic vesicles. We identified a novel SR-containing protein, named GSRP-56 (Golgi-localized SR-containing protein-56), by a yeast two-hybrid method, using a member of the transient receptor potential channel family, TRPV2, as bait. GSRP-56 is an isoform derived from a giant SR-containing protein, Syne-1 (synaptic nuclear envelope protein-1, also referred to as Nesprin-1 or Enaptin), predicted to be produced by alternative splicing. Immunological analysis demonstrated that this isoform is a 56-kDa protein,more » which is localized predominantly in the Golgi apparatus in cardiomyocytes and C2C12 myoblasts/myotubes, and we found that two SR domains were required both for Golgi targeting and for interaction with TRPV2. Interestingly, overexpression of GSRP-56 resulted in a morphological change in the Golgi structure, characterized by its enlargement of cis-Golgi marker antibody-staining area, which would result partly from fragmentation of Golgi membranes. Our findings indicate that GSRP-56 is a novel, particularly small Golgi-localized member of the spectrin family, which possibly play a role in maintenance of the Golgi structure.« less

Two Novel Rab2 Interactors Regulate Dense-core Vesicle Maturation

PubMed Central

Ailion, Michael; Hannemann, Mandy; Dalton, Susan; Pappas, Andrea; Watanabe, Shigeki; Hegermann, Jan; Liu, Qiang; Han, Hsiao-Fen; Gu, Mingyu; Goulding, Morgan Q.; Sasidharan, Nikhil; Schuske, Kim; Hullett, Patrick; Eimer, Stefan; Jorgensen, Erik M.

2014-01-01

Summary Peptide neuromodulators are released from a unique organelle: the dense-core vesicle. Dense-core vesicles are generated at the trans-Golgi, and then sort cargo during maturation before being secreted. To identify proteins that act in this pathway, we performed a genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We identified two conserved Rab2-binding proteins: RUND-1, a RUN domain protein, and CCCP-1, a coiled-coil protein. RUND-1 and CCCP-1 colocalize with RAB-2 at the Golgi, and rab-2, rund-1 and cccp-1 mutants have similar defects in sorting soluble and transmembrane dense-core vesicle cargos. RUND-1 also interacts with the Rab2 GAP protein TBC-8 and the BAR domain protein RIC-19, a RAB-2 effector. In summary, a new pathway of conserved proteins controls the maturation of dense-core vesicles at the trans-Golgi network. PMID:24698274

Entry and Exit Mechanisms at the cis-Face of the Golgi Complex

PubMed Central

Lorente-Rodríguez, Andrés; Barlowe, Charles

2011-01-01

Vesicular transport of protein and lipid cargo from the endoplasmic reticulum (ER) to cis-Golgi compartments depends on coat protein complexes, Rab GTPases, tethering factors, and membrane fusion catalysts. ER-derived vesicles deliver cargo to an ER-Golgi intermediate compartment (ERGIC) that then fuses with and/or matures into cis-Golgi compartments. The forward transport pathway to cis-Golgi compartments is balanced by a retrograde directed pathway that recycles transport machinery back to the ER. How trafficking through the ERGIC and cis-Golgi is coordinated to maintain organelle structure and function is poorly understood and highlights central questions regarding trafficking routes and organization of the early secretory pathway. PMID:21482742

The Golgi in Cell Migration: Regulation by Signal Transduction and Its Implications for Cancer Cell Metastasis

PubMed Central

Millarte, Valentina; Farhan, Hesso

2012-01-01

Migration and invasion are fundamental features of metastatic cancer cells. The Golgi apparatus, an organelle involved in posttranslational modification and sorting of proteins, is widely accepted to regulate directional cell migration. In addition, mounting evidence suggests that the Golgi is a hub for different signaling pathways. In this paper we will give an overview on how polarized secretion and microtubule nucleation at the Golgi regulate directional cell migration. We will review different signaling pathways that signal to and from the Golgi. Finally, we will discuss how these signaling pathways regulate the role of the Golgi in cell migration and invasion. We propose that by identifying regulators of the Golgi, we might be able to uncover unappreciated modulators of cell migration. Uncovering the regulatory network that orchestrates cell migration is of fundamental importance for the development of new therapeutic strategies against cancer cell metastasis. PMID:22623902

Retrograde traffic from the Golgi to the endoplasmic reticulum.

PubMed

Spang, Anne

2013-06-01

Proteins to be secreted are transported from the endoplasmic reticulum (ER) to the Golgi apparatus. The transport of these proteins requires the localization and activity of proteins that create ER exit sites, coat proteins to collect cargo and to reshape the membrane into a transport container, and address labels--SNARE proteins--to target the vesicles specifically to the Golgi apparatus. In addition some proteins may need export chaperones or export receptors to enable their exit into transport vesicles. ER export factors, SNAREs, and misfolded Golgi-resident proteins must all be retrieved from the Golgi to the ER again. This retrieval is also part of the organellar homeostasis pathway essential to maintaining the identity of the ER and of the Golgi apparatus. In this review, I will discuss the different processes in retrograde transport from the Golgi to the ER and highlight the mechanistic insights we have obtained in the last couple of years.

Retrograde Traffic from the Golgi to the Endoplasmic Reticulum

PubMed Central

Spang, Anne

2013-01-01

Proteins to be secreted are transported from the endoplasmic reticulum (ER) to the Golgi apparatus. The transport of these proteins requires the localization and activity of proteins that create ER exit sites, coat proteins to collect cargo and to reshape the membrane into a transport container, and address labels—SNARE proteins—to target the vesicles specifically to the Golgi apparatus. In addition some proteins may need export chaperones or export receptors to enable their exit into transport vesicles. ER export factors, SNAREs, and misfolded Golgi-resident proteins must all be retrieved from the Golgi to the ER again. This retrieval is also part of the organellar homeostasis pathway essential to maintaining the identity of the ER and of the Golgi apparatus. In this review, I will discuss the different processes in retrograde transport from the Golgi to the ER and highlight the mechanistic insights we have obtained in the last couple of years. PMID:23732476

Golgi Enrichment and Proteomic Analysis of Developing Pinus radiata Xylem by Free-Flow Electrophoresis

PubMed Central

Macdonald, Lucy J.; Adams, Paul D.; Petzold, Christopher J.; Strabala, Timothy J.; Wagner, Armin; Heazlewood, Joshua L.

2013-01-01

Our understanding of the contribution of Golgi proteins to cell wall and wood formation in any woody plant species is limited. Currently, little Golgi proteomics data exists for wood-forming tissues. In this study, we attempted to address this issue by generating and analyzing Golgi-enriched membrane preparations from developing xylem of compression wood from the conifer Pinus radiata. Developing xylem samples from 3-year-old pine trees were harvested for this purpose at a time of active growth and subjected to a combination of density centrifugation followed by free flow electrophoresis, a surface charge separation technique used in the enrichment of Golgi membranes. This combination of techniques was successful in achieving an approximately 200-fold increase in the activity of the Golgi marker galactan synthase and represents a significant improvement for proteomic analyses of the Golgi from conifers. A total of thirty known Golgi proteins were identified by mass spectrometry including glycosyltransferases from gene families involved in glucomannan and glucuronoxylan biosynthesis. The free flow electrophoresis fractions of enriched Golgi were highly abundant in structural proteins (actin and tubulin) indicating a role for the cytoskeleton during compression wood formation. The mass spectrometry proteomics data associated with this study have been deposited to the ProteomeXchange with identifier PXD000557. PMID:24416096

Mena-GRASP65 interaction couples actin polymerization to Golgi ribbon linking.

PubMed

Tang, Danming; Zhang, Xiaoyan; Huang, Shijiao; Yuan, Hebao; Li, Jie; Wang, Yanzhuang

2016-01-01

In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking. © 2016 Tang et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Rab6a/a’ Are Important Golgi Regulators of Pro-Inflammatory TNF Secretion in Macrophages

PubMed Central

Micaroni, Massimo; Stanley, Amanda C.; Khromykh, Tatiana; Venturato, Juliana; Wong, Colin X. F.; Lim, Jet P.; Marsh, Brad J.; Storrie, Brian; Gleeson, Paul A.; Stow, Jennifer L.

2013-01-01

Lipopolysaccharide (LPS)-activated macrophages secrete pro-inflammatory cytokines, including tumor necrosis factor (TNF) to elicit innate immune responses. Secretion of these cytokines is also a major contributing factor in chronic inflammatory disease. In previous studies we have begun to elucidate the pathways and molecules that mediate the intracellular trafficking and secretion of TNF. Rab6a and Rab6a' (collectively Rab6) are trans-Golgi-localized GTPases known for roles in maintaining Golgi structure and Golgi-associated trafficking. We found that induction of TNF secretion by LPS promoted the selective increase of Rab6 expression. Depletion of Rab6 (via siRNA and shRNA) resulted in reorganization of the Golgi ribbon into more compact structures that at the resolution of electron microcopy consisted of elongated Golgi stacks that likely arose from fusion of smaller Golgi elements. Concomitantly, the delivery of TNF to the cell surface and subsequent release into the media was reduced. Dominant negative mutants of Rab6 had similar effects in disrupting TNF secretion. In live cells, Rab6–GFP were localized on trans-Golgi network (TGN)-derived tubular carriers demarked by the golgin p230. Rab6 depletion and inactive mutants altered carrier egress and partially reduced p230 membrane association. Our results show that Rab6 acts on TNF trafficking at the level of TGN exit in tubular carriers and our findings suggest Rab6 may stabilize p230 on the tubules to facilitate TNF transport. Both Rab6 isoforms are needed in macrophages for Golgi stack organization and for the efficient post-Golgi transport of TNF. This work provides new insights into Rab6 function and into the role of the Golgi complex in cytokine secretion in inflammatory macrophages. PMID:23437303

Isolation and Proteomic Characterization of the Arabidopsis Golgi Defines Functional and Novel Components Involved in Plant Cell Wall Biosynthesis1[W][OA

PubMed Central

Parsons, Harriet T.; Christiansen, Katy; Knierim, Bernhard; Carroll, Andrew; Ito, Jun; Batth, Tanveer S.; Smith-Moritz, Andreia M.; Morrison, Stephanie; McInerney, Peter; Hadi, Masood Z.; Auer, Manfred; Mukhopadhyay, Aindrila; Petzold, Christopher J.; Scheller, Henrik V.; Loqué, Dominique; Heazlewood, Joshua L.

2012-01-01

The plant Golgi plays a pivotal role in the biosynthesis of cell wall matrix polysaccharides, protein glycosylation, and vesicle trafficking. Golgi-localized proteins have become prospective targets for reengineering cell wall biosynthetic pathways for the efficient production of biofuels from plant cell walls. However, proteomic characterization of the Golgi has so far been limited, owing to the technical challenges inherent in Golgi purification. In this study, a combination of density centrifugation and surface charge separation techniques have allowed the reproducible isolation of Golgi membranes from Arabidopsis (Arabidopsis thaliana) at sufficiently high purity levels for in-depth proteomic analysis. Quantitative proteomic analysis, immunoblotting, enzyme activity assays, and electron microscopy all confirm high purity levels. A composition analysis indicated that approximately 19% of proteins were likely derived from contaminating compartments and ribosomes. The localization of 13 newly assigned proteins to the Golgi using transient fluorescent markers further validated the proteome. A collection of 371 proteins consistently identified in all replicates has been proposed to represent the Golgi proteome, marking an appreciable advancement in numbers of Golgi-localized proteins. A significant proportion of proteins likely involved in matrix polysaccharide biosynthesis were identified. The potential within this proteome for advances in understanding Golgi processes has been demonstrated by the identification and functional characterization of the first plant Golgi-resident nucleoside diphosphatase, using a yeast complementation assay. Overall, these data show key proteins involved in primary cell wall synthesis and include a mixture of well-characterized and unknown proteins whose biological roles and importance as targets for future research can now be realized. PMID:22430844

Rab6a/a' are important Golgi regulators of pro-inflammatory TNF secretion in macrophages.

PubMed

Micaroni, Massimo; Stanley, Amanda C; Khromykh, Tatiana; Venturato, Juliana; Wong, Colin X F; Lim, Jet P; Marsh, Brad J; Storrie, Brian; Gleeson, Paul A; Stow, Jennifer L

2013-01-01

Lipopolysaccharide (LPS)-activated macrophages secrete pro-inflammatory cytokines, including tumor necrosis factor (TNF) to elicit innate immune responses. Secretion of these cytokines is also a major contributing factor in chronic inflammatory disease. In previous studies we have begun to elucidate the pathways and molecules that mediate the intracellular trafficking and secretion of TNF. Rab6a and Rab6a' (collectively Rab6) are trans-Golgi-localized GTPases known for roles in maintaining Golgi structure and Golgi-associated trafficking. We found that induction of TNF secretion by LPS promoted the selective increase of Rab6 expression. Depletion of Rab6 (via siRNA and shRNA) resulted in reorganization of the Golgi ribbon into more compact structures that at the resolution of electron microcopy consisted of elongated Golgi stacks that likely arose from fusion of smaller Golgi elements. Concomitantly, the delivery of TNF to the cell surface and subsequent release into the media was reduced. Dominant negative mutants of Rab6 had similar effects in disrupting TNF secretion. In live cells, Rab6-GFP were localized on trans-Golgi network (TGN)-derived tubular carriers demarked by the golgin p230. Rab6 depletion and inactive mutants altered carrier egress and partially reduced p230 membrane association. Our results show that Rab6 acts on TNF trafficking at the level of TGN exit in tubular carriers and our findings suggest Rab6 may stabilize p230 on the tubules to facilitate TNF transport. Both Rab6 isoforms are needed in macrophages for Golgi stack organization and for the efficient post-Golgi transport of TNF. This work provides new insights into Rab6 function and into the role of the Golgi complex in cytokine secretion in inflammatory macrophages.

Nordihydroguaiaretic acid blocks protein transport in the secretory pathway causing redistribution of Golgi proteins into the endoplasmic reticulum.

PubMed

Fujiwara, T; Takami, N; Misumi, Y; Ikehara, Y

1998-01-30

We have investigated the effect of nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, on the intracellular protein transport and the structure of the Golgi complex. Pulse-chase experiments and immunoelectron microscopy showed that NDGA strongly inhibits the transport of newly synthesized secretory proteins to the Golgi complex resulting in their accumulation in the endoplasmic reticulum (ER). Despite their retention in the ER, oligosaccharides of secretory and ER-resident proteins were processed to endoglycosidase H-resistant forms, raising the possibility that oligosaccharide-processing enzymes are redistributed from the Golgi to the ER. Morphological observations further revealed that alpha-mannosidase II (a cis/medial-Golgi marker), but not TGN38 (a trans-Golgi network marker), rapidly redistributes to the ER in the presence of NDGA, resulting in the disappearance of the characteristic Golgi structure. Upon removal of the drug, the Golgi complex was reassembled into the normal structure as judged by perinuclear staining of alpha-mannosidase II and by restoration of the secretion. These effects of NDGA are quite similar to those of brefeldin A. However, unlike brefeldin A, NDGA did not cause a dissociation of beta-coatomer protein, a subunit of coatomer, from the Golgi membrane. On the contrary, NDGA exerted the stabilizing effect on beta-coatomer protein/membrane interaction against the dissociation caused by brefeldin A and ATP depletion. Taken together, these results indicate that NDGA is a potent agent disrupting the structure and function of the Golgi complex with a mechanism different from those known for other drugs reported so far.

Characterization of a mammalian Golgi-localized protein complex, COG, that is required for normal Golgi morphology and function

PubMed Central

Ungar, Daniel; Oka, Toshihiko; Brittle, Elizabeth E.; Vasile, Eliza; Lupashin, Vladimir V.; Chatterton, Jon E.; Heuser, John E.; Krieger, Monty; Waters, M. Gerard

2002-01-01

Multiprotein complexes are key determinants of Golgi apparatus structure and its capacity for intracellular transport and glycoprotein modification. Three complexes that have previously been partially characterized include (a) the Golgi transport complex (GTC), identified in an in vitro membrane transport assay, (b) the ldlCp complex, identified in analyses of CHO cell mutants with defects in Golgi-associated glycosylation reactions, and (c) the mammalian Sec34 complex, identified by homology to yeast Sec34p, implicated in vesicular transport. We show that these three complexes are identical and rename them the conserved oligomeric Golgi (COG) complex. The COG complex comprises four previously characterized proteins (Cog1/ldlBp, Cog2/ldlCp, Cog3/Sec34, and Cog5/GTC-90), three homologues of yeast Sec34/35 complex subunits (Cog4, -6, and -8), and a previously unidentified Golgi-associated protein (Cog7). EM of ldlB and ldlC mutants established that COG is required for normal Golgi morphology. “Deep etch” EM of purified COG revealed an ∼37-nm-long structure comprised of two similarly sized globular domains connected by smaller extensions. Consideration of biochemical and genetic data for mammalian COG and its yeast homologue suggests a model for the subunit distribution within this complex, which plays critical roles in Golgi structure and function. PMID:11980916

GOLGI IN COPPER HOMEOSTASIS: A VIEW FROM THE MEMBRANE TRAFFICKING FIELD

PubMed Central

Polishchuk, Roman; Lutsenko, Svetlana

2013-01-01

Copper is essential for a variety of important biological processes as a cofactor and regulator of many enzymes. Incorporation of copper into the secreted and plasma membrane-targeted cuproenzymes takes place in Golgi, a compartment central for normal copper homeostasis. The Golgi complex harbors copper-transporting ATPases, ATP7A and ATP7B, that transfer copper from the cytosol into Golgi lumen for incorporation into copper-dependent enzymes. The Golgi complex also sends these ATPases to appropriate post-Golgi destinations to ensure correct Cu fluxes in the body and to avoid potentially toxic copper accumulation. Mutations in ATP7A or ATP7B or in the proteins that regulate their trafficking affect their exit from Golgi or subsequent retrieval to this organelle. This, in turn, disrupts the homeostatic Cu balance, resulting in copper deficiency (Menkes disease) or copper overload (Wilson disease). Research over the last decade has yielded significant insights into the enzymatic properties and cell biology of the copper-ATPases. However, the mechanisms through which the Golgi regulates trafficking of ATP7A/7B and, therefore, maintain Cu homeostasis remain unclear. This review summarizes current data on the role of the Golgi in Cu metabolism and outlines questions and challenges that should be addressed to understand ATP7A and ATP7B trafficking mechanisms in health and disease. PMID:23846821

GOLGI FRACTIONS PREPARED FROM RAT LIVER HOMOGENATES

PubMed Central

Ehrenreich, J. H.; Bergeron, J. J. M.; Siekevitz, P.; Palade, G. E.

1973-01-01

In devising a new procedure for the isolation of Golgi fractions from rat liver homogenates, we have taken advantage of the overloading with very low density lipoprotein (VLDL) particles that occurs in the Golgi elements of hepatocytes ∼90 min after ethanol is administered (0.6 g/100 g body weight) by stomach tube to the animals. The VLDLs act as morphological markers as well as density modifiers of these elements. The starting preparation is a total microsomal fraction prepared from liver homogenized (1:5) in 0.25 M sucrose. This fraction is resuspended in 1.15 M sucrose and loaded at the bottom of a discontinuous sucrose density gradient. Centrifugation at ∼13 x 106 g·min yields by flotation three Golgi fractions of density >1.041 and <1.173. The light and intermediate fractions consist essentially of VLDL-loaded Golgi vacuoles and cisternae. Nearly empty, often collapsed, Golgi cisternae are the main component of the heavy fraction. A procedure which subjects the Golgi fractions to hypotonic shock and shearing in a French press at pH 8.5 allows the extraction of the content of the Golgi elements and the subsequent isolation of their membranes by differential centrifugation. PMID:4356571

MTCL1 crosslinks and stabilizes non-centrosomal microtubules on the Golgi membrane.

PubMed

Sato, Yoshinori; Hayashi, Kenji; Amano, Yoshiko; Takahashi, Mikiko; Yonemura, Shigenobu; Hayashi, Ikuko; Hirose, Hiroko; Ohno, Shigeo; Suzuki, Atsushi

2014-11-04

Recent studies have revealed the presence of a microtubule subpopulation called Golgi-derived microtubules that support Golgi ribbon formation, which is required for maintaining polarized cell migration. CLASPs and AKAP450/CG-NAP are involved in their formation, but the underlying molecular mechanisms remain unclear. Here, we find that the microtubule-crosslinking protein, MTCL1, is recruited to the Golgi membranes through interactions with CLASPs and AKAP450/CG-NAP, and promotes microtubule growth from the Golgi membrane. Correspondingly, MTCL1 knockdown specifically impairs the formation of the stable perinuclear microtubule network to which the Golgi ribbon tethers and extends. Rescue experiments demonstrate that besides its crosslinking activity mediated by the N-terminal microtubule-binding region, the C-terminal microtubule-binding region plays essential roles in these MTCL1 functions through a novel microtubule-stabilizing activity. These results suggest that MTCL1 cooperates with CLASPs and AKAP450/CG-NAP in the formation of the Golgi-derived microtubules, and mediates their development into a stable microtubule network.

ACBD3 functions as a scaffold to organize the Golgi stacking proteins and a Rab33b-GAP.

PubMed

Yue, Xihua; Bao, Mengjing; Christiano, Romain; Li, Siyang; Mei, Jia; Zhu, Lianhui; Mao, Feifei; Yue, Qiang; Zhang, Panpan; Jing, Shuaiyang; Rothman, James E; Qian, Yi; Lee, Intaek

2017-09-01

Golgin45 plays important roles in Golgi stack assembly and is known to bind both the Golgi stacking protein GRASP55 and Rab2 in the medial-Golgi cisternae. In this study, we sought to further characterize the cisternal adhesion complex using a proteomics approach. We report here that Acyl-CoA binding domain containing 3 (ACBD3) is likely to be a novel binding partner of Golgin45. ACBD3 interacts with Golgin45 via its GOLD domain, while its co-expression significantly increases Golgin45 targeting to the Golgi. Furthermore, ACBD3 recruits TBC1D22, a Rab33b GTPase activating protein (GAP), to a large multi-protein complex containing Golgin45 and GRASP55. These results suggest that ACBD3 may provide a scaffolding to organize the Golgi stacking proteins and a Rab33b-GAP at the medial-Golgi. © 2017 Federation of European Biochemical Societies.

Phospho-eNOS Ser-1176 is associated with the nucleoli and the Golgi complex in C6 rat glioma cells.

PubMed

Klinz, Franz-Josef; Herberg, Natalie; Arnhold, Stefan; Addicks, Klaus; Bloch, Wilhelm

2007-06-29

Enzymatic activity of endothelial nitric oxide synthase (eNOS) is controlled by posttranslational modifications, protein-protein interactions, and subcellular localization. For example, N-terminal fatty acid modifications target eNOS to the Golgi complex where it becomes phosphorylated. We show here by immunofluorescence analysis that phospho-eNOS Ser-1176 is enriched in the perinuclear region of interphase C6 rat glioma cells. Confocal double immunofluorescence microscopy with the Golgi marker protein 58K revealed that phospho-eNOS Ser-1176 is associated with the Golgi complex. Surprisingly, we observed several spots in the nucleus of C6 cells that were positive for phospho-eNOS Ser-1176. Confocal double immunofluorescence analysis with the nucleolus marker protein fibrillarin revealed that within the nucleus phospho-eNOS Ser-1176 is exclusively associated with the nucleoli. It is known that in mitotic cells nucleoli are lost during prophase and rebuild during telophase. In agreement with this, we find no nucleoli-like distribution of phospho-eNOS Ser-1176 in metaphase and anaphase C6 glioma cells. Our finding that phospho-eNOS Ser-1176 is selectively associated with the nucleoli points to a so far unknown role for eNOS in interphase glioma cells.

Acute Depletion of Diacylglycerol from the Cis-Golgi Affects Localised Nuclear Envelope Morphology During Mitosis.

PubMed

Chung, Gary Hong Chun; Domart, Marie Charlotte; Peddie, Christopher; Mantell, Judith; Mclaverty, Kieran; Arabiotorre, Angela; Hodgson, Lorna; Byrne, Richard D; Verkade, Paul; Arkill, Kenton; Collinson, Lucy M; Larijani, Banafshe

2018-06-12

Dysregulation of nuclear envelope (NE) assembly results in various cancers; for example, renal and some lung carcinomas ensue due to NE malformation. The NE is a dynamic membrane compartment and its completion during mitosis, is a highly regulated process but the detailed mechanism still remains incompletely understood. Previous studies have found isolated diacylglycerol (DAG) containing vesicles are essential for completing the fusion of NE in non-somatic cells. We investigated the impact of DAG depletion from cis-Golgi in mammalian cells on NE reassembly. Using advanced electron microscopy, we observed an enriched DAG population of vesicles at the vicinity of the NE gaps of telophase mammalian cells. We applied a miniSOG-C1-domain tag that localized DAG-enriched vesicles at the perinuclear region, which suggested the existence of NE fusogenic vesicles. We quantified the impact of Golgi-DAG depletion by measuring the in situ NE rim curvature of the re-forming NE. The rim curvature in these cells was significantly reduced compared with controls, which indicated a localized defect in NE morphology. Our novel results demonstrate the significance of the role of DAG from cis-Golgi for the regulation of NE assembly. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Study of ethanol-induced Golgi disorganization reveals the potential mechanism of alcohol-impaired N-glycosylation

PubMed Central

Casey, Carol A.; Bhat, Ganapati; Holzapfel, Melissa S.; Petrosyan, Armen

2016-01-01

Background It is known that ethanol (EtOH) and its metabolites have a negative effect on protein glycosylation. The fragmentation of the Golgi apparatus induced by alteration of the structure of largest Golgi matrix protein, giantin, is the major consequence of damaging effects of EtOH-metabolism on the Golgi, however, the link between this and abnormal glycosylation remains unknown. Because previously we have shown that Golgi morphology dictates glycosylation, we examined the effect EtOH administration has on function of Golgi residential enzymes involved in N-glycosylation. Methods HepG2 cells transfected with mouse ADH1 (VA-13 cells) were treated with 35 mM ethanol for 72 h. Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8 weeks. Characterization of Golgi-associated mannosyl (α-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (MGAT1), α-1,2-mannosidase (Man-I) and α-mannosidase II (Man-II) were performed in VA-13 cells and rat hepatocytes followed by 3D Structured Illumination Microscopy (SIM). Results First, we detected that EtOH administration results in the loss of sialylated N-glycans on asialoglycoprotein receptor, however the high mannose-type N-glycans are increased. Further analysis by 3D SIM microscopy revealed that EtOH treatment despite Golgi disorganization does not change cis-Golgi localization for Man-I, but does induce medial-to-cis relocation of MGAT1 and Man-II. Using different approaches, including electron microscopy, we revealed that EtOH treatment results in dysfunction of Arf1 GTPase followed by a deficiency in COPI vesicles at the Golgi. Silencing beta-COP or expression of GDP-bound mutant Arf1(T31N) mimics the EtOH effect on retaining MGAT1 and Man-II at the cis-Golgi, suggesting that (a) EtOH specifically blocks activation of Arf1, and (b) EtOH alters the proper localization of Golgi enzymes through impairment of COPI. Importantly, the level of MGAT1 was reduced, because likely MGAT1, contrary to Man-I and Man-II, is giantin-sensitive. Conclusions Thus, we provide the mechanism by which EtOH-induced Golgi remodeling may significantly modify formation of N-glycans. PMID:27748959

The extent of polylactosamine glycosylation of MDCK LAMP-2 is determined by its Golgi residence time.

PubMed

Nabi, I R; Dennis, J W

1998-09-01

The increased polylactosamine glycosylation of LAMP-2 in MDCK cells cultured for 1 day relative to cells cultured for 3 days has been correlated with its slower rate of Golgi transit (Nabi and Rodriguez-Boulan, 1993, Mol. Biol. Cell., 4, 627-635). To determine if the differential polylactosamine glycosylation of LAMP-2 is a consequence of glycosyltransferase expression levels, the activities of beta1-6GlcNAc-TV, beta1-3GlcNAc-T(i), beta1-2GlcNAc-TI, beta1, 4Gal-T, alpha2-6sialyl-T, and alpha2-3sialyl-T were assayed and no significant differences in the activities of these enzymes in 1 and 3 day cell extracts were detected. During MDCK epithelial polarization, the Golgi apparatus undergoes morphological changes and apiconuclear Golgi networks were more evident in 3 day cells. Treatment with nocodazole disrupted Golgi networks and generated numerous Golgi clusters in both 1 day and 3 day cells. In the presence of nocodazole the differential migration of LAMP-2 in 1 and 3 day MDCK cells was maintained and could be eliminated by treatment with endo-beta-galactosidase, indicating that gross Golgi morphology did not influence the extent of LAMP-2 polylactosamine glycosylation. Nocodazole treatment did, however, result in the faster migration of LAMP-2 which was not due to modification of core N-glycans as the precursor form of the glycoprotein migrated with an identical molecular size. Following incubation at 20 degrees C, which prevents the exit of proteins from the trans-Golgi network, the molecular size of LAMP-2 increased to a similar extent in both 1 and 3 day MDCK cells. Extending the time of incubation at 20 degrees C did not influence the size of LAMP-2, demonstrating that its glycosylation is modified not by its retention within the Golgi but rather by its equivalent slower Golgi passage at the lower temperature in both 1 and 3 day cells. An identical effect was observed in nocodazole treated cells, demonstrating that Golgi residence time determines the extent of LAMP-2 polylactosamine glycosylation, even in isolated Golgi clusters.

Oncogenic signaling by Kit tyrosine kinase occurs selectively on the Golgi apparatus in gastrointestinal stromal tumors

PubMed Central

Obata, Y; Horikawa, K; Takahashi, T; Akieda, Y; Tsujimoto, M; Fletcher, J A; Esumi, H; Nishida, T; Abe, R

2017-01-01

Gastrointestinal stromal tumors (GISTs) are caused by gain-of-function mutations in the Kit receptor tyrosine kinase. Most primary GIST patients respond to the Kit inhibitor imatinib, but this drug often becomes ineffective because of secondary mutations in the Kit kinase domain. The characteristic intracellular accumulation of imatinib-sensitive and -resistant Kit protein is well documented, but its relationship to oncogenic signaling remains unknown. Here, we show that in cancer tissue from primary GIST patients as well as in cell lines, mutant Kit accumulates on the Golgi apparatus, whereas normal Kit localizes to the plasma membrane (PM). In imatinib-resistant GIST with a secondary Kit mutation, Kit localizes predominantly on the Golgi apparatus. Both imatinib-sensitive and imatinib-resistant Kit (Kit(mut)) become fully auto-phosphorylated only on the Golgi and only if in a complex-glycosylated form. Kit(mut) accumulates on the Golgi during the early secretory pathway, but not after endocytosis. The aberrant kinase activity of Kit(mut) prevents its export from the Golgi to the PM. Furthermore, Kit(mut) on the Golgi signals and activates the phosphatidylinositol 3-kinase–Akt (PI3K–Akt) pathway, signal transducer and activator of transcription 5 (STAT5), and the Mek–Erk pathway. Blocking the biosynthetic transport of Kit(mut) to the Golgi from the endoplasmic reticulum inhibits oncogenic signaling. PM localization of Kit(mut) is not required for its signaling. Activation of Src-family tyrosine kinases on the Golgi is essential for oncogenic Kit signaling. These results suggest that the Golgi apparatus serves as a platform for oncogenic Kit signaling. Our study demonstrates that Kit(mut)’s pathogenicity is related to its mis-localization, and may offer a new strategy for treating imatinib-resistant GISTs. PMID:28192400

Arabidopsis COG Complex Subunits COG3 and COG8 Modulate Golgi Morphology, Vesicle Trafficking Homeostasis and Are Essential for Pollen Tube Growth

PubMed Central

Li, Yingxin; Li, Pengxiang; Gao, Caiji; Ding, Yu; Lan, Zhiyi; Shi, Zhixuan; Rui, Qingchen; Feng, Yihong; Liu, Yulong; Zhao, Yanxue; Wu, Chengyun; Zhang, Qian; Li, Yan; Jiang, Liwen

2016-01-01

Spatially and temporally regulated membrane trafficking events incorporate membrane and cell wall materials into the pollen tube apex and are believed to underlie the rapid pollen tube growth. In plants, the molecular mechanisms and physiological functions of intra-Golgi transport and Golgi integrity maintenance remain largely unclear. The conserved oligomeric Golgi (COG) complex has been implicated in tethering of retrograde intra-Golgi vesicles in yeast and mammalian cells. Using genetic and cytologic approaches, we demonstrate that T-DNA insertions in Arabidopsis COG complex subunits, COG3 and COG8, cause an absolute, male-specific transmission defect that can be complemented by expression of COG3 and COG8 from the LAT52 pollen promoter, respectively. No obvious abnormalities in the microgametogenesis of the two mutants are observed, but in vitro and in vivo pollen tube growth are defective. COG3 or COG8 proteins fused to green fluorescent protein (GFP) label the Golgi apparatus. In pollen of both mutants, Golgi bodies exhibit altered morphology. Moreover, γ-COP and EMP12 proteins lose their tight association with the Golgi. These defects lead to the incorrect deposition of cell wall components and proteins during pollen tube growth. COG3 and COG8 interact directly with each other, and a structural model of the Arabidopsis COG complex is proposed. We believe that the COG complex helps to modulate Golgi morphology and vesicle trafficking homeostasis during pollen tube tip growth. PMID:27448097

An investigation of the role of transmembrane domains in Golgi protein retention.

PubMed Central

Munro, S

1995-01-01

The single transmembrane domains (TMDs) of the resident glycosylation enzymes of the Golgi apparatus are involved in preventing these proteins moving beyond the Golgi. It has been proposed that either the TMDs associate, resulting in the formation of large oligomers of Golgi enzymes, or that they mediate the lateral segregation of the enzymes between lipid microdomains. Evidence for either type of interaction has been sought by examining the retention of sialyltransferase (ST), an enzyme of the mammalian trans Golgi. No evidence could be obtained for specific interactions or 'kin recognition' between ST and other proteins of the trans Golgi. Moreover, it is shown that the previously described kin recognition between enzymes of the medial Golgi involves the lumenal portions of these proteins rather than their TMDs. To investigate further the role of the ST TMD, the effects on Golgi retention of various alterations in the TMD were examined. The addition or removal of residues showed that the efficiency of retention of ST is related to TMD length. Moreover, when a type I plasma membrane protein was expressed with a synthetic TMD of 23 leucines it appeared on the cell surface, but when the TMD was shortened to 17 leucines accumulation in the Golgi was observed. These observations are more consistent with lipid-based sorting of ST TMD, but they also allow for reconciliation with the kin recognition model which appears to act on sequences outside of the TMD. Images PMID:7588599

The golgin protein Coy1 functions in intra-Golgi retrograde transport and interacts with the COG complex and Golgi SNAREs

PubMed Central

Anderson, Nadine S.; Mukherjee, Indrani; Bentivoglio, Christine M.; Barlowe, Charles

2017-01-01

Extended coiled-coil proteins of the golgin family play prominent roles in maintaining the structure and function of the Golgi complex. Here we further investigate the golgin protein Coy1 and document its function in retrograde transport between early Golgi compartments. Cells that lack Coy1 displayed a reduced half-life of the Och1 mannosyltransferase, an established cargo of intra-Golgi retrograde transport. Combining the coy1Δ mutation with deletions in other putative retrograde golgins (sgm1Δ and rud3Δ) caused strong glycosylation and growth defects and reduced membrane association of the conserved oligomeric Golgi (COG) complex. In contrast, overexpression of COY1 inhibited the growth of mutant strains deficient in fusion activity at the Golgi (sed5-1 and sly1-ts). To map Coy1 protein interactions, coimmunoprecipitation experiments revealed an association with the COG complex and with intra-Golgi SNARE proteins. These physical interactions are direct, as Coy1 was efficiently captured in vitro by Lobe A of the COG complex and the purified SNARE proteins Gos1, Sed5, and Sft1. Thus our genetic, in vivo, and biochemical data indicate a role for Coy1 in regulating COG complex-dependent fusion of retrograde-directed COPI vesicles. PMID:28794270

Functional involvement of TMF/ARA160 in Rab6-dependent retrograde membrane traffic.

PubMed

Yamane, Junko; Kubo, Akiharu; Nakayama, Kazuhisa; Yuba-Kubo, Akiko; Katsuno, Tatsuya; Tsukita, Shoichiro; Tsukita, Sachiko

2007-10-01

The small GTPase Rab6 regulates retrograde membrane traffic from endosomes to the Golgi apparatus and from the Golgi to the endoplasmic reticulum (ER). We examined the role of a Rab6-binding protein, TMF/ARA160 (TATA element modulatory factor/androgen receptor-coactivator of 160 kDa), in this process. High-resolution immunofluorescence imaging revealed that TMF signal surrounded Rab6-positive Golgi structures and immunoelectron microscopy revealed that TMF is concentrated at the budding structures localized at the tips of cisternae. The knockdown of either TMF or Rab6 by RNA interference blocked retrograde transport of endocytosed Shiga toxin from early/recycling endosomes to the trans-Golgi network, causing missorting of the toxin to late endosomes/lysosomes. However, the TMF knockdown caused Rab6-dependent displacement of N-acetylgalactosaminyltransferase-2 (GalNAc-T2), but not beta1,4-galactosyltransferase (GalT), from the Golgi. Analyses using chimeric proteins, in which the cytoplasmic regions of GalNAc-T2 and GalT were exchanged, revealed that the cytoplasmic region of GalNAc-T2 plays a crucial role in its TMF-dependent Golgi retention. These observations suggest critical roles for TMF in two Rab6-dependent retrograde transport processes: one from endosomes to the Golgi and the other from the Golgi to the ER.

ER/Golgi trafficking is facilitated by unbranched actin filaments containing Tpm4.2.

PubMed

Kee, Anthony J; Bryce, Nicole S; Yang, Lingyan; Polishchuk, Elena; Schevzov, Galina; Weigert, Roberto; Polishchuk, Roman; Gunning, Peter W; Hardeman, Edna C

2017-10-01

We have identified novel actin filaments defined by tropomyosin Tpm4.2 at the ER. EM analysis of mouse embryo fibroblasts (MEFs) isolated from mice expressing a mutant Tpm4.2 (Tpm4 Plt53/Plt53 ), incapable of incorporating into actin filaments, revealed swollen ER structures compared with wild-type (WT) MEFs (Tpm4 +/+ ). ER-to-Golgi, but not Golgi-to-ER trafficking was altered in the Tpm4 Plt53/Plt53 MEFs following the transfection of the temperature sensitive ER-associated ts045-VSVg construct. Exogenous Tpm4.2 was able to rescue the ER-to-Golgi trafficking defect in the Tpm4 Plt53/Plt53 cells. The treatment of WT MEFs with the myosin II inhibitor, blebbistatin, blocked the Tpm4.2-dependent ER-to-Golgi trafficking. The lack of an effect on ER-to-Golgi trafficking following treatment of MEFs with CK666 indicates that branched Arp2/3-containing actin filaments are not involved in anterograde vesicle trafficking. We propose that unbranched, Tpm4.2-containing filaments have an important role in maintaining ER/Golgi structure and that these structures, in conjunction with myosin II motors, mediate ER-to-Golgi trafficking. © 2017 Wiley Periodicals, Inc.

Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes

PubMed Central

Patwardhan, Anand; Bardin, Sabine; Miserey-Lenkei, Stéphanie; Larue, Lionel; Goud, Bruno; Raposo, Graça; Delevoye, Cédric

2017-01-01

Exocytic carriers convey neo-synthesized components from the Golgi apparatus to the cell surface. While the release and anterograde movement of Golgi-derived vesicles require the small GTPase RAB6, its effector ELKS promotes the targeting and docking of secretory vesicles to particular areas of the plasma membrane. Here, we show that specialized cell types exploit and divert the secretory pathway towards lysosome related organelles. In cultured melanocytes, the secretory route relies on RAB6 and ELKS to directly transport and dock Golgi-derived carriers to melanosomes. By delivering specific cargos, such as MART-1 and TYRP2/ DCT, the RAB6/ELKS-dependent secretory pathway controls the formation and maturation of melanosomes but also pigment synthesis. In addition, pigmentation defects are observed in RAB6 KO mice. Our data together reveal for the first time that the secretory pathway can be directed towards intracellular organelles of endosomal origin to ensure their biogenesis and function. PMID:28607494

Fluorescent kapakahines serve as non-toxic probes for live cell Golgi imaging.

PubMed

Rocha, Danilo D; Espejo, Vinson R; Rainier, Jon D; La Clair, James J; Costa-Lotufo, Letícia V

2015-09-01

There is an ongoing need for fluorescent probes that specifically-target select organelles within mammalian cells. This study describes the development of probes for the selective labeling of the Golgi apparatus and offers applications for live cell and fixed cell imaging. The kapakahines, characterized by a common C(3)-N(1') dimeric tryptophan linkage, comprise a unique family of bioactive marine depsipeptide natural products. We describe the uptake and subcellular localization of fluorescently-labeled analogs of kapakahine E. Using confocal microscopy, we identify a rapid and selective localization within the Golgi apparatus. Comparison with commercial Golgi stains indicates a unique localization pattern, which differs from currently available materials, therein offering a new tool to monitor the Golgi in live cells without toxic side effects. This study identifies a fluorescent analog of kapakahine E that is rapidly uptaken in cells and localizes within the Golgi apparatus. The advance of microscopic methods is reliant on the parallel discovery of next generation molecular probes. This study describes the advance of stable and viable probe for staining the Golgi apparatus. Copyright © 2015 Elsevier Inc. All rights reserved.

Coupling fission and exit of RAB6 vesicles at Golgi hotspots through kinesin-myosin interactions.

PubMed

Miserey-Lenkei, Stéphanie; Bousquet, Hugo; Pylypenko, Olena; Bardin, Sabine; Dimitrov, Ariane; Bressanelli, Gaëlle; Bonifay, Raja; Fraisier, Vincent; Guillou, Catherine; Bougeret, Cécile; Houdusse, Anne; Echard, Arnaud; Goud, Bruno

2017-11-01

The actin and microtubule cytoskeletons play important roles in Golgi structure and function, but how they are connected remain poorly known. In this study, we investigated whether RAB6 GTPase, a Golgi-associated RAB involved in the regulation of several transport steps at the Golgi level, and two of its effectors, Myosin IIA and KIF20A participate in the coupling between actin and microtubule cytoskeleton. We have previously shown that RAB6-Myosin IIA interaction is critical for the fission of RAB6-positive transport carriers from Golgi/TGN membranes. Here we show that KIF20A is also involved in the fission process and serves to anchor RAB6 on Golgi/TGN membranes near microtubule nucleating sites. We provide evidence that the fission events occur at a limited number of hotspots sites. Our results suggest that coupling between actin and microtubule cytoskeletons driven by Myosin II and KIF20A ensures the spatial coordination between RAB6-positive vesicles fission from Golgi/TGN membranes and their exit along microtubules.

Golgi enzymes do not cycle through the endoplasmic reticulum during protein secretion or mitosis

PubMed Central

Villeneuve, Julien; Duran, Juan; Scarpa, Margherita; Bassaganyas, Laia; Van Galen, Josse; Malhotra, Vivek

2017-01-01

Golgi-specific sialyltransferase (ST) expressed as a chimera with the rapamycin-binding domain of mTOR, FRB, relocates to the endoplasmic reticulum (ER) in cells exposed to rapamycin that also express invariant chain (Ii)-FKBP in the ER. This result has been taken to indicate that Golgi-resident enzymes cycle to the ER constitutively. We show that ST-FRB is trapped in the ER even without Ii-FKBP upon rapamycin addition. This is because ER-Golgi–cycling FKBP proteins contain a C-terminal KDEL-like sequence, bind ST-FRB in the Golgi, and are transported together back to the ER by KDEL receptor–mediated retrograde transport. Moreover, depletion of KDEL receptor prevents trapping of ST-FRB in the ER by rapamycin. Thus ST-FRB cycles artificially by binding to FKBP domain–containing proteins. In addition, Golgi-specific O-linked glycosylation of a resident ER protein occurs only upon artificial fusion of Golgi membranes with ER. Together these findings support the consensus view that there is no appreciable mixing of Golgi-resident enzymes with ER under normal conditions. PMID:27807044

A Distinct Pathway for Polar Exocytosis in Plant Cell Wall Formation1[OPEN

PubMed Central

Wang, Hao; Zhuang, Xiaohong; Wang, Xiangfeng; Law, Angus Ho Yin; Zhao, Teng; Du, Shengwang; Loy, Michael M.T.; Jiang, Liwen

2016-01-01

Post-Golgi protein sorting and trafficking to the plasma membrane (PM) is generally believed to occur via the trans-Golgi network (TGN). In this study using Nicotiana tabacum pectin methylesterase (NtPPME1) as a marker, we have identified a TGN-independent polar exocytosis pathway that mediates cell wall formation during cell expansion and cytokinesis. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that Golgi-derived secretory vesicles (GDSVs) labeled by NtPPME1-GFP are distinct from those organelles belonging to the conventional post-Golgi exocytosis pathway. In addition, pharmaceutical treatments, superresolution imaging, and dynamic studies suggest that NtPPME1 follows a polar exocytic process from Golgi-GDSV-PM/cell plate (CP), which is distinct from the conventional Golgi-TGN-PM/CP secretion pathway. Further studies show that ROP1 regulates this specific polar exocytic pathway. Taken together, we have demonstrated an alternative TGN-independent Golgi-to-PM polar exocytic route, which mediates secretion of NtPPME1 for cell wall formation during cell expansion and cytokinesis and is ROP1-dependent. PMID:27531442

The effects of brefeldin-A on the high mannose oligosaccharides of mouse thyrotropin, free alpha-subunits, and total glycoproteins.

PubMed

Perkel, V S; Liu, A Y; Miura, Y; Magner, J A

1988-07-01

We have studied the effects of Brefeldin-A (BFA) on the processing of high mannose (Man) oligosaccharides of TSH. BFA is a drug that inhibits the intracellular translocation of newly synthesized glycoproteins and causes dilatation of the rough endoplasmic reticulum (RER) as well as mild swelling of the Golgi apparatus. Mouse pituitary thyrotropic tumor tissue was incubated with [3H]Man for a 2-h pulse, with and without a 3-h chase; BFA (5 micrograms/ml) was included during selected pulse and selected chase incubations. TSH and free alpha-subunits were obtained from detergent lysates of tissue by immunoprecipitation using specific antisera. Total glycoproteins were obtained by trichloroacetic acid precipitation. Endoglycosidase-H-released [3H]oligosaccharides were analyzed by paper chromatography. BFA inhibited carbohydrate processing of TSH, free alpha-subunits, and total glycoproteins, resulting in the accumulation of Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2, especially during the chase period. Subcellular fractions enriched in RER, heavy (proximal) Golgi, and light (distal) Golgi were prepared by centrifugation in discontinuous sucrose gradients. [3H]Man-labeled oligosaccharides of TSH and total glycoproteins in the subcellular fractions were analyzed. In contrast to oligosaccharides with eight or nine Man residues found in control incubations, BFA caused the accumulation of oligosaccharides containing five to eight Man residues. These BFA-induced oligosaccharide alterations began in the RER and proximal Golgi with the 2-h pulse and extended into the distal Golgi during the chase incubations. Thus, BFA blocks the normal intracellular transport and processing of TSH, free alpha-subunits, and total glycoproteins within thyrotrophs, causing species with smaller than normal high Man oligosaccharides to appear in subcellular compartments as early as the RER. The translocation block between RER and Golgi produced by BFA may prevent the processing of Man8GlcNAc2 to Man5GlcNAc2 by Golgi (alpha,1-2)mannosidase I, yet the species retained within the RER may be subject to ongoing processing by endoplasmic reticulum (alpha,1-2)mannosidase, resulting in the accumulation of Man5-8GlcNAc2 within the RER.

PITPNC1 recruits RAB1B to the Golgi network to drive malignant secretion

PubMed Central

Halberg, Nils; Sengelaub, Caitlin A.; Navrazhina, Kristina; Molina, Henrik; Uryu, Kunihiro; Tavazoie, Sohail F.

2017-01-01

SUMMARY Enhanced secretion of tumorigenic effector proteins is a feature of malignant cells. The molecular and cellular mechanisms underlying this feature are poorly defined. We identify PITPNC1 as a gene amplified in a large fraction of human breast cancer and over-expressed in metastatic breast, melanoma and colon cancer. Biochemical, molecular, and cell-biological studies reveal that PITPNC1 promotes malignant secretion by binding Golgi resident PI4P and localizing RAB1B to the Golgi. RAB1B localization to the Golgi allows for the recruitment of GOLPH3 to the trans-Golgi, which facilitates Golgi extension and enhanced vesicular release. PITPNC1-mediated vesicular release drives metastasis by increasing the secretion of pro-invasive and pro-angiogenic mediators HTRA1, MMP1, FAM3C, PDGFA, and ADAM10. We establish PITPNC1 as a PI4P-binding protein that enhances vesicular secretion capacity in malignancy. PMID:26977884

Golgi polarization plays a role in the directional migration of neonatal dermal fibroblasts induced by the direct current electric fields.

PubMed

Kim, Min Sung; Lee, Mi Hee; Kwon, Byeong-Ju; Koo, Min-Ah; Seon, Gyeung Mi; Park, Jong-Chul

2015-05-01

Directional cell migration requires cell polarization. The reorganization of the Golgi apparatus is an important phenomenon in the polarization and migration of many types of cells. Direct current electric fields (dc (EF) induced directional cell migration in a wide variety of cells. Here nHDFs migrated toward cathode under 1 V/cm dc EF, however 1 μM of brefeldin A (BFA) inhibited the dc EF induced directional migration. BFA (1 μM) did not cause the complete Golgi dispersal for 2 h. When the Golgi polarization maintained their direction of polarity, the direction of cell migration also kept toward the same direction of the Golgi polarization even though the dc EF was reversed. In this study, the importance of the Golgi polarization in the directional migration of nHDf under dc EF was identified. Copyright © 2015 Elsevier Inc. All rights reserved.

PI3K class II α regulates δ-opioid receptor export from the trans-Golgi network.

PubMed

Shiwarski, Daniel J; Darr, Marlena; Telmer, Cheryl A; Bruchez, Marcel P; Puthenveedu, Manojkumar A

2017-08-01

The interplay between signaling and trafficking by G protein-coupled receptors (GPCRs) has focused mainly on endocytic trafficking. Whether and how surface delivery of newly synthesized GPCRs is regulated by extracellular signals is less understood. Here we define a signaling-regulated checkpoint at the trans -Golgi network (TGN) that controls the surface delivery of the delta opioid receptor (δR). In PC12 cells, inhibition of phosphoinositide-3 kinase (PI3K) activity blocked export of newly synthesized δR from the Golgi and delivery to the cell surface, similar to treatment with nerve growth factor (NGF). Depletion of class II phosphoinositide-3 kinase α (PI3K C2A), but not inhibition of class I PI3K, blocked δR export to comparable levels and attenuated δR-mediated cAMP inhibition. NGF treatment displaced PI3K C2A from the Golgi and optogenetic recruitment of the PI3K C2A kinase domain to the TGN-induced δR export downstream of NGF. Of importance, PI3K C2A expression promotes export of endogenous δR in primary trigeminal ganglion neurons. Taken together, our results identify PI3K C2A as being required and sufficient for δR export and surface delivery in neuronal cells and suggest that it could be a key modulator of a novel Golgi export checkpoint that coordinates GPCR delivery to the surface. © 2017 Shiwarski et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Molecular and Phenotypic Analysis of CaVRG4, Encoding an Essential Golgi Apparatus GDP-Mannose Transporter

PubMed Central

Nishikawa, Akiko; Poster, Jay B.; Jigami, Yoshifumi; Dean, Neta

2002-01-01

Cell surface mannan is implicated in almost every aspect of pathogenicity of Candida albicans. In Saccharomyces cerevisiae, the Vrg4 protein acts as a master regulator of mannan synthesis through its role in substrate provision. The substrate for mannosylation of proteins and lipids in the Golgi apparatus is GDP-mannose, whose lumenal transport is catalyzed by Vrg4p. This nucleotide sugar is synthesized in the cytoplasm by pathways that are highly conserved in all eukaryotes, but its lumenal transport (and hence Golgi apparatus-specific mannosylation) is a fungus-specific process. To begin to study the role of Golgi mannosylation in C. albicans, we isolated the CaVRG4 gene and analyzed the effects of loss of its function. CaVRG4 encodes a functional homologue of the S. cerevisiae GDP-mannose transporter. CaVrg4p localized to punctate spots within the cytoplasm of C. albicans in a pattern reminiscent of localization of Vrg4p in the Golgi apparatus in S. cerevisiae. Like partial loss of ScVRG4 function, partial loss of CaVRG4 function resulted in mannosylation defects, which in turn led to a number of cell wall-associated phenotypes. While heterozygotes displayed no growth phenotypes, a hemizygous strain, containing a single copy of CaVRG4 under control of the methionine-repressible MET3 promoter, did not grow in the presence of methionine and cysteine, demonstrating that CaVRG4 is essential for viability. Mutant Candida vrg4 strains were defective in hyphal formation but exhibited a constitutive polarized mode of pseudohyphal growth. Because the VRG4 gene is essential for yeast viability but does not have a mammalian homologue, it is a particularly attractive target for development of antifungal therapies. PMID:11741841

Proteoglycan biosynthesis in chondrocytes: protein A-gold localization of proteoglycan protein core and chondroitin sulfate within Golgi subcompartments

PubMed Central

1985-01-01

The intracellular pathway of cartilage proteoglycan biosynthesis was investigated in isolated chondrocytes using a protein A-gold electron microscopy immunolocalization procedure. Proteoglycans contain a protein core to which chondroitin sulfate and keratan sulfate chains and oligosaccharides are added in posttranslational processing. Specific antibodies have been used in this study to determine separately the distribution of the protein core and chondroitin sulfate components. In normal chondrocytes, proteoglycan protein core was readily localized only in smooth-membraned vesicles which co-labeled with ricin, indicating them to be galactose-rich medial/trans-Golgi cisternae, whereas there was only a low level of labeling in the rough endoplasmic reticulum. Chondroitin sulfate was also localized in medial/trans-Golgi cisternae of control chondrocytes but was not detected in other cellular compartments. In cells treated with monensin (up to 1.0 microM), which strongly inhibits proteoglycan secretion (Burditt, L.J., A. Ratcliffe, P. R. Fryer, and T. Hardingham, 1985, Biochim. Biophys. Acta., 844:247-255), there was greatly increased intracellular localization of proteoglycan protein core in both ricin- positive vesicles, and in ricin-negative vesicles (derived from cis- Golgi stacks) and in the distended rough endoplasmic reticulum. Chondroitin sulfate also increased in abundance after monensin treatment, but continued to be localized only in ricin-positive vesicles. The results suggested that the synthesis of chondroitin sulfate on proteoglycan only occurs in medial/trans-Golgi cisternae as a late event in proteoglycan biosynthesis. This also suggests that glycosaminoglycan synthesis on proteoglycans takes place in a compartment in common with events in the biosynthesis of both O-linked and N-linked oligosaccharides on other secretory glycoproteins. PMID:3934179

Live-cell imaging of dual-labeled Golgi stacks in tobacco BY-2 cells reveals similar behaviors for different cisternae during movement and brefeldin A treatment.

PubMed

Madison, Stephanie L; Nebenführ, Andreas

2011-09-01

In plant cells, the Golgi apparatus consists of numerous stacks that, in turn, are composed of several flattened cisternae with a clear cis-to-trans polarity. During normal functioning within living cells, this unusual organelle displays a wide range of dynamic behaviors such as whole stack motility, constant membrane flux through the cisternae, and Golgi enzyme recycling through the ER. In order to further investigate various aspects of Golgi stack dynamics and integrity, we co-expressed pairs of established Golgi markers in tobacco BY-2 cells to distinguish sub-compartments of the Golgi during monensin treatments, movement, and brefeldin A (BFA)-induced disassembly. A combination of cis and trans markers revealed that Golgi stacks remain intact as they move through the cytoplasm. The Golgi stack orientation during these movements showed a slight preference for the cis side moving ahead, but trans cisternae were also found at the leading edge. During BFA treatments, the different sub-compartments of about half of the observed stacks fused with the ER sequentially; however, no consistent order could be detected. In contrast, the ionophore monensin resulted in swelling of trans cisternae while medial and particularly cis cisternae were mostly unaffected. Our results thus demonstrate a remarkable equivalence of the different cisternae with respect to movement and BFA-induced fusion with the ER. In addition, we propose that a combination of dual-label fluorescence microscopy and drug treatments can provide a simple alternative approach to the determination of protein localization to specific Golgi sub-compartments.

Sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-Golgi network of AtT20 cells.

PubMed

Tooze, J; Tooze, S A; Fuller, S D

1987-09-01

Murine hepatitis virus (strain A59), (MHV-A59) is a coronavirus that buds into pre-Golgi compartments and then exploits the exocytic pathway of the host cell to reach the exterior. The fibroblastic cells in which replication of this virus is usually studied have only a constitutive exocytic pathway that the virus uses. MHV-A59 also infects, albeit inefficiently, AtT20 cells, murine pituitary tumor cells with a regulated as well as a constitutive exocytic pathway. Here we examine AtT20 cells at early times after the infection, when the Golgi apparatus retains its morphological and biochemical integrity. We observe that progeny coronavirus and secretory protein destined for the secretory granules of the regulated exocytic pathway traverse the same Golgi stacks and accumulate in the trans-Golgi network. Their pathways diverge at this site, the condensed secretory proteins including the ACTH going to the secretory granules and the coronavirus to post-Golgi transport vesicles devoid of ACTH. On very rare occasions there is missorting such that aggregates of condensed secretory proteins and viruses occur together in post-Golgi vesicles. We conclude that the constitutive and regulated exocytic pathways, identified respectively by the progeny virions and the secretory protein ACTH, diverge at the exit from the trans-Golgi network.

Discrete, continuous, and stochastic models of protein sorting in the Golgi apparatus

PubMed Central

Gong, Haijun; Guo, Yusong; Linstedt, Adam

2017-01-01

The Golgi apparatus plays a central role in processing and sorting proteins and lipids in eukaryotic cells. Golgi compartments constantly exchange material with each other and with other cellular components, allowing them to maintain and reform distinct identities despite dramatic changes in structure and size during cell division, development, and osmotic stress. We have developed three minimal models of membrane and protein exchange in the Golgi—a discrete, stochastic model, a continuous ordinary differential equation model, and a continuous stochastic differential equation model—each based on two fundamental mechanisms: vesicle-coat-mediated selective concentration of cargoes and soluble N-ethylmaleimide-sensitive factor attachment protein receptor SNARE proteins during vesicle formation and SNARE-mediated selective fusion of vesicles. By exploring where the models differ, we hope to discover whether the discrete, stochastic nature of vesicle-mediated transport is likely to have appreciable functional consequences for the Golgi. All three models show similar ability to restore and maintain distinct identities over broad parameter ranges. They diverge, however, in conditions corresponding to collapse and reassembly of the Golgi. The results suggest that a continuum model provides a good description of Golgi maintenance but that considering the discrete nature of vesicle-based traffic is important to understanding assembly and disassembly of the Golgi. Experimental analysis validates a prediction of the models that altering guanine nucleotide exchange factor expression levels will modulate Golgi size. PMID:20365406

Secretory granule formation and membrane recycling by the trans-Golgi network in adipokinetic cells of Locusta migratoria in relation to flight and rest.

PubMed

Diederen, J H; Vullings, H G

1995-03-01

The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the trans-Golgi network in the peptidergic neurosecretory adipokinetic cells of Locusta migratoria was investigated by means of ultrastructural morphometric methods. The patterns of labelling of the trans-Golgi network by the exogenous adsorptive endocytotic tracer wheat-germ agglutinin-conjugated horse-radish peroxidase and by the endogenous marker enzyme acid phosphatase were used as parameters and were measured by an automatic image analysis system. The results show that endocytosed fragments of plasma membrane with bound peroxidase label were transported to the trans-Golgi network and used to build new secretory granules. The amounts of peroxidase and especially of acid phosphatase within the trans-Golgi network showed a strong tendency to be smaller in flight-stimulated cells than in non-stimulated cells. The amounts of acid phosphatase in the immature secretory granules originating from the trans-Golgi network were significantly smaller in stimulated cells. The number of immature secretory granules positive for acid phosphatase tended to be higher in stimulated cells. Thus, flight stimulation of adipokinetic cells for 1 h influences the functioning of the trans-Golgi network; this most probably results in a slight enhancement of the production of secretory granules by the trans-Golgi network.

The Arf GEF GBF1 and Arf4 synergize with the sensory receptor cargo, rhodopsin, to regulate ciliary membrane trafficking.

PubMed

Wang, Jing; Fresquez, Theresa; Kandachar, Vasundhara; Deretic, Dusanka

2017-12-01

The small GTPase Arf4 and the Arf GTPase-activating protein (GAP) ASAP1 cooperatively sequester sensory receptor cargo into transport carriers targeted to primary cilia, but the input that drives Arf4 activation in this process remains unknown. Here, we show, by using frog retinas and recombinant human proteins, that during the carrier biogenesis from the photoreceptor Golgi/ trans -Golgi network (TGN) a functional complex is formed between Arf4, the Arf guanine nucleotide exchange factor (GEF) GBF1 and the light-sensing receptor, rhodopsin. Rhodopsin and Arf4 bind the regulatory N-terminal dimerization and cyclophillin-binding (DCB)-homology upstream of Sec7 (HUS) domain of GBF1. The complex is sensitive to Golgicide A (GCA), a selective inhibitor of GBF1 that accordingly blocks rhodopsin delivery to the cilia, without disrupting the photoreceptor Golgi. The emergence of newly synthesized rhodopsin in the endomembrane system is essential for GBF1-Arf4 complex formation in vivo Notably, GBF1 interacts with the Arf GAP ASAP1 in a GCA-resistant manner. Our findings indicate that converging signals on GBF1 from the influx of cargo into the Golgi/TGN and the feedback from Arf4, combined with input from ASAP1, control Arf4 activation during sensory membrane trafficking to primary cilia. © 2017. Published by The Company of Biologists Ltd.

Golgi and plasma membrane pools of PI(4)P contribute to plasma membrane PI(4,5)P2 and maintenance of KCNQ2/3 ion channel current

PubMed Central

Dickson, Eamonn J.; Jensen, Jill B.; Hille, Bertil

2014-01-01

Plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] regulates the activity of many ion channels and other membrane-associated proteins. To determine precursor sources of the PM PI(4,5)P2 pool in tsA-201 cells, we monitored KCNQ2/3 channel currents and translocation of PHPLCδ1 domains as real-time indicators of PM PI(4,5)P2, and translocation of PHOSH2×2, and PHOSH1 domains as indicators of PM and Golgi phosphatidylinositol 4-phosphate [PI(4)P], respectively. We selectively depleted PI(4)P pools at the PM, Golgi, or both using the rapamycin-recruitable lipid 4-phosphatases. Depleting PI(4)P at the PM with a recruitable 4-phosphatase (Sac1) results in a decrease of PI(4,5)P2 measured by electrical or optical indicators. Depleting PI(4)P at the Golgi with the 4-phosphatase or disrupting membrane-transporting motors induces a decline in PM PI(4,5)P2. Depleting PI(4)P simultaneously at both the Golgi and the PM induces a larger decrease of PI(4,5)P2. The decline of PI(4,5)P2 following 4-phosphatase recruitment takes 1–2 min. Recruiting the endoplasmic reticulum (ER) toward the Golgi membranes mimics the effects of depleting PI(4)P at the Golgi, apparently due to the trans actions of endogenous ER Sac1. Thus, maintenance of the PM pool of PI(4,5)P2 appears to depend on precursor pools of PI(4)P both in the PM and in the Golgi. The decrease in PM PI(4,5)P2 when Sac1 is recruited to the Golgi suggests that the Golgi contribution is ongoing and that PI(4,5)P2 production may be coupled to important cell biological processes such as membrane trafficking or lipid transfer activity. PMID:24843134

Phospholipase D2 Is Involved in the Formation of Golgi Tubules and ArfGAP1 Recruitment

PubMed Central

Martínez-Martínez, Narcisa; Martínez-Alonso, Emma; Ballesta, José; Martínez-Menárguez, José A.

2014-01-01

Lipids and lipid-modifying enzymes play a key role in the biogenesis, maintenance and fission of transport carriers in the secretory and endocytic pathways. In the present study we demonstrate that phosphatidic acid generated by phospholipase D2 (PLD2) is involved in the formation of Golgi tubules. The main evidence to support this is: 1) inhibitors of phosphatidic acid formation and PLD2 depletion inhibit the formation of tubules containing resident enzymes and regulators of intra-Golgi transport in a low temperature (15°C) model of Golgi tubulation but do not affect brefeldin A-induced tubules, 2) inhibition of PLD2 enzymatic activity and PLD2 depletion in cells cultured under physiological conditions (37°C) induce the formation of tubules specifically containing Golgi matrix proteins, and, 3) over-expression of PLD2 induces the formation of a tubular network. In addition, it was found that the generation of this lipid by the isoenzyme is necessary for ArfGAP1 recruitment to Golgi membranes. These results suggest that both proteins are involved in the molecular mechanisms which drive the formation of different types of Golgi tubules. PMID:25354038

Functional selectivity of GPCR-directed drug action through location bias

PubMed Central

Irannejad, Roshanak; Pessino, Veronica; Mika, Delphine; Huang, Bo; Wedegaertner, Philip B.; Conti, Marco; von Zastrow, Mark

2017-01-01

G protein-coupled receptors (GPCRs) are increasingly recognized to operate from intracellular membranes as well as the plasma membrane. The β2-adrenergic GPCR can activate Gs-linkedcyclic AMP (cAMP) signaling from endosomes. We show here that the homologous human β1-adrenergic receptor initiates an internal Gs-cAMP signal from the Golgi apparatus. By developing a chemical method to acutely squelch G protein coupling at defined membrane locations, we demonstrate that Golgi activation contributes significantly to the overall cellular cAMP response. Golgi signalling utilizes a pre-existing receptor pool rather than receptors delivered from the cell surface, requiring separate access of extracellular ligands. Epinephrine, a hydrophilic endogenous ligand, accesses the Golgi-localized receptor pool by facilitated transport requiring the organic cation transporter 3 (OCT3) whereas drugs can access the Golgi pool by passive diffusion according to hydrophobicity. We demonstrate marked differences among both agonist and antagonist drugs in Golgi-localized receptor access, and show that β-blocker drugs presently used in the clinic differ markedly in ability to antagonize the Golgi signal. We propose ’location bias’ as a new principle for achieving functional selectivity of GPCR-directed drug action. PMID:28553949

CREB3L1-mediated functional and structural adaptation of the secretory pathway in hormone-stimulated thyroid cells.

PubMed

García, Iris A; Torres Demichelis, Vanina; Viale, Diego L; Di Giusto, Pablo; Ezhova, Yulia; Polishchuk, Roman S; Sampieri, Luciana; Martinez, Hernán; Sztul, Elizabeth; Alvarez, Cecilia

2017-12-15

Many secretory cells increase the synthesis and secretion of cargo proteins in response to specific stimuli. How cells couple increased cargo load with a coordinate rise in secretory capacity to ensure efficient transport is not well understood. We used thyroid cells stimulated with thyrotropin (TSH) to demonstrate a coordinate increase in the production of thyroid-specific cargo proteins and ER-Golgi transport factors, and a parallel expansion of the Golgi complex. TSH also increased expression of the CREB3L1 transcription factor, which alone caused amplified transport factor levels and Golgi enlargement. Furthermore, CREB3L1 potentiated the TSH-induced increase in Golgi volume. A dominant-negative CREB3L1 construct hampered the ability of TSH to induce Golgi expansion, implying that this transcription factor contributes to Golgi expansion. Our findings support a model in which CREB3L1 acts as a downstream effector of TSH to regulate the expression of cargo proteins, and simultaneously increases the synthesis of transport factors and the expansion of the Golgi to synchronize the rise in cargo load with the amplified capacity of the secretory pathway. © 2017. Published by The Company of Biologists Ltd.

The Arf-GDP-regulated recruitment of GBF1 to Golgi membranes requires domains HDS1 and HDS2 and a Golgi-localized protein receptor.

PubMed

Quilty, Douglas; Chan, Calvin J; Yurkiw, Katherine; Bain, Alexandra; Babolmorad, Ghazal; Melançon, Paul

2018-04-19

We previously proposed a novel mechanism by which the enzyme Golgi-specific Brefeldin A resistance factor 1 (GBF1) is recruited to the membranes of the cis -Golgi, based on in vivo experiments. Here, we extended our in vivo analysis on the production of regulatory Arf-GDP and observed that ArfGAP2 and ArfGAP3 do not play a role in GBF1 recruitment. We confirm that Arf-GDP localization is critical, as a TGN-localized Arf-GDP mutant protein fails to promote GBF1 recruitment. We also reported the establishment of an in vitro GBF1 recruitment assay that supports the regulation of GBF1 recruitment by Arf-GDP. This in vitro assay yielded further evidence for the requirement of a Golgi-localized protein because heat denaturation or protease treatment of Golgi membranes abrogated GBF1 recruitment. Finally, combined in vivo and in vitro measurements indicated that the recruitment to Golgi membranes via a putative receptor requires only the HDS1 and HDS2 domains in the C-terminal half of GBF1. © 2018. Published by The Company of Biologists Ltd.

COPI-mediated retrograde trafficking from the Golgi to the ER regulates EGFR nuclear transport

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ying-Nai; Wang, Hongmei; Yamaguchi, Hirohito

2010-09-03

Research highlights: {yields} ARF1 activation is involved in the EGFR transport to the ER and the nucleus. {yields} Assembly of {gamma}-COP coatomer mediates EGFR transport to the ER and the nucleus. {yields} Golgi-to-ER retrograde trafficking regulates nuclear transport of EGFR. -- Abstract: Emerging evidence indicates that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus. A retrograde route from the Golgi to the endoplasmic reticulum (ER) is postulated to be involved in the EGFR trafficking to the nucleus; however, the molecular mechanism in this proposed model remains unexplored.more » Here, we demonstrate that membrane-embedded vesicular trafficking is involved in the nuclear transport of EGFR. Confocal immunofluorescence reveals that in response to EGF, a portion of EGFR redistributes to the Golgi and the ER, where its NH{sub 2}-terminus resides within the lumen of Golgi/ER and COOH-terminus is exposed to the cytoplasm. Blockage of the Golgi-to-ER retrograde trafficking by brefeldin A or dominant mutants of the small GTPase ADP-ribosylation factor, which both resulted in the disassembly of the coat protein complex I (COPI) coat to the Golgi, inhibit EGFR transport to the ER and the nucleus. We further find that EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with {gamma}-COP, one of the subunits of the COPI coatomer. Our findings experimentally provide a comprehensive pathway that nuclear transport of EGFR is regulated by COPI-mediated vesicular trafficking from the Golgi to the ER, and may serve as a general mechanism in regulating the nuclear transport of other cell surface receptors.« less

Integrated self-organization of transitional ER and early Golgi compartments.

PubMed

Glick, Benjamin S

2014-02-01

COPII coated vesicles bud from an ER domain termed the transitional ER (tER), but the mechanism that clusters COPII vesicles at tER sites is unknown. tER sites are closely associated with early Golgi or pre-Golgi structures, suggesting that the clustering of nascent COPII vesicles could be achieved by tethering to adjacent membranes. This model challenges the prevailing view that COPII vesicles are clustered by a scaffolding protein at the ER surface. Although Sec16 was proposed to serve as such a scaffolding protein, recent data suggest that rather than organizing COPII into higher-order structures, Sec16 acts at the level of individual COPII vesicles to regulate COPII turnover. A plausible synthesis is that tER sites are created by tethering to Golgi membranes and are regulated by Sec16. Meanwhile, the COPII vesicles that bud from tER sites are thought to nucleate new Golgi cisternae. Thus, an integrated self-organization process may generate tER-Golgi units. © 2014 WILEY Periodicals, Inc.

An inducible ER–Golgi tether facilitates ceramide transport to alleviate lipotoxicity

PubMed Central

Choudhary, Vineet

2017-01-01

Ceramides are key intermediates in sphingolipid biosynthesis and potent signaling molecules. However, excess ceramide is toxic, causing growth arrest and apoptosis. In this study, we identify a novel mechanism by which cells prevent the toxic accumulation of ceramides; they facilitate nonvesicular ceramide transfer from the endoplasmic reticulum (ER) to the Golgi complex, where ceramides are converted to complex sphingolipids. We find that the yeast protein Nvj2p promotes the nonvesicular transfer of ceramides from the ER to the Golgi complex. The protein is a tether that generates close contacts between these compartments and may directly transport ceramide. Nvj2p normally resides at contacts between the ER and other organelles, but during ER stress, it relocalizes to and increases ER–Golgi contacts. ER–Golgi contacts fail to form during ER stress in cells lacking Nvj2p. Our findings demonstrate that cells regulate ER–Golgi contacts in response to stress and reveal that nonvesicular ceramide transfer out of the ER prevents the buildup of toxic amounts of ceramides. PMID:28011845

The impossible interview with the man of the hidden biological structures. Interview by Paolo Mazzarello.

PubMed

Golgi, Camillo

2006-12-01

This paper presents an "impossible interview" to Professor Camillo Golgi, placed in time in December 1906. The Italian Professor Golgi from Pavia has been awarded the Nobel Prize for Physiology or Medicine ex aequo with the Spanish anatomist Santiago Ramón y Cajal. Both scientists have obtained the award for their work on the anatomy of the nervous system. However, they have opposite views on the mechanisms underlying nervous functions. Golgi believes that the axons stained by his "black reaction" form a continuous anatomical or functional network along which nervous impulses propagate. Ramón y Cajal is the paladin of the neuron theory, a hypothesis questioned by Golgi in his Nobel lecture of Tuesday, December 11. After the ceremony, an independent journalist has interviewed Professor Golgi in the Grand Hotel in Stockholm. Excerpts about his education, his main scientific discoveries, and his personal life are here given (reconstructing the "impossible interview" on the basis of Golgi's original writings).

ZIO impregnation and cytochemical localization of thiamine pyrophosphatase and acid phosphatase activities in small granule-containing (SGC) cells of rat superior cervical ganglia.

PubMed

Chau, Y P; Lu, K S

1994-10-01

Cytochemical relationship between Golgi complex and dense-cored granules (DCGs) of small granule-containing (SGC) cells in rat superior cervical ganglia was examined in electron microscopy by zinc-iodide-osmium tetroxide (ZIO) method and by enzyme cytochemistry for thiamine pyrophosphatase (TPPase) and acid phosphatase (ACPase). After ZIO impregnation, all the saccules of Golgi apparatus and some of tubular rough endoplasmic reticulum (rER) were stained. DCGs in periphery of SGC cells were not stained, but varying degrees of dense deposits occurred in the DCGs in vicinity of Golgi trans-saccules. Both TPPase and ACPase activities were localized in one or two stacked layers of saccules on the trans side of the Golgi complex. No reaction products were demonstrated in the DCGs. From these results, we suggest that the DCGs of SGC cells in rat superior cervical ganglia are derived from the Golgi complex, and that lysosomal cleavage of protein contents in the DCGs may occur in the trans Golgi saccules.

STAM Adaptor Proteins Interact with COPII Complexes and Function in ER-to-Golgi Trafficking

PubMed Central

Rismanchi, Neggy; Puertollano, Rosa; Blackstone, Craig

2009-01-01

Signal transducing adaptor molecules (STAMs) are involved in growth factor and cytokine signaling as well as receptor degradation, and they form complexes with a number of endocytic proteins, including Hrs and Eps15. Here we demonstrate that STAM proteins also localize prominently to early exocytic compartments and profoundly regulate Golgi morphology. Upon STAM overexpression in cells the Golgi apparatus becomes extensively fragmented and dispersed, but when STAMs are depleted the Golgi becomes highly condensed. Under both scenarios, vesicular stomatitis virus G protein (VSVG)-GFP trafficking to the plasma membrane is markedly inhibited, and recovery of Golgi morphology after brefeldin A treatment is substantially impaired in STAM-depleted cells. Furthermore, STAM proteins interact with COPII proteins, probably at endoplasmic reticulum (ER) exit sites, and Sar1 activity is required to maintain the localization of STAMs at discrete sites. Thus, in addition to their roles in signaling and endocytosis, STAMs function prominently in ER-to-Golgi trafficking, most likely through direct interactions with the COPII complex. PMID:19054391

The Future of Golgi Research

PubMed Central

2010-01-01

This essay looks backward on the past three decades of research toward understanding the mechanism of macromolecular traffic through and within the Golgi apparatus with an eye to the future. I also explain why I feel the Golgi should continue to hold the attention of molecular cell biologists. PMID:21079007

The Cirque du Soleil of Golgi membrane dynamics

PubMed Central

2009-01-01

The role of lipid metabolic enzymes in Golgi membrane remodeling is a subject of intense interest. Now, in this issue, Schmidt and Brown (2009. J. Cell Biol. doi:10.1083/jcb.200904147) report that lysophosphatidic acid–specific acyltransferase, LPAAT3, contributes to Golgi membrane dynamics by suppressing tubule formation. PMID:19635838

The Cirque du Soleil of Golgi membrane dynamics.

PubMed

Bankaitis, Vytas A

2009-07-27

The role of lipid metabolic enzymes in Golgi membrane remodeling is a subject of intense interest. Now, in this issue, Schmidt and Brown (2009. J. Cell Biol. doi:10.1083/jcb.200904147) report that lysophosphatidic acid-specific acyltransferase, LPAAT3, contributes to Golgi membrane dynamics by suppressing tubule formation.

Golgi-Colonnier method: correlation of the degree of chromium reduction and pH change with quality of staining.

PubMed

Angulo, A; Merchán, J A; Molina, M

1994-03-01

We examined the role of chromium reduction in the Golgi-Colonnier method, correlating the quality of neuronal impregnation with the levels of hexavalent (CrVI) and trivalent (CrIII) chromium in the tissue and in the chromation fluid (CF). The concentrations of both chromium species were assessed by measuring spectrophotometrically the CrVI before and after oxidizing the sample and by calculating the ratio of CrVI to total chromium (chromium ratio, CrR). The CrR was almost identical in the tissue and the CF, decreasing exponentially during chromation due to a progressive consumption of CrVI to form CrIII. Satisfactory cell impregnation was obtained only when the CrR was 0.45-0.7, regardless of other factors. The CrR values could be accurately predicted by the pH increase of the CF; this increase has proven to be a most reliable criterion to decide the endpoint of the chromation process. The dependence of cell staining on the [CrIII], together with the well-known ability of this species to bridge proteins, suggests that the key event for cell impregnation is the cross-linking of neuronal proteins by CrIII polymers.

Super-Resolution Imaging of the Golgi in Live Cells with a Bio-orthogonal Ceramide Probe**

PubMed Central

Erdmann, Roman S.; Takakura, Hideo; Thompson, Alexander D.; Rivera-Molina, Felix; Allgeyer, Edward S.; Bewersdorf, Joerg; Toomre, Derek K.; Schepartz, Alanna

2014-01-01

We report a lipid-based strategy to visualize Golgi structure and dynamics at super-resolution in live cells. The method is based on two novel reagents: a trans-cyclooctene-containing ceramide lipid (Cer-TCO) and a highly reactive, tetrazine-tagged near-IR dye (SiR-Tz). These reagents assemble via an extremely rapid ‘tetrazine-click’ reaction into Cer-SiR, a highly photostable ‘vital dye’ that enables prolonged live cell imaging of the Golgi apparatus by 3D confocal and STED microscopy. Cer-SiR is non-toxic at concentrations as high as 2 μM and does not perturb the mobility of Golgi-resident enzymes or the traffic of cargo from the endoplasmic reticulum through the Golgi and to the plasma membrane. PMID:25081303

Isolation of a matrix that binds medial Golgi enzymes

PubMed Central

1994-01-01

Rat liver Golgi stacks were extracted with Triton X-100 at neutral pH. After centrifugation the low speed pellet contained two medial-Golgi enzymes, N-acetylglucosaminyltransferase I and mannosidase II, but no enzymes or markers from other parts of the Golgi apparatus. Both were present in the same structures which appeared, by electron microscopy, to be small remnants of cisternal membranes. The enzymes could be removed by treatment with low salt, leaving behind a salt pellet, which we term the matrix. Removal of salt caused specific re-binding of both enzymes to the matrix, with an apparent dissociation constant of 3 nM for mannosidase II. Re-binding was abolished by pretreatment of intact Golgi stacks with proteinase K, suggesting that the matrix was present between the cisternae. PMID:8106542

Creating Knock-outs of Conserved Oligomeric Golgi complex subunits using CRISPR-mediated gene editing paired with a selection strategy based on glycosylation defects associated with impaired COG complex function

PubMed Central

Blackburn, Jessica Bailey; Lupashin, Vladimir V.

2017-01-01

Summary The Conserved Oligomeric Golgi (COG) complex is a key evolutionally conserved multisubunit protein machinery that regulates tethering and fusion of intra-Golgi transport vesicles. The Golgi apparatus specifically promotes sorting and complex glycosylation of glycoconjugates. Without proper glycosylation and processing, proteins and lipids will be mislocalized and/or have impaired function. The Golgi glycosylation machinery is kept in homeostasis by a careful balance of anterograde and retrograde trafficking to ensure proper localization of the glycosylation enzymes and their substrates. This balance, like other steps of membrane trafficking, is maintained by vesicle trafficking machinery that includes COPI vesicular coat proteins, SNAREs, Rabs, and both coiled-coil and multi-subunit vesicular tethers. COG complex interacts with other membrane trafficking components and is essential for proper localization of Golgi glycosylation machinery. Here we describe using CRISPR-mediated gene editing coupled with a phenotype-based selection strategy directly linked to the COG complex’s role in glycosylation homeostasis to obtain COG complex subunit knock-outs (KOs). This has resulted in clonal KOs for each COG subunit in HEK293T cells and gives the ability to further probe the role of the COG complex in Golgi homeostasis. PMID:27632008

Microtubule actin crosslinking factor 1b: a novel plakin that localizes to the Golgi complex.

PubMed

Lin, Chung-Ming; Chen, Hui-Jye; Leung, Conrad L; Parry, David A D; Liem, Ronald K H

2005-08-15

MACF1 (microtubule actin crosslinking factor), also called ACF7 (actin crosslinking family 7) is a cytoskeletal linker protein that can associate with both actin filaments and microtubules. We have identified a novel alternatively spliced isoform of MACF1. We named this isoform MACF1b and renamed the original isoform MACF1a. MACF1b is identical to MACF1a, except that it has a region containing plakin (or plectin) repeats in the middle of the molecule. MACF1b is ubiquitously expressed in adult tissues with especially high levels in the lung. We studied the subcellular localization of MACF1b proteins in mammalian cell lines. In two lung cell lines, MACF1b was chiefly localized to the Golgi complex. Upon treatments that disrupt the Golgi complex, MACF1b redistributed into the cytosol, but remained co-localized with the dispersed Golgi ministacks. MACF1b proteins can be detected in the enriched Golgi fraction by western blotting. The domain of MACF1b that targets it to the Golgi was found at the N-terminal part of the region that contains the plakin repeats. Reducing the level of MACF1 proteins by small-interfering RNA resulted in the dispersal of the Golgi complex.

The trans-Golgi Network and the Golgi Stacks Behave Independently During Regeneration After Brefeldin A Treatment in Tobacco BY-2 Cells.

PubMed

Ito, Yoko; Toyooka, Kiminori; Fujimoto, Masaru; Ueda, Takashi; Uemura, Tomohiro; Nakano, Akihiko

2017-04-01

The trans-Golgi network (TGN) plays an essential role in intracellular membrane trafficking. In plant cells, recent live-cell imaging studies have revealed the dynamic behavior of the TGN independent from the Golgi apparatus. In order to better understand the relationships between the two organelles, we examined their dynamic responses to the reagent brefeldin A (BFA) and their recovery after BFA removal. Golgi markers responded to BFA similarly over a range of concentrations, whereas the behavior of the TGN was BFA concentration dependent. The TGN formed aggregates at high concentrations of BFA; however, TGN proteins relocalized to numerous small vesicular structures dispersed throughout the cytoplasm at lower BFA concentrations. During recovery from weak BFA treatment, the TGN started to regenerate earlier than the completion of the Golgi. The regeneration of the two organelles proceeded independently of each other for a while, and eventually was completed by their association. Our data suggest that there is some degree of autonomy for the regeneration of the TGN and the Golgi in tobacco BY-2 cells. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Human Rhinovirus 16 Causes Golgi Apparatus Fragmentation without Blocking Protein Secretion

PubMed Central

Mousnier, Aurelie; Swieboda, Dawid; Pinto, Anaïs; Guedán, Anabel; Rogers, Andrew V.; Walton, Ross; Johnston, Sebastian L.

2014-01-01

ABSTRACT The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion. IMPORTANCE The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be differences between different members of the family and inconsistent results when comparing infection with live virus to expression of individual nonstructural proteins. We demonstrate that individual nonstructural HRV16 proteins, when expressed in HeLa cells, can both fragment the Golgi apparatus and block secretion, whereas viral infection fragments the Golgi apparatus without blocking secretion. This has major implications for how we interpret mechanistic evidence derived from the expression of single viral proteins. PMID:25100828

Macromolecular differentiation of Golgi stacks in root tips of Arabidopsis and Nicotiana seedlings as visualized in high pressure frozen and freeze-substituted samples

NASA Technical Reports Server (NTRS)

Staehelin, L. A.; Giddings, T. H. Jr; Kiss, J. Z.; Sack, F. D.

1990-01-01

The plant root tip represents a fascinating model system for studying changes in Golgi stack architecture associated with the developmental progression of meristematic cells to gravity sensing columella cells, and finally to "young" and "old", polysaccharide-slime secreting peripheral cells. To this end we have used high pressure freezing in conjunction with freeze-substitution techniques to follow developmental changes in the macromolecular organization of Golgi stacks in root tips of Arabidopsis and Nicotiana. Due to the much improved structural preservation of all cells under investigation, our electron micrographs reveal both several novel structural features common to all Golgi stacks, as well as characteristic differences in morphology between Golgi stacks of different cell types. Common to all Golgi stacks are clear and discrete differences in staining patterns and width of cis, medial and trans cisternae. Cis cisternae have the widest lumina (approximately 30 nm) and are the least stained. Medial cisternae are narrower (approximately 20 nm) and filled with more darkly staining products. Most trans cisternae possess a completely collapsed lumen in their central domain, giving rise to a 4-6 nm wide dark line in cross-sectional views. Numerous vesicles associated with the cisternal margins carry a non-clathrin type of coat. A trans Golgi network with clathrin coated vesicles is associated with all Golgi stacks except those of old peripheral cells. It is easily distinguished from trans cisternae by its blebbing morphology and staining pattern. The zone of ribosome exclusion includes both the Golgi stack and the trans Golgi network. Intercisternal elements are located exclusively between trans cisternae of columella and peripheral cells, but not meristematic cells. In older peripheral cells only trans cisternae exhibit slime-related staining. Golgi stacks possessing intercisternal elements also contain parallel rows of freeze-fracture particles in their trans cisternal membranes. We propose that intercisternal elements serve as anchors of enzyme complexes involved in the synthesis of polysaccharide slime molecules to prevent the complexes from being dragged into the forming secretory vesicles by the very large slime molecules. In addition, we draw attention to the similarities in composition and apparent site of synthesis of xyloglucans and slime molecules.

Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain.

PubMed

Aoki, D; Lee, N; Yamaguchi, N; Dubois, C; Fukuda, M N

1992-05-15

Galactosyltransferase (GT; UDPgalactose:beta-D-N-acetylglucosaminide beta-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membrane-anchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was designed to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin alpha subunit (hCG alpha) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, delta tail and delta stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29----Ser29 and His32----Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31----Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32----Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrane domain of GT contributes to the Golgi retention signal and that particularly Cys29 and His32 in this region are critical for GT to be retained in the Golgi.

Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain.

PubMed Central

Aoki, D; Lee, N; Yamaguchi, N; Dubois, C; Fukuda, M N

1992-01-01

Galactosyltransferase (GT; UDPgalactose:beta-D-N-acetylglucosaminide beta-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membrane-anchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was designed to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin alpha subunit (hCG alpha) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, delta tail and delta stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29----Ser29 and His32----Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31----Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32----Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrane domain of GT contributes to the Golgi retention signal and that particularly Cys29 and His32 in this region are critical for GT to be retained in the Golgi. Images PMID:1584766

PPFIA1 drives active α5β1 integrin recycling and controls fibronectin fibrillogenesis and vascular morphogenesis

PubMed Central

Mana, Giulia; Clapero, Fabiana; Panieri, Emiliano; Panero, Valentina; Böttcher, Ralph T.; Tseng, Hui-Yuan; Saltarin, Federico; Astanina, Elena; Wolanska, Katarzyna I.; Morgan, Mark R.; Humphries, Martin J.; Santoro, Massimo M.; Serini, Guido; Valdembri, Donatella

2016-01-01

Basolateral polymerization of cellular fibronectin (FN) into a meshwork drives endothelial cell (EC) polarity and vascular remodelling. However, mechanisms coordinating α5β1 integrin-mediated extracellular FN endocytosis and exocytosis of newly synthesized FN remain elusive. Here we show that, on Rab21-elicited internalization, FN-bound/active α5β1 is recycled to the EC surface. We identify a pathway, comprising the regulators of post-Golgi carrier formation PI4KB and AP-1A, the small GTPase Rab11B, the surface tyrosine phosphatase receptor PTPRF and its adaptor PPFIA1, which we propose acts as a funnel combining FN secretion and recycling of active α5β1 integrin from the trans-Golgi network (TGN) to the EC surface, thus allowing FN fibrillogenesis. In this framework, PPFIA1 interacts with active α5β1 integrin and localizes close to EC adhesions where post-Golgi carriers are targeted. We show that PPFIA1 is required for FN polymerization-dependent vascular morphogenesis, both in vitro and in the developing zebrafish embryo. PMID:27876801

A Novel Feature Extraction Method with Feature Selection to Identify Golgi-Resident Protein Types from Imbalanced Data

PubMed Central

Yang, Runtao; Zhang, Chengjin; Gao, Rui; Zhang, Lina

2016-01-01

The Golgi Apparatus (GA) is a major collection and dispatch station for numerous proteins destined for secretion, plasma membranes and lysosomes. The dysfunction of GA proteins can result in neurodegenerative diseases. Therefore, accurate identification of protein subGolgi localizations may assist in drug development and understanding the mechanisms of the GA involved in various cellular processes. In this paper, a new computational method is proposed for identifying cis-Golgi proteins from trans-Golgi proteins. Based on the concept of Common Spatial Patterns (CSP), a novel feature extraction technique is developed to extract evolutionary information from protein sequences. To deal with the imbalanced benchmark dataset, the Synthetic Minority Over-sampling Technique (SMOTE) is adopted. A feature selection method called Random Forest-Recursive Feature Elimination (RF-RFE) is employed to search the optimal features from the CSP based features and g-gap dipeptide composition. Based on the optimal features, a Random Forest (RF) module is used to distinguish cis-Golgi proteins from trans-Golgi proteins. Through the jackknife cross-validation, the proposed method achieves a promising performance with a sensitivity of 0.889, a specificity of 0.880, an accuracy of 0.885, and a Matthew’s Correlation Coefficient (MCC) of 0.765, which remarkably outperforms previous methods. Moreover, when tested on a common independent dataset, our method also achieves a significantly improved performance. These results highlight the promising performance of the proposed method to identify Golgi-resident protein types. Furthermore, the CSP based feature extraction method may provide guidelines for protein function predictions. PMID:26861308

Spatial and Functional Aspects of ER-Golgi Rabs and Tethers

PubMed Central

Saraste, Jaakko

2016-01-01

Two conserved Rab GTPases, Rab1 and Rab2, play important roles in biosynthetic-secretory trafficking between the endoplasmic reticulum (ER) and the Golgi apparatus in mammalian cells. Both are expressed as two isoforms that regulate anterograde transport via the intermediate compartment (IC) to the Golgi, but are also required for transport in the retrograde direction. Moreover, Rab1 has been implicated in the formation of autophagosomes. Rab1 and Rab2 have numerous effectors or partners that function in membrane tethering, but also have other roles. These include the coiled-coil proteins p115, GM130, giantin, golgin-84, and GMAP-210, as well as the multisubunit COG (conserved oligomeric Golgi) and TRAPP (transport protein particle) tethering complexes. TRAPP also acts as the GTP exchange factor (GEF) in the activation of Rab1. According to the traditional view of the IC elements as motile, transient structures, the functions of the Rabs could take place at the two ends of the ER-Golgi itinerary, i.e., at ER exit sites (ERES) and/or cis-Golgi. However, there is considerable evidence for their specific association with the IC, including its recently identified pericentrosomal domain (pcIC), where many of the effectors turn out to be present, thus being able to exert their functions at the pre-Golgi level. The IC localization of these proteins is of particular interest based on the imaging of Rab1 dynamics, indicating that the IC is a stable organelle that bidirectionally communicates with the ER and Golgi, and is functionally linked to the endosomal system via the pcIC. PMID:27148530

Regulation of traffic and organelle architecture of the ER-Golgi interface by signal transduction.

PubMed

Tillmann, Kerstin D; Millarte, Valentina; Farhan, Hesso

2013-09-01

The components that control trafficking between organelles of the secretory pathway as well as their architecture were uncovered to a reasonable extent in the past decades. However, only recently did we begin to explore the regulation of the secretory pathway by cellular signaling. In the current review, we focus on trafficking between the endoplasmic reticulum and the Golgi apparatus. We highlight recent advances that have been made toward a better understanding of how the secretory pathway is regulated by signaling and discuss how this knowledge is important to obtain an integrative view of secretion in the context of other homeostatic processes such as growth and proliferation.

Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae

PubMed Central

Beznoussenko, Galina V; Parashuraman, Seetharaman; Rizzo, Riccardo; Polishchuk, Roman; Martella, Oliviano; Di Giandomenico, Daniele; Fusella, Aurora; Spaar, Alexander; Sallese, Michele; Capestrano, Maria Grazia; Pavelka, Margit; Vos, Matthijn R; Rikers, Yuri GM; Helms, Volkhard; Mironov, Alexandre A; Luini, Alberto

2014-01-01

The mechanism of transport through the Golgi complex is not completely understood, insofar as no single transport mechanism appears to account for all of the observations. Here, we compare the transport of soluble secretory proteins (albumin and α1-antitrypsin) with that of supramolecular cargoes (e.g., procollagen) that are proposed to traverse the Golgi by compartment progression–maturation. We show that these soluble proteins traverse the Golgi much faster than procollagen while moving through the same stack. Moreover, we present kinetic and morphological observations that indicate that albumin transport occurs by diffusion via intercisternal continuities. These data provide evidence for a transport mechanism that applies to a major class of secretory proteins and indicate the co-existence of multiple intra-Golgi trafficking modes. DOI: http://dx.doi.org/10.7554/eLife.02009.001 PMID:24867214

Shifted Golgi targeting of glycosyltransferases and α-mannosidase IA from giantin to GM130-GRASP65 results in formation of high mannose N-glycans in aggressive prostate cancer cells.

PubMed

Bhat, Ganapati; Hothpet, Vishwanath-Reddy; Lin, Ming-Fong; Cheng, Pi-Wan

2017-11-01

There is a pressing need for biomarkers that can distinguish indolent from aggressive prostate cancer to prevent over-treatment of patients with indolent tumor. Golgi targeting of glycosyltransferases was characterized by confocal microscopy after knockdown of GM130, giantin, or both. N-glycans on a trans-Golgi enzyme β4galactosyltransferase-1 isolated by immunoprecipitation from androgen-sensitive and independent prostate cancer cells were determined by matrix-assisted laser desorption-time of flight-mass spectrometry. In situ proximity ligation assay was employed to determine co-localization of (a) α-mannosidase IA, an enzyme required for processing Man 8 GlcNAc 2 down to Man 5 GlcNAc 2 to enable synthesis of complex-type N-glycans, with giantin, GM130, and GRASP65, and (b) trans-Golgi glycosyltransferases with high mannose N-glycans terminated with α3-mannose. Defective giantin in androgen-independent prostate cancer cells results in a shift of Golgi targeting of glycosyltransferases and α-mannosidase IA from giantin to GM130-GRASP65. Consequently, trans-Golgi enzymes and cell surface glycoproteins acquire high mannose N-glycans, which are absent in cells with functional giantin. In situ proximity ligation assays of co-localization of α-mannosidase IA with GM130 and GRASP65, and trans-Golgi glycosyltransferases with high mannose N-glycans are negative in androgen-sensitive LNCaP C-33 cells but positive in androgen-independent LNCaP C-81 and DU145 cells, and LNCaP C-33 cells devoid of giantin. In situ proximity ligation assays of Golgi localization of α-mannosidase IA at giantin versus GM130-GRASP65 site, and absence or presence of N-glycans terminated with α3-mannose on trans-Golgi glycosyltransferases may be useful for distinguishing indolent from aggressive prostate cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Dependence of Golgi apparatus integrity on nitric oxide in vascular cells: implications in pulmonary arterial hypertension

PubMed Central

Lee, Jason E.; Patel, Kirit; Almodóvar, Sharilyn; Tuder, Rubin M.; Flores, Sonia C.

2011-01-01

Although reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of pulmonary arterial hypertension (PAH), its consequences on organellar structure and function within vascular cells is largely unexplored. We investigated the effect of reduced NO on the structure of the Golgi apparatus as assayed by giantin or GM130 immunofluorescence in human pulmonary arterial endothelial (HPAECs) and smooth muscle (HPASMCs) cells, bovine PAECs, and human EA.hy926 endothelial cells. Golgi structure was also investigated in cells in tissue sections of pulmonary vascular lesions in idiopathic PAH (IPAH) and in macaques infected with a chimeric simian immunodeficiency virus containing the human immunodeficiency virus (HIV)-nef gene (SHIV-nef) with subcellular three-dimensional (3D) immunoimaging. Compounds with NO scavenging activity including 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), methylene blue, N-acetylcysteine, and hemoglobin markedly fragmented the Golgi in all cell types evaluated as did monocrotaline pyrrole, while LY-83583, sildenafil, fasudil, Y-27632, Tiron, Tempol, or H2O2 did not. Golgi fragmentation by NO scavengers was inhibited by diethylamine NONOate, was evident in HPAECs after selective knockdown of endothelial nitric oxide synthase using small interfering RNA (siRNA), was independent of microtubule organization, required the GTPase dynamin 2, and was accompanied by depletion of α-soluble N-ethylmaleimide-sensitive factor (NSF) acceptor protein (α-SNAP) from Golgi membranes and codispersal of the SNAP receptor (SNARE) Vti1a with giantin. Golgi fragmentation was confirmed in endothelial and smooth muscle cells in pulmonary arterial lesions in IPAH and the SHIV-nef-infected macaque with subcellular 3D immunoimaging. In SHIV-nef-infected macaques Golgi fragmentation was observed in cells containing HIV-nef-bearing endosomes. The observed Golgi fragmentation suggests that NO plays a significant role in modulating global protein trafficking patterns that contribute to changes in the cell surface landscape and functional signaling in vascular cells. PMID:21217069

N-Glycomic and Microscopic Subcellular Localization Analyses of NPP1, 2 and 6 Strongly Indicate that trans-Golgi Compartments Participate in the Golgi to Plastid Traffic of Nucleotide Pyrophosphatase/Phosphodiesterases in Rice

PubMed Central

Kaneko, Kentaro; Takamatsu, Takeshi; Inomata, Takuya; Oikawa, Kazusato; Itoh, Kimiko; Hirose, Kazuko; Amano, Maho; Nishimura, Shin-Ichiro; Toyooka, Kiminori; Matsuoka, Ken; Pozueta-Romero, Javier; Mitsui, Toshiaki

2016-01-01

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1–NPP6. Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)–Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2–GFP and NPP6–GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER–Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs. PMID:27335351

The Prenylated Rab GTPase Receptor PRA1.F4 Contributes to Protein Exit from the Golgi Apparatus.

PubMed

Lee, Myoung Hui; Yoo, Yun-Joo; Kim, Dae Heon; Hanh, Nguyen Hong; Kwon, Yun; Hwang, Inhwan

2017-07-01

Prenylated Rab acceptor1 (PRA1) functions in the recruitment of prenylated Rab proteins to their cognate organelles. Arabidopsis ( Arabidopsis thaliana ) contains a large number of proteins belonging to the AtPRA1 family. However, their physiological roles remain largely unknown. Here, we investigated the physiological role of AtPRA1.F4, a member of the AtPRA1 family. A T-DNA insertion knockdown mutant of AtPRA1.F4 , atpra1.f4 , was smaller in stature than parent plants and possessed shorter roots, whereas transgenic plants overexpressing HA:AtPRA1.F4 showed enhanced development of secondary roots and root hairs. However, both overexpression and knockdown plants exhibited increased sensitivity to high-salt stress, lower vacuolar Na + /K + -ATPase and plasma membrane ATPase activities, lower and higher pH in the vacuole and apoplast, respectively, and highly vesiculated Golgi apparatus. HA:AtPRA1.F4 localized to the Golgi apparatus and assembled into high-molecular-weight complexes. atpra1.f4 plants displayed a defect in vacuolar trafficking, which was complemented by low but not high levels of HA : AtPRA1.F4 Overexpression of HA:AtPRA1.F4 also inhibited protein trafficking at the Golgi apparatus, albeit differentially depending on the final destination or type of protein: trafficking of vacuolar proteins, plasma membrane proteins, and trans-Golgi network (TGN)-localized SYP61 was strongly inhibited; trafficking of TGN-localized SYP51 was slightly inhibited; and trafficking of secretory proteins and TGN-localized SYP41 was negligibly or not significantly inhibited. Based on these results, we propose that Golgi-localized AtPRA1.F4 is involved in the exit of many but not all types of post-Golgi proteins from the Golgi apparatus. Additionally, an appropriate level of AtPRA1.F4 is crucial for its function at the Golgi apparatus. © 2017 American Society of Plant Biologists. All Rights Reserved.

Qa-SNAREs localized to the trans-Golgi network regulate multiple transport pathways and extracellular disease resistance in plants

PubMed Central

Uemura, Tomohiro; Kim, Hyeran; Saito, Chieko; Ebine, Kazuo; Ueda, Takashi; Schulze-Lefert, Paul; Nakano, Akihiko

2012-01-01

In all eukaryotic cells, a membrane-trafficking system connects the post-Golgi organelles, such as the trans-Golgi network (TGN), endosomes, vacuoles, and the plasma membrane. This complex network plays critical roles in several higher-order functions in multicellular organisms. The TGN, one of the important organelles for protein transport in the post-Golgi network, functions as a sorting station, where cargo proteins are directed to the appropriate post-Golgi compartments. Unlike its roles in animal and yeast cells, the TGN has also been reported to function like early endosomal compartments in plant cells. However, the physiological roles of the TGN functions in plants are not understood. Here, we report a study of the SYP4 group (SYP41, SYP42, and SYP43), which represents the plant orthologs of the Tlg2/syntaxin16 Qa-SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) that localizes on the TGN in yeast and animal cells. The SYP4 group regulates the secretory and vacuolar transport pathways in the post-Golgi network and maintains the morphology of the Golgi apparatus and TGN. Consistent with a secretory role, SYP4 proteins are required for extracellular resistance responses to a fungal pathogen. We also reveal a plant cell-specific higher-order role of the SYP4 group in the protection of chloroplasts from salicylic acid-dependent biotic stress. PMID:22307646

Biogenesis of Golgi Stacks in Imaginal Discs of Drosophila melanogaster

PubMed Central

Kondylis, Vangelis; Goulding, Sarah E.; Dunne, Jonathan C.; Rabouille, Catherine

2001-01-01

We provide a detailed description of Golgi stack biogenesis that takes place in vivo during one of the morphogenetic events in the lifespan of Drosophila melanogaster. In early third-instar larvae, small clusters consisting mostly of vesicles and tubules were present in epithelial imaginal disk cells. As larvae progressed through mid- and late-third instar, these larval clusters became larger but also increasingly formed cisternae, some of which were stacked. In white pupae, the typical Golgi stack was observed. We show that larval clusters are Golgi stack precursors by 1) localizing various Golgi-specific markers to the larval clusters by electron and immunofluorescence confocal microscopy, 2) driving this conversion in wild-type larvae incubated at 37°C for 2 h, and 3) showing that this conversion does not take place in an NSF1 mutant (comt 17). The biological significance of this conversion became clear when we found that the steroid hormone 20-hydroxyecdysone (ecdysone) is critically involved in this conversion. In its absence, Golgi stack biogenesis did not occur and the larval clusters remained unaltered. We showed that dGM130 and sec23p expression increases approximately three- and fivefold, respectively, when discs are exposed to ecdysone in vivo and in vitro. Taken together, these results suggest that we have developed an in vivo system to study the ecdysone-triggered Golgi stack biogenesis. PMID:11514618

Yif1 associates with Yip1 on Golgi and regulates dendrite pruning in sensory neurons during Drosophila metamorphosis.

PubMed

Wang, Qiwei; Wang, Yan; Yu, Fengwei

2018-05-16

Pruning that selectively removes unnecessary neurites without causing neuronal death is essential for sculpting the mature nervous system during development. In Drosophila , ddaC sensory neurons specifically prune their larval dendrites with intact axons during metamorphosis. However, it remains unknown about an important role of ER-to-Golgi transport in dendrite pruning. Here, in a clonal screen we identified Yif1, an uncharacterized Drosophila homologue of Yif1p that is known as a regulator of ER-to-Golgi transport in yeast. We show that Yif1 is required for dendrite pruning of ddaC neurons but not for apoptosis of ddaF neurons. We further identified the Yif1-binding partner Yip1 which is also crucial for dendrite pruning. Yif1 forms a protein complex with Yip1 in S2 cells and ddaC neurons. Yip1 and Yif1 colocalize on ER/Golgi and are required for the integrity of Golgi apparatus and outposts. Moreover, we show that two GTPases Rab1 and Sar1, known to regulate ER-to-Golgi transport, are essential for dendrite pruning of ddaC neurons. Finally, our data reveal that ER-to-Golgi transport promotes endocytosis and downregulation of cell adhesion molecule Neuroglian and thereby dendrite pruning. © 2018. Published by The Company of Biologists Ltd.

Nonrandom γ-TuNA-dependent spatial pattern of microtubule nucleation at the Golgi

PubMed Central

Sanders, Anna A. W. M.; Chang, Kevin; Zhu, Xiaodong; Thoppil, Roslin J.; Holmes, William R.; Kaverina, Irina

2017-01-01

Noncentrosomal microtubule (MT) nucleation at the Golgi generates MT network asymmetry in motile vertebrate cells. Investigating the Golgi-derived MT (GDMT) distribution, we find that MT asymmetry arises from nonrandom nucleation sites at the Golgi (hotspots). Using computational simulations, we propose two plausible mechanistic models of GDMT nucleation leading to this phenotype. In the “cooperativity” model, formation of a single GDMT promotes further nucleation at the same site. In the “heterogeneous Golgi” model, MT nucleation is dramatically up-regulated at discrete and sparse locations within the Golgi. While MT clustering in hotspots is equally well described by both models, simulating MT length distributions within the cooperativity model fits the data better. Investigating the molecular mechanism underlying hotspot formation, we have found that hotspots are significantly smaller than a Golgi subdomain positive for scaffolding protein AKAP450, which is thought to recruit GDMT nucleation factors. We have further probed potential roles of known GDMT-promoting molecules, including γ-TuRC-mediated nucleation activator (γ-TuNA) domain-containing proteins and MT stabilizer CLASPs. While both γ-TuNA inhibition and lack of CLASPs resulted in drastically decreased GDMT nucleation, computational modeling revealed that only γ-TuNA inhibition suppressed hotspot formation. We conclude that hotspots require γ-TuNA activity, which facilitates clustered GDMT nucleation at distinct Golgi sites. PMID:28931596

Calcium-containing phosphopeptides pave the secretory pathway for efficient protein traffic and secretion in fungi.

PubMed

Martín, Juan F

2014-09-10

Casein phosphopeptides (CPPs) containing chelated calcium drastically increase the secretion of extracellular homologous and heterologous proteins in filamentous fungi. Casein phosphopeptides released by digestion of alpha - and beta-casein are rich in phosphoserine residues (SerP). They stimulate enzyme secretion in the gastrointestinal tract and enhance the immune response in mammals, and are used as food supplements. It is well known that casein phosphopeptides transport Ca2+ across the membranes and play an important role in Ca2+ homeostasis in the cells. Addition of CPPs drastically increases the production of heterologous proteins in Aspergillus as host for industrial enzyme production. Recent proteomics studies showed that CPPs alter drastically the vesicle-mediated secretory pathway in filamentous fungi, apparently because they change the calcium concentration in organelles that act as calcium reservoirs. In the organelles calcium homeostasis a major role is played by the pmr1 gene, that encodes a Ca2+/Mn2+ transport ATPase, localized in the Golgi complex; this transporter controls the balance between intra-Golgi and cytoplasmic Ca2+ concentrations. A Golgi-located casein kinase (CkiA) governs the ER to Golgi directionality of the movement of secretory proteins by interacting with the COPII coat of secretory vesicles when they reach the Golgi. Mutants defective in the casein-2 kinase CkiA show abnormal targeting of some secretory proteins, including cytoplasmic membrane amino acid transporters that in ckiA mutants are miss-targeted to vacuolar membranes. Interestingly, addition of CPPs increases a glyceraldehyde-3-phpshate dehydrogenase protein that is known to associate with microtubules and act as a vesicle/membrane fusogenic agent. In summary, CPPs alter the protein secretory pathway in fungi adapting it to a deregulated protein traffic through the organelles and vesicles what results in a drastic increase in secretion of heterologous and also of some homologous proteins.

Sec34 is implicated in traffic from the endoplasmic reticulum to the Golgi and exists in a complex with GTC-90 and ldlBp.

PubMed

Loh, Eva; Hong, Wanjin

2002-06-14

Sec34p/Grd20p has been implicated in endoplasmic reticulum (ER)-to-Golgi transport and/or post-Golgi trafficking events and exists in a protein complex consisting of at least eight subunits in yeast. Although the mammalian counterpart (Sec34) of Sec34p has been molecularly identified, its role and interacting partners remain undefined. In this study, we have prepared antibodies specifically against the recombinant N-terminal fragment of Sec34 that recognize a polypeptide of about 93 kDa and label the Golgi apparatus. In a well-characterized semi-intact cell assay that reconstitutes transport of the envelope glycoprotein (VSVG) of vesicular stomatitis virus from the ER to the Golgi apparatus, anti-Sec34 antibodies inhibited the transport in a dose-dependent manner. The inhibition by anti-Sec34 antibodies could be neutralized by a noninhibitory amount of the antigen. Large-scale immunoprecipitation of rat liver cytosol with immobilized anti-Sec34 antibodies has co-immunoprecipitated GTC-90 and ldlBp, two peripheral Golgi proteins previously shown to exist in separate protein complexes. Two mammalian homologues (Dor1 and Cod1) of the yeast Sec34 complex were similarly recovered in the Sec34 immunoprecipitates. When expressed in transfected cells, epitope-tagged ldlCp and Cod2 were co-immunoprecipitated with anti-Sec34 antibodies with efficiencies comparable to that observed for tagged ldlBp, Dor1, and Cod1. Direct interactions of Sec34 with ldlBp and ldlCp were further demonstrated in vitro. These results suggest that Sec34, GTC-90, and ldlBp/ldlCp are part of the same protein complex(es) that regulates diverse aspects of Golgi function, including transport from the ER to the Golgi apparatus.

Nonrandom γ-TuNA-dependent spatial pattern of microtubule nucleation at the Golgi.

PubMed

Sanders, Anna A W M; Chang, Kevin; Zhu, Xiaodong; Thoppil, Roslin J; Holmes, William R; Kaverina, Irina

2017-11-07

Noncentrosomal microtubule (MT) nucleation at the Golgi generates MT network asymmetry in motile vertebrate cells. Investigating the Golgi-derived MT (GDMT) distribution, we find that MT asymmetry arises from nonrandom nucleation sites at the Golgi (hotspots). Using computational simulations, we propose two plausible mechanistic models of GDMT nucleation leading to this phenotype. In the "cooperativity" model, formation of a single GDMT promotes further nucleation at the same site. In the "heterogeneous Golgi" model, MT nucleation is dramatically up-regulated at discrete and sparse locations within the Golgi. While MT clustering in hotspots is equally well described by both models, simulating MT length distributions within the cooperativity model fits the data better. Investigating the molecular mechanism underlying hotspot formation, we have found that hotspots are significantly smaller than a Golgi subdomain positive for scaffolding protein AKAP450, which is thought to recruit GDMT nucleation factors. We have further probed potential roles of known GDMT-promoting molecules, including γ-TuRC-mediated nucleation activator (γ-TuNA) domain-containing proteins and MT stabilizer CLASPs. While both γ-TuNA inhibition and lack of CLASPs resulted in drastically decreased GDMT nucleation, computational modeling revealed that only γ-TuNA inhibition suppressed hotspot formation. We conclude that hotspots require γ-TuNA activity, which facilitates clustered GDMT nucleation at distinct Golgi sites. © 2017 Sanders et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Interorganellar Regulation of Lysosome Positioning by the Golgi Apparatus through Rab34 Interaction with Rab-interacting Lysosomal Protein

PubMed Central

Wang, Tuanlao; Hong, Wanjin

2002-01-01

We present evidence to suggest the existence of a regulatory pathway for the Golgi apparatus to modulate the spatial positioning of otherwise distantly located lysosomes. Rab34, a new member of the Rab GTPase family, is associated primarily with the Golgi apparatus. Expression of wild-type or GTP-restricted but not GDP-restricted versions of Rab34 causes spatial redistribution of lysosomes from the periphery to the peri-Golgi region. The regulation of lysosomal positioning by Rab34 depends on its association with the membrane mediated by prenylation and its direct interaction with Rab-interacting lysosomal protein (RILP). This biological activity, mediated by Rab34-RILP interaction, is dependent on Lys82 in the switch I region. Our results have uncovered a novel mechanism for the Golgi apparatus to regulate the spatial distribution of another organelle. PMID:12475955

Agonist-biased trafficking of somatostatin receptor 2A in enteric neurons.

PubMed

Zhao, Peishen; Canals, Meritxell; Murphy, Jane E; Klingler, Diana; Eriksson, Emily M; Pelayo, Juan-Carlos; Hardt, Markus; Bunnett, Nigel W; Poole, Daniel P

2013-09-06

Somatostatin (SST) 14 and SST 28 activate somatostatin 2A receptors (SSTR2A) on enteric neurons to control gut functions. SST analogs are treatments of neuroendocrine and bleeding disorders, cancer, and diarrhea, with gastrointestinal side effects of constipation, abdominal pain, and nausea. How endogenous agonists and drugs differentially regulate neuronal SSTR2A is unexplored. We evaluated SSTR2A trafficking in murine myenteric neurons and neuroendocrine AtT-20 cells by microscopy and determined whether agonist degradation by endosomal endothelin-converting enzyme 1 (ECE-1) controls SSTR2A trafficking and association with β-arrestins, key regulators of receptors. SST-14, SST-28, and peptide analogs (octreotide, lanreotide, and vapreotide) stimulated clathrin- and dynamin-mediated internalization of SSTR2A, which colocalized with ECE-1 in endosomes and the Golgi. After incubation with SST-14, SSTR2A recycled to the plasma membrane, which required active ECE-1 and an intact Golgi. SSTR2A activated by SST-28, octreotide, lanreotide, or vapreotide was retained within the Golgi and did not recycle. Although ECE-1 rapidly degraded SST-14, SST-28 was resistant to degradation, and ECE-1 did not degrade SST analogs. SST-14 and SST-28 induced transient interactions between SSTR2A and β-arrestins that were stabilized by an ECE-1 inhibitor. Octreotide induced sustained SSTR2A/β-arrestin interactions that were not regulated by ECE-1. Thus, when activated by SST-14, SSTR2A internalizes and recycles via the Golgi, which requires ECE-1 degradation of SST-14 and receptor dissociation from β-arrestins. After activation by ECE-1-resistant SST-28 and analogs, SSTR2A remains in endosomes because of sustained β-arrestin interactions. Therapeutic SST analogs are ECE-1-resistant and retain SSTR2A in endosomes, which may explain their long-lasting actions.

Agonist-biased Trafficking of Somatostatin Receptor 2A in Enteric Neurons*

PubMed Central

Zhao, Peishen; Canals, Meritxell; Murphy, Jane E.; Klingler, Diana; Eriksson, Emily M.; Pelayo, Juan-Carlos; Hardt, Markus; Bunnett, Nigel W.; Poole, Daniel P.

2013-01-01

Somatostatin (SST) 14 and SST 28 activate somatostatin 2A receptors (SSTR2A) on enteric neurons to control gut functions. SST analogs are treatments of neuroendocrine and bleeding disorders, cancer, and diarrhea, with gastrointestinal side effects of constipation, abdominal pain, and nausea. How endogenous agonists and drugs differentially regulate neuronal SSTR2A is unexplored. We evaluated SSTR2A trafficking in murine myenteric neurons and neuroendocrine AtT-20 cells by microscopy and determined whether agonist degradation by endosomal endothelin-converting enzyme 1 (ECE-1) controls SSTR2A trafficking and association with β-arrestins, key regulators of receptors. SST-14, SST-28, and peptide analogs (octreotide, lanreotide, and vapreotide) stimulated clathrin- and dynamin-mediated internalization of SSTR2A, which colocalized with ECE-1 in endosomes and the Golgi. After incubation with SST-14, SSTR2A recycled to the plasma membrane, which required active ECE-1 and an intact Golgi. SSTR2A activated by SST-28, octreotide, lanreotide, or vapreotide was retained within the Golgi and did not recycle. Although ECE-1 rapidly degraded SST-14, SST-28 was resistant to degradation, and ECE-1 did not degrade SST analogs. SST-14 and SST-28 induced transient interactions between SSTR2A and β-arrestins that were stabilized by an ECE-1 inhibitor. Octreotide induced sustained SSTR2A/β-arrestin interactions that were not regulated by ECE-1. Thus, when activated by SST-14, SSTR2A internalizes and recycles via the Golgi, which requires ECE-1 degradation of SST-14 and receptor dissociation from β-arrestins. After activation by ECE-1-resistant SST-28 and analogs, SSTR2A remains in endosomes because of sustained β-arrestin interactions. Therapeutic SST analogs are ECE-1-resistant and retain SSTR2A in endosomes, which may explain their long-lasting actions. PMID:23913690

Viewing Golgi structure and function from a different perspective--insights from electron tomography.

PubMed

Marsh, Brad J; Pavelka, Margit

2013-01-01

Historically, ultrastructural investigations, which have focused on elucidating the biological idiosyncrasies of the Golgi apparatus, have tended towards oversimplified or fallacious hypotheses when postulating how the Golgi apparatus reorganizes itself both structurally and functionally to fulfill the plethora of cellular processes underpinned by this complex organelle. Key questions are still unanswered with regard to how changes in Golgi architecture correlate so reproducibly to changes in its functional priorities under different physiological conditions or experimental perturbations. This fact alone serves to highlight how the technical limitations associated with conventional two-dimensional imaging approaches employed in the past failed to adequately capture the extraordinary complexity of the Golgi's three-dimensional (3D) structure-now a hallmark of this challenging organelle. Consequently, this has hampered progress towards developing a clear understanding of how changes in its structure and function typically occur in parallel. In this chapter, we highlight but a few of the significant new insights regarding variations in the Golgi's structure-function relationships that have been afforded over recent years through advanced electron microscopic techniques for 3D image reconstruction, commonly referred to as electron tomography. Copyright © 2013 Elsevier Inc. All rights reserved.

The oncolytic compound LTX-401 targets the Golgi apparatus

PubMed Central

Zhou, Heng; Sauvat, Allan; Gomes-da-Silva, Lígia C; Durand, Sylvère; Forveille, Sabrina; Iribarren, Kristina; Yamazaki, Takahiro; Souquere, Sylvie; Bezu, Lucillia; Müller, Kevin; Leduc, Marion; Liu, Peng; Zhao, Liwei; Marabelle, Aurélien; Zitvogel, Laurence; Rekdal, Øystein; Kepp, Oliver; Kroemer, Guido

2016-01-01

LTX-401 is an oncolytic amino acid derivative with potential immunogenic properties. Here, we demonstrate that LTX-401 selectively destroys the structure of the Golgi apparatus, as determined by means of ultrastructural analyses and fluorescence microscopic observation of cells expressing Golgi-targeted GFP reporters. Subcellular fractionation followed by mass spectrometric detection revealed that LTX-401 selectively enriched in the Golgi rather than in mitochondria or in the cytosol. The Golgi-dissociating agent Brefeldin A (BFA) reduced cell killing by LTX-401 as it partially inhibited LTX-401-induced mitochondrial release of cytochrome c and the activation of BAX. The cytotoxic effect of LTX-401 was attenuated by the double knockout of BAX and BAK, as well as the mitophagy-enforced depletion of mitochondria, yet was refractory to caspase inhibition. LTX-401 induced all major hallmarks of immunogenic cell death detectable with biosensor cell lines including calreticulin exposure, ATP release, HMGB1 exodus and a type-1 interferon response. Moreover, LTX-401-treated tumors manifested a strong lymphoid infiltration. Altogether these results support the contention that LTX-401 can stimulate immunogenic cell death through a pathway in which Golgi-localized LTX-401 operates upstream of mitochondrial membrane permeabilization. PMID:27588704

Quantifying Golgi structure using EM: combining volume-SEM and stereology for higher throughput.

PubMed

Ferguson, Sophie; Steyer, Anna M; Mayhew, Terry M; Schwab, Yannick; Lucocq, John Milton

2017-06-01

Investigating organelles such as the Golgi complex depends increasingly on high-throughput quantitative morphological analyses from multiple experimental or genetic conditions. Light microscopy (LM) has been an effective tool for screening but fails to reveal fine details of Golgi structures such as vesicles, tubules and cisternae. Electron microscopy (EM) has sufficient resolution but traditional transmission EM (TEM) methods are slow and inefficient. Newer volume scanning EM (volume-SEM) methods now have the potential to speed up 3D analysis by automated sectioning and imaging. However, they produce large arrays of sections and/or images, which require labour-intensive 3D reconstruction for quantitation on limited cell numbers. Here, we show that the information storage, digital waste and workload involved in using volume-SEM can be reduced substantially using sampling-based stereology. Using the Golgi as an example, we describe how Golgi populations can be sensed quantitatively using single random slices and how accurate quantitative structural data on Golgi organelles of individual cells can be obtained using only 5-10 sections/images taken from a volume-SEM series (thereby sensing population parameters and cell-cell variability). The approach will be useful in techniques such as correlative LM and EM (CLEM) where small samples of cells are treated and where there may be variable responses. For Golgi study, we outline a series of stereological estimators that are suited to these analyses and suggest workflows, which have the potential to enhance the speed and relevance of data acquisition in volume-SEM.

trans-Golgi retention of a plasma membrane protein: mutations in the cytoplasmic domain of the asialoglycoprotein receptor subunit H1 result in trans-Golgi retention

PubMed Central

1995-01-01

Unlike the wild-type asialoglycoprotein receptor subunit H1 which is transported to the cell surface, endocytosed and recycled, a mutant lacking residues 4-33 of the 40-amino acid cytoplasmic domain was found to be retained intracellularly upon expression in different cell lines. The mutant protein accumulated in the trans-Golgi, as judged from the acquisition of trans-Golgi-specific modifications of the protein and from the immunofluorescence staining pattern. It was localized to juxtanuclear, tubular structures that were also stained by antibodies against galactosyltransferase and gamma-adaptin. The results of further mutagenesis in the cytoplasmic domain indicated that the size rather than the specific sequence of the cytoplasmic domain determines whether H1 is retained in the trans-Golgi or transported to the cell surface. Truncation to less than 17 residues resulted in retention, and extension of a truncated tail by an unrelated sequence restored surface transport. The transmembrane segment of H1 was not sufficient for retention of a reporter molecule and it could be replaced by an artificial apolar sequence without affecting Golgi localization. The cytoplasmic domain thus appears to inhibit interaction(s) of the exoplasmic portion of H1 with trans-Golgi component(s) for example by steric hindrance or by changing the positioning of the protein in the membrane. This mechanism may also be functional in other proteins. PMID:7615632

Stimulus-Dependent State Transition between Synchronized Oscillation and Randomly Repetitive Burst in a Model Cerebellar Granular Layer

PubMed Central

Tanaka, Shigeru; Nagao, Soichi; Nishino, Tetsuro

2011-01-01

Information processing of the cerebellar granular layer composed of granule and Golgi cells is regarded as an important first step toward the cerebellar computation. Our previous theoretical studies have shown that granule cells can exhibit random alternation between burst and silent modes, which provides a basis of population representation of the passage-of-time (POT) from the onset of external input stimuli. On the other hand, another computational study has reported that granule cells can exhibit synchronized oscillation of activity, as consistent with observed oscillation in local field potential recorded from the granular layer while animals keep still. Here we have a question of whether an identical network model can explain these distinct dynamics. In the present study, we carried out computer simulations based on a spiking network model of the granular layer varying two parameters: the strength of a current injected to granule cells and the concentration of Mg2+ which controls the conductance of NMDA channels assumed on the Golgi cell dendrites. The simulations showed that cells in the granular layer can switch activity states between synchronized oscillation and random burst-silent alternation depending on the two parameters. For higher Mg2+ concentration and a weaker injected current, granule and Golgi cells elicited spikes synchronously (synchronized oscillation state). In contrast, for lower Mg2+ concentration and a stronger injected current, those cells showed the random burst-silent alternation (POT-representing state). It is suggested that NMDA channels on the Golgi cell dendrites play an important role for determining how the granular layer works in response to external input. PMID:21779155

Golgi sorting regulates organization and activity of GPI-proteins at apical membranes

PubMed Central

Tivodar, Simona; Formiggini, Fabio; Ossato, Giulia; Gratton, Enrico; Tramier, Marc; Coppey-Moisan, Maïté; Zurzolo, Chiara

2014-01-01

Here, we combined classical biochemistry with novel biophysical approaches to study with high spatial and temporal resolution the organization of GPI-anchored proteins (GPI-APs) at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, following sorting in the Golgi, each GPI-AP reaches the apical surface in homo-clusters. Golgi-derived homo-clusters are required for their subsequent plasma membrane organization into cholesterol-dependent hetero-clusters. By contrast, in non-polarized MDCK cells GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form hetero-clusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, different from fibroblasts, in polarized epithelial cells a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and the function of GPI-APs at the apical surface. PMID:24681536

Stapled Golgi cisternae remain in place as cargo passes through the stack

PubMed Central

Lavieu, Gregory; Zheng, Hong; Rothman, James E

2013-01-01

We have designed a membrane ‘staple’, which consists of membrane-anchored repeats of the trans-aggregating FM domain that face the lumen of the secretory pathway. In the presence of the disaggregating drug these proteins transit the secretory pathway. When the drug is removed these proteins form electron-dense plaques which we term staples. Unexpectedly, when initially positioned within the cis-Golgi, staples remained at the cis face of the Golgi even after many hours. By contrast, soluble FM-aggregates transited the Golgi. Staples and soluble aggregates placed in cis-Golgi cisternae therefore have different fates. Whereas the membrane staples are located in the flattened, stacked central regions of the cisternae, the soluble aggregates are in the dilated rims. This suggests that while the cisternae are static on the time scale of protein traffic, the dilated rims are mobile and progress in the cis → trans direction via a mechanism that we term ‘Rim Progression’. DOI: http://dx.doi.org/10.7554/eLife.00558.001 PMID:23755362

High-resolution mapping reveals topologically distinct cellular pools of phosphatidylserine

PubMed Central

Fairn, Gregory D.; Schieber, Nicole L.; Ariotti, Nicholas; Murphy, Samantha; Kuerschner, Lars; Webb, Richard I.; Grinstein, Sergio

2011-01-01

Phosphatidylserine (PS) plays a central role in cell signaling and in the biosynthesis of other lipids. To date, however, the subcellular distribution and transmembrane topology of this crucial phospholipid remain ill-defined. We transfected cells with a GFP-tagged C2 domain of lactadherin to detect by light and electron microscopy PS exposed on the cytosolic leaflet of the plasmalemma and organellar membranes. Cytoplasmically exposed PS was found to be clustered on the plasma membrane, and to be associated with caveolae, the trans-Golgi network, and endocytic organelles including intraluminal vesicles of multivesicular endosomes. This labeling pattern was compared with the total cellular distribution of PS as visualized using a novel on-section technique. These complementary methods revealed PS in the interior of the ER, Golgi complex, and mitochondria. These results indicate that PS in the lumenal monolayer of the ER and Golgi complex becomes exposed cytosolically at the trans-Golgi network. Transmembrane flipping of PS may contribute to the exit of cargo from the Golgi complex. PMID:21788369

Cell biology. ER-to-Golgi traffic--this bud's for you.

PubMed

Brittle, E E; Waters, M G

2000-07-21

How do protein-transporting vesicles, which bud from the endoplasmic reticulum (ER), specifically dock to, and fuse with, the Golgi apparatus? In their Perspective, Brittle and Waters discuss new work (Allan et al.) suggesting that some vesicle-associated docking and fusion proteins are "programmed" during vesicle budding from the ER and direct downstream events that occur during fusion of these transport vesicles with the membranes of the Golgi.

Dissecting the Role of Disturbed ER-Golgi Trafficking in Antivirals and Alcohol Abuse-Induced Pathogenesis of Liver Disorders.

PubMed

Ji, Cheng

2017-01-01

Antiviral drugs and alcohol abuse-induced organelle stresses have been linked to many disorders and the underlying molecular mechanisms are under intense investigations. This brief review communicates emerging evidence and research trends on how certain antivirals and alcohol affect ER-Golgi trafficking, which potentially impacts the function and integrity of the Golgi apparatus contributing to endoplasmic reticulum stress and cellular injury.

Determinants for membrane association and permeabilization of the coxsackievirus 2B protein and the identification of the Golgi complex as the target organelle.

PubMed

de Jong, Arjan S; Wessels, Els; Dijkman, Henri B P M; Galama, Jochem M D; Melchers, Willem J G; Willems, Peter H G M; van Kuppeveld, Frank J M

2003-01-10

The 2B protein of enterovirus is responsible for the alterations in the permeability of secretory membranes and the plasma membrane in infected cells. The structural requirements for the membrane association and the subcellular localization of this essential virus protein, however, have not been defined. Here, we provide evidence that the 2B protein is an integral membrane protein in vivo that is predominantly localized at the Golgi complex upon individual expression. Addition of organelle-specific targeting signals to the 2B protein revealed that the Golgi localization is an absolute prerequisite for the ability of the protein to modify plasma membrane permeability. Expression of deletion mutants and heterologous proteins containing specific domains of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in the Golgi complex and the possible mechanism by which a Golgi-localized virus protein modifies plasma membrane permeability are discussed.

Cholesterol and Fatty Acids Regulate Dynamic Caveolin Trafficking through the Golgi Complex and between the Cell Surface and Lipid BodiesV⃞

PubMed Central

Pol, Albert; Martin, Sally; Fernández, Manuel A.; Ingelmo-Torres, Mercedes; Ferguson, Charles; Enrich, Carlos; Parton, Robert G.

2005-01-01

Caveolins are a crucial component of plasma membrane (PM) caveolae but have also been localized to intracellular compartments, including the Golgi complex and lipid bodies. Mutant caveolins associated with human disease show aberrant trafficking to the PM and Golgi accumulation. We now show that the Golgi pool of mainly newly synthesized protein is detergent-soluble and predominantly in a monomeric state, in contrast to the surface pool. Caveolin at the PM is not recognized by specific caveolin antibodies unless PM cholesterol is depleted. Exit from the Golgi complex of wild-type caveolin-1 or -3, but not vesicular stomatitis virus-G protein, is modulated by changing cellular cholesterol levels. In contrast, a muscular dystrophy-associated mutant of caveolin-3, Cav3P104L, showed increased accumulation in the Golgi complex upon cholesterol treatment. In addition, we demonstrate that in response to fatty acid treatment caveolin can follow a previously undescribed pathway from the PM to lipid bodies and can move from lipid bodies to the PM in response to removal of fatty acids. The results suggest that cholesterol is a rate-limiting component for caveolin trafficking. Changes in caveolin flux through the exocytic pathway can therefore be an indicator of cellular cholesterol and fatty acid levels. PMID:15689493

Identification of inhibitors using a cell-based assay for monitoring Golgi-resident protease activity.

PubMed

Coppola, Julia M; Hamilton, Christin A; Bhojani, Mahaveer S; Larsen, Martha J; Ross, Brian D; Rehemtulla, Alnawaz

2007-05-01

Noninvasive real-time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease's natural milieu. We developed a protease activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, on protease-mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV as well as in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high-throughput screening of 39,000-compound small molecule libraries, leading to identification of furin inhibitors. Furthermore, this strategy was used to identify inhibitors of another Golgi protease, the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE). BACE cleavage of the APP leads to formation of the Abeta peptide, a key event that leads to Alzheimer's disease. In conclusion, we describe a customizable noninvasive technology for real-time assessment of Golgi protease activity used to identify inhibitors of furin and BACE.

Auxin Deprivation Induces Synchronous Golgi Differentiation in Suspension-Cultured Tobacco BY-2 Cells1

PubMed Central

Winicur, Zev M.; Feng Zhang, Guo; Andrew Staehelin, L.

1998-01-01

To date, the lack of a method for inducing plant cells and their Golgi stacks to differentiate in a synchronous manner has made it difficult to characterize the nature and extent of Golgi retailoring in biochemical terms. Here we report that auxin deprivation can be used to induce a uniform population of suspension-cultured tobacco (Nicotiana tabacum cv BY-2) cells to differentiate synchronously during a 4-d period. Upon removal of auxin, the cells stop dividing, undergo elongation, and differentiate in a manner that mimics the formation of slime-secreting epidermal and peripheral root-cap cells. The morphological changes to the Golgi apparatus include a proportional increase in the number of trans-Golgi cisternae, a switch to larger-sized secretory vesicles that bud from the trans-Golgi cisternae, and an increase in osmium staining of the secretory products. Biochemical alterations include an increase in large, fucosylated, mucin-type glycoproteins, changes in the types of secreted arabinogalactan proteins, and an increase in the amounts and types of molecules containing the peripheral root-cap-cell-specific epitope JIM 13. Taken together, these findings support the hypothesis that auxin deprivation can be used to induce tobacco BY-2 cells to differentiate synchronously into mucilage-secreting cells. PMID:9625703

Physiological Roles of Plant Post-Golgi Transport Pathways in Membrane Trafficking.

PubMed

Uemura, Tomohiro

2016-10-01

Membrane trafficking is the fundamental system through which proteins are sorted to their correct destinations in eukaryotic cells. Key regulators of this system include RAB GTPases and soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs). Interestingly, the numbers of RAB GTPases and SNAREs involved in post-Golgi transport pathways in plant cells are larger than those in animal and yeast cells, suggesting that plants have evolved unique and complex post-Golgi transport pathways. The trans-Golgi network (TGN) is an important organelle that acts as a sorting station in the post-Golgi transport pathways of plant cells. The TGN also functions as the early endosome, which is the first compartment to receive endocytosed proteins. Several endocytosed proteins on the plasma membrane (PM) are initially targeted to the TGN/EE, then recycled back to the PM or transported to the vacuole for degradation. The recycling and degradation of the PM localized proteins is essential for the development and environmental responses in plant. The present review describes the post-Golgi transport pathways that show unique physiological functions in plants. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The golgin GMAP-210 is required for efficient membrane trafficking in the early secretory pathway

PubMed Central

Roboti, Peristera; Sato, Keisuke; Lowe, Martin

2015-01-01

Golgins are coiled-coil proteins that participate in membrane-tethering events at the Golgi complex. Golgin-mediated tethering is thought to be important for vesicular trafficking and Golgi organization. However, the degree to which individual golgins contribute to these processes is poorly defined, and it has been proposed that golgins act in a largely redundant manner. Previous studies on the golgin GMAP-210 (also known as TRIP11), which is mutated in the rare skeletal disorder achondrogenesis type 1A, have yielded conflicting results regarding its involvement in trafficking. Here, we re-investigated the trafficking role of GMAP-210, and found that it is indeed required for efficient trafficking in the secretory pathway. GMAP-210 acts at both the endoplasmic reticulum (ER)-to-Golgi intermediate compartment (ERGIC) and Golgi complex during anterograde trafficking, and is also required for retrograde trafficking to the ER. Using co-depletion experiments, we also found that GMAP-210 acts in a partially redundant manner with the golgin GM130 to ensure efficient anterograde cargo delivery to the cis-Golgi. In summary, our results indicate a role for GMAP-210 in several trafficking steps at the ER–Golgi interface, some of which are partially redundant with another golgin, namely GM130 (also known as GOLGA2). PMID:25717001

DOE Office of Scientific and Technical Information (OSTI.GOV)

Venkatagopalan, Pavithra; School of Life Sciences, Arizona State University, Tempe, AZ 85287-5401; Microbiology Graduate Program, Arizona State University, Tempe, AZ 85287-5401

Coronaviruses (CoVs) assemble at endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes and egress from cells in cargo vesicles. Only a few molecules of the envelope (E) protein are assembled into virions. The role of E in morphogenesis is not fully understood. The cellular localization and dynamics of mouse hepatitis CoV A59 (MHV) E protein were investigated to further understanding of its role during infection. E protein localized in the ERGIC and Golgi with the amino and carboxy termini in the lumen and cytoplasm, respectively. E protein does not traffic to the cell surface. MHV was genetically engineered with a tetracysteinemore » tag at the carboxy end of E. Fluorescence recovery after photobleaching (FRAP) showed that E is mobile in ERGIC/Golgi membranes. Correlative light electron microscopy (CLEM) confirmed the presence of E in Golgi cisternae. The results provide strong support that E proteins carry out their function(s) at the site of budding/assembly. - Highlights: • Mouse hepatitis coronavirus (MHV-CoV) E protein localizes in the ERGIC and Golgi. • MHV-CoV E does not transport to the cell surface. • MHV-CoV can be genetically engineered with a tetracysteine tag appended to E. • First FRAP and correlative light electron microscopy of a CoV E protein. • Live-cell imaging shows that E is mobile in ERGIC/Golgi membranes.« less

Identification of inhibitors using a cell based assay for monitoring golgi-resident protease activity

PubMed Central

Coppola, Julia M.; Hamilton, Christin A.; Bhojani, Mahaveer S.; Larsen, Martha J.; Ross, Brian D.; Rehemtulla, Alnawaz

2007-01-01

Non-invasive real time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease’s natural milieu. We developed a protease-activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, upon protease mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV, and in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high throughput screening of 30,000 compound small molecule libraries, leading to identification of furin inhibitors. Further, this strategy was utilized to identify inhibitors of another Golgi protease, the β-site APP-cleaving enzyme (BACE). BACE cleavage of the amyloid precursor protein leads to formation of the Aβ peptide, a key event that leads to Alzheimer’s disease. In conclusion, we describe a customizable, non-invasive technology for real time assessment of Golgi protease activity used to identify inhibitors of furin and BACE. PMID:17316541

The elaborate route for UDP-arabinose delivery into the Golgi of plants

DOE PAGES

Rautengarten, Carsten; Birdseye, Devon; Pattathil, Sivakumar; ...

2017-04-03

In plants, L-Arabinose (Ara) is a key component of cell wall polymers, glycoproteins, as well as flavonoids, and signaling peptides. Whereas the majority of Ara found in plant glycans occurs as a furanose ring (Araf), the activated precursor has a pyranose ring configuration (UDP-Arap). The biosynthesis of UDP-Arap mainly occurs via the epimerization of UDP-xylose (UDP-Xyl) in the Golgi lumen. Given that the predominant Ara form found in plants is Araf, UDP-Arap must exit the Golgi to be interconverted into UDPAraf by UDP-Ara mutases that are located outside on the cytosolic surface of the Golgi. Subsequently, UDP-Araf must be transportedmore » back into the lumen. During this step it is vital because glycosyltransferases, the enzymes mediating the glycosylation reactions, are located within the Golgi lumen, and UDP-Arap, synthesized within the Golgi, is not their preferred substrate. Therefore, the transport of UDP-Araf into the Golgi is a prerequisite. Although this step is critical for cell wall biosynthesis and the glycosylation of proteins and signaling peptides, the identification of these transporters has remained elusive. In this study, we present data demonstrating the identification and characterization of a family of Golgilocalized UDP-Araf transporters in Arabidopsis. The application of a proteoliposome-based transport assay revealed that four members of the nucleotide sugar transporter (NST) family can efficiently transport UDP-Araf in vitro. Subsequent analysis of mutant lines affected in the function of these NSTs confirmed their role as UDP-Araf transporters in vivo.« less

Golgi-type I and Golgi-type II neurons in the ventral anterior thalamic nucleus of the adult human: morphological features and quantitative analysis.

PubMed

Al-Hussain Bani Hani, Saleh M; El-Dwairi, Qasim A; Bataineh, Ziad M; Al-Haidari, Mohammad S; Al-Alami, Jamil

2008-05-01

The morphological and quantitative features of neurons in the adult human ventral anterior thalamic nucleus were studied in Golgi preparations. Two neuronal types were found and their quantitative features were studied. Golgi-type I neurons were medium to large cells with dense dendritic trees and dendritic protrusions and short hair-like appendages. They have somatic mean diameter of 30.8 microm (+/-9.4, n = 85). They have an average 100.3 dendritic branches, 48.97 dendritic branching points, and 58.85 dendritic tips. The mean diameters of their primary, secondary, and tertiary dendrites were 3.1 microm (+/-1, n = 80), 1.85 microm (+/-0.8, n = 145), and 1.5 microm (+/-0.4, n = 160), respectively. Golgi-type II neurons were small to medium cells with few sparsely branching dendrites and dendritic stalked appendages with or without terminal swellings. They have somatic mean diameters of 22.2 microm (+/-5.8, n = 120). They have an average 33.76 dendritic branches, 16.49 dendritic branching points, and 21.97 dendritic tips. The mean diameters of their primary, secondary, and tertiary dendrites were 1.6 microm (+/-0.86, n = 70), 1.15 microm (+/-0.55, n = 118), and 1 microm (+/-0.70, n = 95), respectively. These quantitative data may form the basis for further quantitative studies involving aging or some degenerative diseases that may affect cell bodies and/or dendritic trees of the Golgi-type I and/or Golgi-type II thalamic neurons.

The elaborate route for UDP-arabinose delivery into the Golgi of plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rautengarten, Carsten; Birdseye, Devon; Pattathil, Sivakumar

In plants, L-Arabinose (Ara) is a key component of cell wall polymers, glycoproteins, as well as flavonoids, and signaling peptides. Whereas the majority of Ara found in plant glycans occurs as a furanose ring (Araf), the activated precursor has a pyranose ring configuration (UDP-Arap). The biosynthesis of UDP-Arap mainly occurs via the epimerization of UDP-xylose (UDP-Xyl) in the Golgi lumen. Given that the predominant Ara form found in plants is Araf, UDP-Arap must exit the Golgi to be interconverted into UDPAraf by UDP-Ara mutases that are located outside on the cytosolic surface of the Golgi. Subsequently, UDP-Araf must be transportedmore » back into the lumen. During this step it is vital because glycosyltransferases, the enzymes mediating the glycosylation reactions, are located within the Golgi lumen, and UDP-Arap, synthesized within the Golgi, is not their preferred substrate. Therefore, the transport of UDP-Araf into the Golgi is a prerequisite. Although this step is critical for cell wall biosynthesis and the glycosylation of proteins and signaling peptides, the identification of these transporters has remained elusive. In this study, we present data demonstrating the identification and characterization of a family of Golgilocalized UDP-Araf transporters in Arabidopsis. The application of a proteoliposome-based transport assay revealed that four members of the nucleotide sugar transporter (NST) family can efficiently transport UDP-Araf in vitro. Subsequent analysis of mutant lines affected in the function of these NSTs confirmed their role as UDP-Araf transporters in vivo.« less

OSBP-related protein 11 (ORP11) dimerizes with ORP9 and localizes at the Golgi-late endosome interface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, You; Li, Shiqian; Maeyraenpaeae, Mikko I.

2010-11-15

We characterize here ORP11, a member of the oxysterol-binding protein family. ORP11 is present at highest levels in human ovary, testis, kidney, liver, stomach, brain, and adipose tissue. Immunohistochemistry demonstrates abundant ORP11 in the epithelial cells of kidney tubules, testicular tubules, caecum, and skin. ORP11 in HEK293 cells resides on Golgi complex and LE, co-localizing with GFP-Rab9, TGN46, GFP-Rab7, and a fluorescent medial-trans-Golgi marker. Under electron microscopic observation, cells overexpressing ORP11 displayed lamellar lipid bodies associated with vacuolar structures or the Golgi complex, indicating a disturbance of lipid trafficking. N-terminal fragment of ORP11 (aa 1-292) localized partially to Golgi, butmore » displayed enhanced localization on Rab7- and Rab9-positive LE, while the C-terminal ligand-binding domain (aa 273-747) was cytosolic, demonstrating that the membrane targeting determinants are N-terminal. Yeast two-hybrid screen revealed interaction of ORP11 with the related ORP9. The interacting region was delineated within aa 98-372 of ORP9 and aa 154-292 of ORP11. Overexpressed ORP9 was able to recruit EGFP-ORP11 to membranes, and ORP9 silencing inhibited ORP11 Golgi association. The results identify ORP11 as an OSBP homologue distributing at the Golgi-LE interface and define the ORP9-ORP11 dimer as a functional unit that may act as an intracellular lipid sensor or transporter.« less

Retrograde transport from the yeast Golgi is mediated by two ARF GAP proteins with overlapping function.

PubMed Central

Poon, P P; Cassel, D; Spang, A; Rotman, M; Pick, E; Singer, R A; Johnston, G C

1999-01-01

ARF proteins, which mediate vesicular transport, have little or no intrinsic GTPase activity. They rely on the actions of GTPase-activating proteins (GAPs) for their function. The in vitro GTPase activity of the Saccharomyces cerevisiae ARF proteins Arf1 and Arf2 is stimulated by the yeast Gcs1 protein, and in vivo genetic interactions between arf and gcs1 mutations implicate Gcs1 in vesicular transport. However, the Gcs1 protein is dispensable, indicating that additional ARF GAP proteins exist. We show that the structurally related protein Glo3, which is also dispensable, also exhibits ARF GAP activity. Genetic and in vitro approaches reveal that Glo3 and Gcs1 have an overlapping essential function at the endoplasmic reticulum (ER)-Golgi stage of vesicular transport. Mutant cells deficient for both ARF GAPs cannot proliferate, undergo a dramatic accumulation of ER and are defective for protein transport between ER and Golgi. The glo3Delta and gcs1Delta single mutations each interact with a sec21 mutation that affects a component of COPI, which mediates vesicular transport within the ER-Golgi shuttle, while increased dosage of the BET1, BOS1 and SEC22 genes encoding members of a v-SNARE family that functions within the ER-Golgi alleviates the effects of a glo3Delta mutation. An in vitro assay indicates that efficient retrieval from the Golgi to the ER requires these two proteins. These findings suggest that Glo3 and Gcs1 ARF GAPs mediate retrograde vesicular transport from the Golgi to the ER. PMID:9927415

Inducible Inhibition of Gβγ Reveals Localization-dependent Functions at the Plasma Membrane and Golgi.

PubMed

Klayman, Lauren M; Wedegaertner, Philip B

2017-02-03

Heterotrimeric G proteins signal at a variety of endomembrane locations, in addition to their canonical function at the cytoplasmic surface of the plasma membrane (PM), where they are activated by cell surface G protein-coupled receptors. Here we focus on βγ signaling at the Golgi, where βγ activates a signaling cascade, ultimately resulting in vesicle fission from the trans-Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous βγ in a spatial-temporal manner, we take advantage of a lipid association mutant of the widely used βγ inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system. We show that GRK2ct-KERE cannot inhibit βγ function when expressed in cells, but recruitment to a specific membrane location recovers the ability of GRK2ct-KERE to inhibit βγ signaling. PM-recruited GRK2ct-KERE inhibits lysophosphatidic acid-induced phosphorylation of Akt, whereas Golgi-recruited GRK2ct-KERE inhibits cargo transport from the TGN to the PM. Moreover, we show that Golgi-recruited GRK2ct-KERE inhibits model basolaterally targeted but not apically targeted cargo delivery, for both PM-destined and secretory cargo, providing the first evidence of selectivity in terms of cargo transport regulated by βγ. Last, we show that Golgi fragmentation induced by ilimaquinone and nocodazole is blocked by βγ inhibition, demonstrating that βγ is a key regulator of multiple pathways that impact Golgi morphology. Thus, we have developed a new molecular tool, recruitable GRK2ct-KERE, to modulate βγ signaling at specific subcellular locations, and we demonstrate novel cargo selectivity for βγ regulation of TGN to PM transport and a novel role for βγ in mediating Golgi fragmentation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Subcompartment localization of the side chain xyloglucan-synthesizing enzymes within Golgi stacks of tobacco suspension-cultured cells.

PubMed

Chevalier, Laurence; Bernard, Sophie; Ramdani, Yasmina; Lamour, Romain; Bardor, Muriel; Lerouge, Patrice; Follet-Gueye, Marie-Laure; Driouich, Azeddine

2010-12-01

Xyloglucan is the dominant hemicellulosic polysaccharide of the primary cell wall of dicotyledonous plants that plays a key role in plant development. It is well established that xyloglucan is assembled within Golgi stacks and transported in Golgi-derived vesicles to the cell wall. It is also known that the biosynthesis of xyloglucan requires the action of glycosyltransferases including α-1,6-xylosyltransferase, β-1,2-galactosyltransferase and α-1,2-fucosyltransferase activities responsible for the addition of xylose, galactose and fucose residues to the side chains. There is, however, a lack of knowledge on how these enzymes are distributed within subcompartments of Golgi stacks. We have undertaken a study aiming at mapping these glycosyltransferases within Golgi stacks using immunogold-electron microscopy. To this end, we generated transgenic lines of tobacco (Nicotiana tabacum) BY-2 suspension-cultured cells expressing either the α-1,6-xylosyltransferase, AtXT1, the β-1,2-galactosyltransferase, AtMUR3, or the α-1,2-fucosyltransferase AtFUT1 of Arabidopsis thaliana fused to green-fluorescent protein (GFP). Localization of the fusion proteins within the endomembrane system was assessed using confocal microscopy. Additionally, tobacco cells were high pressure-frozen/freeze-substituted and subjected to quantitative immunogold labelling using anti-GFP antibodies to determine the localization patterns of the enzymes within subtypes of Golgi cisternae. The data demonstrate that: (i) all fusion proteins, AtXT1-GFP, AtMUR3-GFP and AtFUT1-GFP are specifically targeted to the Golgi apparatus; and (ii) AtXT1-GFP is mainly located in the cis and medial cisternae, AtMUR3-GFP is predominantly associated with medial cisternae and AtFUT1-GFP mostly detected over trans cisternae suggesting that initiation of xyloglucan side chains occurs in early Golgi compartments in tobacco cells. The Plant Journal © 2010 Blackwell Publishing Ltd. No claim to original US government works.

Inducible Inhibition of Gβγ Reveals Localization-dependent Functions at the Plasma Membrane and Golgi*

PubMed Central

Klayman, Lauren M.; Wedegaertner, Philip B.

2017-01-01

Heterotrimeric G proteins signal at a variety of endomembrane locations, in addition to their canonical function at the cytoplasmic surface of the plasma membrane (PM), where they are activated by cell surface G protein-coupled receptors. Here we focus on βγ signaling at the Golgi, where βγ activates a signaling cascade, ultimately resulting in vesicle fission from the trans-Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous βγ in a spatial-temporal manner, we take advantage of a lipid association mutant of the widely used βγ inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system. We show that GRK2ct-KERE cannot inhibit βγ function when expressed in cells, but recruitment to a specific membrane location recovers the ability of GRK2ct-KERE to inhibit βγ signaling. PM-recruited GRK2ct-KERE inhibits lysophosphatidic acid-induced phosphorylation of Akt, whereas Golgi-recruited GRK2ct-KERE inhibits cargo transport from the TGN to the PM. Moreover, we show that Golgi-recruited GRK2ct-KERE inhibits model basolaterally targeted but not apically targeted cargo delivery, for both PM-destined and secretory cargo, providing the first evidence of selectivity in terms of cargo transport regulated by βγ. Last, we show that Golgi fragmentation induced by ilimaquinone and nocodazole is blocked by βγ inhibition, demonstrating that βγ is a key regulator of multiple pathways that impact Golgi morphology. Thus, we have developed a new molecular tool, recruitable GRK2ct-KERE, to modulate βγ signaling at specific subcellular locations, and we demonstrate novel cargo selectivity for βγ regulation of TGN to PM transport and a novel role for βγ in mediating Golgi fragmentation. PMID:27994056

Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled alpha-subunits.

PubMed

Valkova, Christina; Albrizio, Marina; Röder, Ira V; Schwake, Michael; Betto, Romeo; Rudolf, Rüdiger; Kaether, Christoph

2011-01-11

The nicotinic acetylcholine receptor of skeletal muscle is composed of five subunits that are assembled in a stepwise manner. Quality control mechanisms ensure that only fully assembled receptors reach the cell surface. Here, we show that Rer1, a putative Golgi-ER retrieval receptor, is involved in the biogenesis of acetylcholine receptors. Rer1 is expressed in the early secretory pathway in the myoblast line C2C12 and in mouse skeletal muscle, and up-regulated during myogenesis. Upon down-regulation of Rer1 in C2C12 cells, unassembled acetylcholine receptor α-subunits escape from the ER and are transported to the plasma membrane and lysosomes, where they are degraded. As a result, the amount of fully assembled receptor at the cell surface is reduced. In vivo Rer1 knockdown and genetic inactivation of one Rer1 allele lead to significantly smaller neuromuscular junctions in mice. Our data show that Rer1 is a functionally important unique factor that controls surface expression of muscle acetylcholine receptors by localizing unassembled α-subunits to the early secretory pathway.

A smart drug: a pH-responsive photothermal ablation agent for Golgi apparatus activated cancer therapy.

PubMed

Xue, Fengfeng; Wen, Ying; Wei, Peng; Gao, Yilin; Zhou, Zhiguo; Xiao, Shuzhang; Yi, Tao

2017-06-13

We report a pH-responsive photothermal ablation agent (pH-PTT) based on cyanine dyes for photothermal therapy (PTT). The nanoparticles formed by BSA and pH-PTT preferentially accumulated in the Golgi apparatus of cancer cells compared to normal cells, and thus can be specifically activated by the acidic Golgi apparatus in cancer cells for effective PTT both ex vivo and in vivo.

Regulation of Glucose Transport in Quiescent, Lactating, and Neoplastic Mammary Epithelia

DTIC Science & Technology

2000-10-01

Manuscripts, Abstracts, Presentations Manuscripts 1. Nemeth, BN, Tsang, ST, Geske , RS, Haney, PM. Golgi targeting of the GLUT 1 glucose transporter in...targeting in lactating mouse mammary gland. Mol. Biol. Cell 1997; 8, 307a (ASCB poster presentation). 6. Geske , S, Haney, PM. Developmental regulation...1995. Characterization of a cis-Golgi matrix protein, GM130. JCellBiol 131:1715-1726. NEMETH BA, TSANG SWY, GESKE RS, HANEY PM, 2000. Golgi targeting

Proliferation of the Golgi apparatus in tobacco BY-2 cells during cell proliferation after release from the stationary phase of growth.

PubMed

Abiodun, Moses; Matsuoka, Ken

2013-08-01

We have recently developed a new method aimed at mass photo-conversion of photo-convertible fluorescence protein (PFP) fluorescence in transformed tobacco BY-2 cells. Using this method we reported recently that the Golgi apparatus is generated by the de novo formation from ER and the division of pre-existing Golgi stacks with similar extents In this work we report that the proliferation of the Golgi apparatus in tobacco cells that enter the growing cycle from the non-dividing cycle is quite similar to that in rapidly growing cells and that de novo formation from the ER and division of pre-existing stacks seems to contribute almost equally to the proliferation.

THE IN VITRO DIFFERENTIATION OF MONONUCLEAR PHAGOCYTES

PubMed Central

Cohn, Zanvil A.; Fedorko, Martha E.; Hirsch, James G.

1966-01-01

A combined morphological, autoradiographic, and cytochemical study at the electron microscope level has been directed towards the formation of electron-opaque granules of cultured macrophages. Labeling of the membrane-bound vesicular structures of pinocytic origin was accomplished with colloidal gold. The initial uptake of gold occurred within micropinocytic vesicles. These electron-lucent vesicles subsequently fused with and discharged their contents into larger pinocytic vacuoles. Colloidal gold was homogeneously distributed in the large pinosomes. In contrast, gold was initially deposited in the periphery of preformed dense granules indicating that these structures were also in constant interaction with the external environment. Colloidal gold was not observed within the cisternae of the endoplasmic reticulum nor within the saccules or vesicles of the Golgi apparatus. There were, however, many small, gold-free vesicles, indistinguishable from Golgi vesicles, which were preferentially aligned about and appeared to fuse with the large pinosomes. The intracellular flow of leucine-H3-labeled protein was followed by electron microscopic autoradiography. After a 15 min pulse of labeled amino acid there was initial labeling of the rough endoplasmic reticulum. Subsequently, much of the label appeared in the Golgi complex. At still later time periods the cytoplasmic dense granules contained the majority of the isotope. Acid phosphatase activity was localized to the dense granules and in the majority of cells to the Golgi apparatus. It is suggested that hydrolytic enzymes are initially synthesized in the endoplasmic reticulum and are then transferred to the Golgi apparatus. Here they are packaged into small Golgi vesicles which represent the primary lysosome of macrophages. The Golgi vesicles subsequently fuse with pinosomes, thereby discharging their hydrolases and forming digestive granules or secondary lysosomes. PMID:5931922

Identification of ER proteins involved in the functional organisation of the early secretory pathway in Drosophila cells by a targeted RNAi screen.

PubMed

Kondylis, Vangelis; Tang, Yang; Fuchs, Florian; Boutros, Michael; Rabouille, Catherine

2011-02-23

In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation. To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for "more and smaller Golgi") upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER. This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation.

Retention in the Golgi apparatus and expression on the cell surface of Cfr/Esl-1/Glg-1/MG-160 are regulated by two distinct mechanisms.

PubMed

Miyaoka, Yuichiro; Kato, Hidenori; Ebato, Kazuki; Saito, Shigeru; Miyata, Naoko; Imamura, Toru; Miyajima, Atsushi

2011-11-15

Cfr (cysteine-rich fibroblast growth factor receptor) is an Fgf (fibroblast growth factor)-binding protein without a tyrosine kinase. We have shown previously that Cfr is involved in Fgf18 signalling via Fgf receptor 3c. However, as Cfr is also known as Glg (Golgi apparatus protein)-1 or MG-160 and occurs in the Golgi apparatus, it remains unknown how the distribution of Cfr is regulated. In the present study, we performed a mutagenic analysis of Cfr to show that two distinct regions contribute to its distribution and stability. First, the C-terminal region retains Cfr in the Golgi apparatus. Secondly, the Cfr repeats in the extracellular juxtamembrane region destabilizes Cfr passed through the Golgi apparatus. This destabilization does not depend on the cleavage and secretion of the extracellular domain of Cfr. Furthermore, we found that Cfr with a GPI (glycosylphosphatidylinositol) anchor was predominantly expressed on the cell surface in Ba/F3 cells and affected Fgf18 signalling in a similar manner to the full-length Cfr, indicating that the interaction of Cfr with Fgfs on the cell surface is important for its function in Fgf signalling. These results suggest that the expression of Cfr in the Golgi apparatus and on the plasma membrane is finely tuned through two distinct mechanisms for exhibiting different functions.

A new use for long-term frozen brain tissue: Golgi impregnation

PubMed Central

Melendez-Ferro, Miguel; Perez-Costas, Emma; Roberts, Rosalinda C.

2009-01-01

The study of dendritic spine shape and number has become a standard in the analysis of synaptic transmission anomalies since a considerable number of neuropsychiatric and neurological diseases have their foundation in alterations in these structures. One of the best ways to study possible alterations of dendritic spines is the use of Golgi impregnation. Although usually the Golgi method implies the use of fresh or fixed tissue, here we report the use of Golgi-Cox for the staining of human and animal brain tissue kept frozen for long periods of time. We successfully applied the Golgi-Cox method to human brain tissue stored for up to 15 years in a freezer. The technique produced reliable and reproducible impregnation of dendrites and dendritic spines in different cortical areas. We also applied the same technique to rat brain frozen for up to one year, obtaining the same satisfactory results. The fact that Golgi-Cox can be successfully applied to this type of tissue adds a new value for hundreds of frozen human or animal brains kept in the freezers of the laboratories, that otherwise would not be useful for anything else. Researchers other than neuroanatomists, i.e. in fields such as biochemistry and molecular biology can also benefit from a simple and reliable technique that can be applied to tissue left from their primary experiments. PMID:18789970

PERMANGANATE FIXATION OF THE GOLGI COMPLEX AND OTHER CYTOPLASMIC STRUCTURES OF MAMMALIAN TESTES

PubMed Central

Mollenhauer, Hilton H.; Zebrun, William

1960-01-01

Observations on the fine structure of KMnO4-fixed testes of small mammals (guinea pig, rat, and mouse) reveal certain morphological differences between the spermatogenic and Sertoli cells which have not been demonstrated in the same tissue fixed with OsO4. Aggregates of minute circular profiles, much smaller than the spherical Golgi vesicles, are described in close association with the Golgi complex of developing spermatids. Groups of dense flattened vesicles, individually surrounded by a membrane of different dimensions than that which bounds most of the other cell organelles, appear dispersed within the cytoplasm of some spermatogenic cells. Flattened vesicles of greater density than those belonging to the Golgi complex are reported confined to the inner Golgi zone of developing guinea pig spermatids between the Golgi cisternae and the head cap. The profiles of endoplasmic reticulum within spermatocytes appear shorter, wider, and more tortuous than those of Sertoli cells. Minute cytoplasmic particles approximately 300 A in diameter and of high electron opacity appear randomly disposed in some Sertoli cells. Groups of irregular-shaped ovoid bodies within the developing spermatids are described as resembling portions of cytoplasm from closely adjacent spermatids. Interpretation is presented regarding the fine structure of KMnO4-fixed testes in view of what has already been reported for mammalian testes fixed in OsO4. PMID:13771855

Subcellular location of secretory proteins retained in the liver during the ethanol-induced inhibition of hepatic protein secretion in the rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Volentine, G.D.; Tuma, D.J.; Sorrell, M.F.

1986-01-01

Ethanol administration inhibits the secretion of proteins by the liver, resulting in their hepatocellular retention. Experiments were designed in this study to determine the subcellular location of the retained secretory proteins. Ethanol was administered acutely to nonfasted rats by gastric intubation, whereas control animals received an isocaloric dose of glucose. Two hours after intubation, when maximum blood ethanol levels (45 mM) were observed, (/sup 3/H)leucine and (/sup 14/C)fucose were injected simultaneously into the dorsal vein of the penis. The labelling of secretory proteins was determined in the liver and plasma at various time periods after label injection. Ethanol treatment decreasedmore » the secretion of both leucine- and fucose-labeled proteins into the plasma. This inhibition of secretion was accompanied by a corresponding increase in the hepatic retention of both leucine- and fucose-labeled immunoprecipitable secretory proteins. At the time of maximum inhibition of secretion, leucine labeled secretory proteins located in the Golgi apparatus represented about 50% of the accumulated secretory proteins in the livers of the ethanol-treated rats, whereas the remainder was essentially equally divided among the rough and smooth endoplasmic reticulum and cytosol. Because fucose is incorporated into secretory proteins almost exclusively in the Golgi complex, fucose-labeled proteins accumulated in the livers of the ethanol-treated rats mainly in the Golgi apparatus, with the remainder located in the cytosol. These results show that ethanol administration causes an impaired movement of secretory proteins along the secretory pathway, and that secretory proteins accumulate mainly, but not exclusively, in the Golgi apparatus.« less

Role of protein kinase D in Golgi exit and lysosomal targeting of the transmembrane protein, Mcoln1

PubMed Central

Marks, David L.; Holicky, Eileen L.; Wheatley, Christine L.; Frumkin, Ayala; Bach, Gideon; Pagano, Richard E.

2012-01-01

The targeting of lysosomal transmembrane proteins from the Golgi apparatus to lysosomes is a complex process that is only beginning to be understood. Here, the lysosomal targeting of Mcoln1, the transmembrane protein defective in the autosomal recessive disease, Mucolipidosis, type IV, was studied by over-expressing full length and truncated forms of the protein in human cells, followed by detection using immunofluorescence and immunoblotting. We demonstrated that a 53 amino acid C-terminal region of Mcoln1 is required for efficient exit from the Golgi. Truncations lacking this region exhibited reduced delivery to lysosomes and decreased proteolytic cleavage of Mcoln1 into characteristic ~35 kDa fragments, suggesting that this cleavage occurs in lysosomes. In addition, we found that co-expression of full length Mcoln1 with kinase-inactive protein kinase D (PKD) 1 or 2 inhibited Mcoln1 Golgi exit and transport to lysosomes and decreased Mcoln1 cleavage. These studies suggest that PKDs play a role in the delivery of some lysosomal resident transmembrane proteins from the Golgi to the lysosomes. PMID:22268962

Golgi-Resident GTPase Rab30 Promotes the Biogenesis of Pathogen-Containing Autophagosomes

PubMed Central

Oda, Seiichiro; Nozawa, Takashi; Nozawa-Minowa, Atsuko; Tanaka, Misako; Aikawa, Chihiro; Harada, Hiroyuki; Nakagawa, Ichiro

2016-01-01

Autophagy acts as a host-defense system against pathogenic microorganisms such as Group A Streptococcus (GAS). Autophagy is a membrane-mediated degradation system that is regulated by intracellular membrane trafficking regulators, including small GTPase Rab proteins. Here, we identified Rab30 as a novel regulator of GAS-containing autophagosome-like vacuoles (GcAVs). We found that Rab30, a Golgi-resident Rab, was recruited to GcAVs in response to autophagy induction by GAS infection in epithelial cells. Rab30 recruitment was dependent upon its GTPase activity. In addition, the knockdown of Rab30 expression significantly reduced GcAV formation efficiency and impaired intracellular GAS degradation. Rab30 normally functions to maintain the structural integrity of the Golgi complex, but GcAV formation occurred even when the Golgi apparatus was disrupted. Although Rab30 also colocalized with a starvation-induced autophagosome, Rab30 was not required for autophagosome formation during starvation. These results suggest that Rab30 mediates autophagy against GAS independently of its normal cellular role in the structural maintenance of the Golgi apparatus, and autophagosome biogenesis during bacterial infection involves specific Rab GTPases. PMID:26771875

A catechol oxidase AcPPO from cherimoya (Annona cherimola Mill.) is localized to the Golgi apparatus.

PubMed

Olmedo, Patricio; Moreno, Adrián A; Sanhueza, Dayan; Balic, Iván; Silva-Sanzana, Christian; Zepeda, Baltasar; Verdonk, Julian C; Arriagada, César; Meneses, Claudio; Campos-Vargas, Reinaldo

2018-01-01

Cherimoya (Annona cherimola) is an exotic fruit with attractive organoleptic characteristics. However, it is highly perishable and susceptible to postharvest browning. In fresh fruit, browning is primarily caused by the polyphenol oxidase (PPO) enzyme catalyzing the oxidation of o-diphenols to quinones, which polymerize to form brown melanin pigment. There is no consensus in the literature regarding a specific role of PPO, and its subcellular localization in different plant species is mainly described within plastids. The present work determined the subcellular localization of a PPO protein from cherimoya (AcPPO). The obtained results revealed that the AcPPO- green fluorescent protein co-localized with a Golgi apparatus marker, and AcPPO activity was present in Golgi apparatus-enriched fractions. Likewise, transient expression assays revealed that AcPPO remained active in Golgi apparatus-enriched fractions obtained from tobacco leaves. These results suggest a putative function of AcPPO in the Golgi apparatus of cherimoya, providing new perspectives on PPO functionality in the secretory pathway, its effects on cherimoya physiology, and the evolution of this enzyme. Copyright © 2017. Published by Elsevier B.V.

Genome-wide siRNA screen identifies UNC50 as a regulator of Shiga toxin 2 trafficking

PubMed Central

Iles, Lakesla R.; Bartholomeusz, Geoffrey

2017-01-01

Shiga toxins 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol. Early endosome-to-Golgi transport allows the toxins to evade degradation in lysosomes. Targeting this trafficking step has therapeutic promise, but the mechanism of trafficking for the more potent toxin STx2 is unclear. To identify host factors required for early endosome-to-Golgi trafficking of STx2, we performed a viability-based genome-wide siRNA screen in HeLa cells. 564, 535, and 196 genes were found to be required for toxicity induced by STx1 only, STx2 only, and both toxins, respectively. We focused on validating endosome/Golgi-localized hits specific for STx2 and found that depletion of UNC50 blocked early endosome-to-Golgi trafficking and induced lysosomal degradation of STx2. UNC50 acted by recruiting GBF1, an ADP ribosylation factor–guanine nucleotide exchange factor (ARF-GEF), to the Golgi. These results provide new information about STx2 trafficking mechanisms and may advance efforts to generate therapeutically viable toxin-trafficking inhibitors. PMID:28883040

Genome-wide siRNA screen identifies UNC50 as a regulator of Shiga toxin 2 trafficking.

PubMed

Selyunin, Andrey S; Iles, Lakesla R; Bartholomeusz, Geoffrey; Mukhopadhyay, Somshuvra

2017-10-02

Shiga toxins 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol. Early endosome-to-Golgi transport allows the toxins to evade degradation in lysosomes. Targeting this trafficking step has therapeutic promise, but the mechanism of trafficking for the more potent toxin STx2 is unclear. To identify host factors required for early endosome-to-Golgi trafficking of STx2, we performed a viability-based genome-wide siRNA screen in HeLa cells. 564, 535, and 196 genes were found to be required for toxicity induced by STx1 only, STx2 only, and both toxins, respectively. We focused on validating endosome/Golgi-localized hits specific for STx2 and found that depletion of UNC50 blocked early endosome-to-Golgi trafficking and induced lysosomal degradation of STx2. UNC50 acted by recruiting GBF1, an ADP ribosylation factor-guanine nucleotide exchange factor (ARF-GEF), to the Golgi. These results provide new information about STx2 trafficking mechanisms and may advance efforts to generate therapeutically viable toxin-trafficking inhibitors. © 2017 Selyunin et al.

The diffuse nervous network of Camillo Golgi: facts and fiction.

PubMed

Raviola, Elio; Mazzarello, Paolo

2011-01-07

The name of Camillo Golgi is inextricably associated, in the mind of most neuroscientists, with the theory that nerve cells communicate with one another by means of an intricate network of anastomosing axonal branches contained in the neuropil intervening between cell bodies in the gray matter of the brain and spinal cord. Examination, however, of Golgi's drawings in the papers published in the decade intervening between publication of his method (1873) and the beginning of his studies on malaria (1885) shows that axonal arborization in the cerebellar cortex and olfactory bulb are depicted as independent of one other. This is in striking contrast with the drawings included by Golgi in his 1906 Nobel lecture where the entire granular layer of the cerebellar cortex is occupied by a network of branching and anastomosing nerve processes. Thus, Golgi in his original papers on the cerebellum represents nerve cells as discrete units and only later in life merges axonal arborizations in the context of a lecture in defense of the reticular theory. Copyright © 2010 Elsevier B.V. All rights reserved.

The Arabidopsis Golgi-localized GDP-L-fucose transporter is required for plant development

DOE PAGES

Rautengarten, Carsten; Ebert, Berit; Liu, Lifeng; ...

2016-07-06

Nucleotide sugar transport across Golgi membranes is essential for the luminal biosynthesis of glycan structures. Here we identify GDP-fucose transporter 1 (GFT1), an Arabidopsis nucleotide sugar transporter that translocates GDP-L-fucose into the Golgi lumen. Using proteo-liposome-based transport assays, we show that GFT preferentially transports GDP-L-fucose over other nucleotide sugars in vitro, while GFT1-silenced plants are almost devoid of L-fucose in cell wall-derived xyloglucan and rhamnogalacturonan II. Furthermore, these lines display reduced L-fucose content in N-glycan structures accompanied by severe developmental growth defects. We conclude that GFT1 is the major nucleotide sugar transporter for import of GDP-L-fucose into the Golgi andmore » is required for proper plant growth and development.« less

A new insight into the three-dimensional architecture of the Golgi complex: Characterization of unusual structures in epididymal principal cells

PubMed Central

Martínez-Martínez, Narcisa; Martínez-Alonso, Emma; Tomás, Mónica; Neumüller, Josef; Pavelka, Margit

2017-01-01

Principal epididymal cells have one of the largest and more developed Golgi complex of mammalian cells. In the present study, we have used this cell as model for the study of the three-dimensional architecture of the Golgi complex of highly secretory and endocytic cells. Electron tomography demonstrated the presence in this cell type of some unknown or very unusual Golgi structures such as branched cisternae, pocket-like cisternal invaginations or tubular connections. In addition, we have used this methodology and immunoelectron microscopy to analyze the close relationship between this organelle and both the endoplasmic reticulum and microtubules, and to describe in detail how these elements interact with compact and non-compact regions of the ribbon. PMID:28957389

The Arabidopsis Golgi-localized GDP-L-fucose transporter is required for plant development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rautengarten, Carsten; Ebert, Berit; Liu, Lifeng

Nucleotide sugar transport across Golgi membranes is essential for the luminal biosynthesis of glycan structures. Here we identify GDP-fucose transporter 1 (GFT1), an Arabidopsis nucleotide sugar transporter that translocates GDP-L-fucose into the Golgi lumen. Using proteo-liposome-based transport assays, we show that GFT preferentially transports GDP-L-fucose over other nucleotide sugars in vitro, while GFT1-silenced plants are almost devoid of L-fucose in cell wall-derived xyloglucan and rhamnogalacturonan II. Furthermore, these lines display reduced L-fucose content in N-glycan structures accompanied by severe developmental growth defects. We conclude that GFT1 is the major nucleotide sugar transporter for import of GDP-L-fucose into the Golgi andmore » is required for proper plant growth and development.« less

Membrane topology of Golgi-localized probable S-adenosylmethionine-dependent methyltransferase in tobacco (Nicotiana tabacum) BY-2 cells.

PubMed

Liu, Jianping; Hayashi, Kyoko; Matsuoka, Ken

2015-01-01

S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) transfer methyl groups to substrates. In this study, a novel putative tobacco SAM-MTase termed Golgi-localized methyl transferase 1 (GLMT1) has been characterized. GLMT1 is comprised of 611 amino acids with short N-terminal region, putative transmembrane region, and C-terminal SAM-MTase domain. Expression of monomeric red fluorescence protein (mRFP)-tagged protein in tobacco BY-2 cell indicated that GLMT1 is a Golgi-localized protein. Analysis of the membrane topology by protease digestion suggested that both C-terminal catalytic region and N-terminal region seem to be located to the cytosolic side of the Golgi apparatus. Therefore, GLMT1 might have a different function than the previously studied SAM-MTases in plants.

The Arabidopsis Golgi-localized GDP-L-fucose transporter is required for plant development

PubMed Central

Rautengarten, Carsten; Ebert, Berit; Liu, Lifeng; Stonebloom, Solomon; Smith-Moritz, Andreia M.; Pauly, Markus; Orellana, Ariel; Scheller, Henrik Vibe; Heazlewood, Joshua L.

2016-01-01

Nucleotide sugar transport across Golgi membranes is essential for the luminal biosynthesis of glycan structures. Here we identify GDP-fucose transporter 1 (GFT1), an Arabidopsis nucleotide sugar transporter that translocates GDP-L-fucose into the Golgi lumen. Using proteo-liposome-based transport assays, we show that GFT preferentially transports GDP-L-fucose over other nucleotide sugars in vitro, while GFT1-silenced plants are almost devoid of L-fucose in cell wall-derived xyloglucan and rhamnogalacturonan II. Furthermore, these lines display reduced L-fucose content in N-glycan structures accompanied by severe developmental growth defects. We conclude that GFT1 is the major nucleotide sugar transporter for import of GDP-L-fucose into the Golgi and is required for proper plant growth and development. PMID:27381418

The Arabidopsis Golgi-localized GDP-L-fucose transporter is required for plant development.

PubMed

Rautengarten, Carsten; Ebert, Berit; Liu, Lifeng; Stonebloom, Solomon; Smith-Moritz, Andreia M; Pauly, Markus; Orellana, Ariel; Scheller, Henrik Vibe; Heazlewood, Joshua L

2016-07-06

Nucleotide sugar transport across Golgi membranes is essential for the luminal biosynthesis of glycan structures. Here we identify GDP-fucose transporter 1 (GFT1), an Arabidopsis nucleotide sugar transporter that translocates GDP-L-fucose into the Golgi lumen. Using proteo-liposome-based transport assays, we show that GFT preferentially transports GDP-L-fucose over other nucleotide sugars in vitro, while GFT1-silenced plants are almost devoid of L-fucose in cell wall-derived xyloglucan and rhamnogalacturonan II. Furthermore, these lines display reduced L-fucose content in N-glycan structures accompanied by severe developmental growth defects. We conclude that GFT1 is the major nucleotide sugar transporter for import of GDP-L-fucose into the Golgi and is required for proper plant growth and development.

Ras-Directed N-Glycoproteins are Novel Early Biomarkers for Tumorigenesis and Malignant Transformation, and Therapeutic Targets of Neurofibromatosis Type I

DTIC Science & Technology

2012-09-01

translocation of receptors from the cytoplasm to the cell surface and retained receptors in the ER and Golgi apparatus , but had no effect on normal...glucose (2-DG) as two potential glycosylation inhibitors. Because proteins travelling to the Golgi apparatus for the consequent steps of...inhibited the transportation of receptors from the cytoplasm to the cell surface and retained receptors in the ER and Golgi apparatus (Fig 3). To

Ras-Directed N-Glycoproteins Are Novel Early Biomarkers for Tumorigenesis and Malignant Transformation and Therapeutic Targets of Neurofibromatosis Type 1

DTIC Science & Technology

2014-08-01

phosphorylates Ser192 in MGAT5B in MPNST cells. Moreover, wild type MGAT5B protein locates in the Golgi apparatus whereas mutated MGAT5BS192A protein...to the Golgi apparatus to attenuate MGAT5B-mediated glycosylation and phosphorylation of kinase receptors. Targeting MGAT5-mediated glycosylation of...tumorigenesis in Nf1+/-;p53+/- mice (bottom). Because proteins travelling to the Golgi apparatus for the consequent steps of glycosylation must be

Liver Rapid Reference Set Application: Gary Norman-INOVA (2012) — EDRN Public Portal

Cancer.gov

We have developed a new and novel assay for the detection of Golgi protein 73 (GP73), also known as Golgi membrane protein 1 (Golm1) or Golgi phosphoprotein 2 (Golph2), in serum/plasma. The clinical question is to determine the clinical utility of gp73 antigen detection by the new assay for early hepatocullular carcinoma (HCC) diagnosis, for risk-assessment of patients at high risk for progression of their liver disease, and for prognosis.

Association of a GPI-anchored protein with detergent-resistant membranes facilitates its trafficking through the early secretory pathway.

PubMed

Hein, Zeynep; Hooper, Nigel M; Naim, Hassan Y

2009-01-15

Membrane microdomains are implicated in the trafficking and sorting of several membrane proteins. In particular GPI-anchored proteins cluster into Triton X-100 resistant, cholesterol- and sphingolipid-rich membrane microdomains and are sorted to the apical membrane. A growing body of evidence has pointed to the existence of other types of microdomains that are insoluble in detergents, such as Lubrol WX and Tween-20. Here, we report on the role of detergent-resistant membranes formed at early stages in the biosynthesis of membrane dipeptidase (MDP), a GPI-anchored protein, on its trafficking and sorting. Pulse-chase experiments revealed a retarded maturation rate of the GPI-anchor deficient mutant (MDPDeltaGPI) as compared to the wild type protein (wtMDP). However, Golgi to cell surface delivery rate did not show a significant difference between the two variants. On the other hand, early biosynthetic forms of wtMDP were partially insoluble in Tween-20, while MDPDeltaGPI was completely soluble. The lack of association of MDPDeltaGPI with detergent-resistant membranes prior to maturation in the Golgi and the reduction in its trafficking rate strongly suggest the existence of an early trafficking control mechanisms for membrane proteins operating at a level between the endoplasmic reticulum and the cis-Golgi.

Vesicles versus Tubes: Is Endoplasmic Reticulum-Golgi Transport in Plants Fundamentally Different from Other Eukaryotes?1

PubMed Central

Robinson, David G.; Brandizzi, Federica; Nakano, Akihiko

2015-01-01

The endoplasmic reticulum (ER) is the gateway to the secretory pathway in all eukaryotic cells. Its products subsequently pass through the Golgi apparatus on the way to the cell surface (true secretion) or to the lytic compartment of the cell (vacuolar protein transport). In animal cells, the Golgi apparatus is present as a stationary larger order complex near the nucleus, and transport between the cortical ER and the Golgi complex occurs via an intermediate compartment which is transported on microtubules. By contrast, higher plant cells have discrete mobile Golgi stacks that move along the cortical ER, and the intermediate compartment is absent. Although many of the major molecular players involved in ER-Golgi trafficking in mammalian and yeast (Saccharomyces cerevisiae) cells have homologs in higher plants, the narrow interface (less than 500 nm) between the Golgi and the ER, together with the motility factor, makes the identification of the transport vectors responsible for bidirectional traffic between these two organelles much more difficult. Over the years, a controversy has arisen over the two major possibilities by which transfer can occur: through vesicles or direct tubular connections. In this article, four leading plant cell biologists attempted to resolve this issue. Unfortunately, their opinions are so divergent and often opposing that it was not possible to reach a consensus. Thus, we decided to let each tell his or her version individually. The review begins with an article by Federica Brandizzi that provides the necessary molecular background on coat protein complexes in relation to the so-called secretory units model for ER-Golgi transport in highly vacuolated plant cells. The second article, written by Chris Hawes, presents the evidence in favor of tubules. It is followed by an article from David Robinson defending the classical notion that transport occurs via vesicles. The last article, by Akihiko Nakano, introduces the reader to possible alternatives to vesicles or tubules, which are now emerging as a result of exciting new developments in high-resolution light microscopy in yeast. PMID:25883241

Golgi-Mediated Synthesis and Secretion of Matrix Polysaccharides of the Primary Cell Wall of Higher Plants

PubMed Central

Driouich, Azeddine; Follet-Gueye, Marie-Laure; Bernard, Sophie; Kousar, Sumaira; Chevalier, Laurence; Vicré-Gibouin, Maïté; Lerouxel, Olivier

2012-01-01

The Golgi apparatus of eukaryotic cells is known for its central role in the processing, sorting, and transport of proteins to intra- and extra-cellular compartments. In plants, it has the additional task of assembling and exporting the non-cellulosic polysaccharides of the cell wall matrix including pectin and hemicelluloses, which are important for plant development and protection. In this review, we focus on the biosynthesis of complex polysaccharides of the primary cell wall of eudicotyledonous plants. We present and discuss the compartmental organization of the Golgi stacks with regards to complex polysaccharide assembly and secretion using immuno-electron microscopy and specific antibodies recognizing various sugar epitopes. We also discuss the significance of the recently identified Golgi-localized glycosyltransferases responsible for the biosynthesis of xyloglucan (XyG) and pectin. PMID:22639665

Golgi-mediated synthesis and secretion of matrix polysaccharides of the primary cell wall of higher plants.

PubMed

Driouich, Azeddine; Follet-Gueye, Marie-Laure; Bernard, Sophie; Kousar, Sumaira; Chevalier, Laurence; Vicré-Gibouin, Maïté; Lerouxel, Olivier

2012-01-01

The Golgi apparatus of eukaryotic cells is known for its central role in the processing, sorting, and transport of proteins to intra- and extra-cellular compartments. In plants, it has the additional task of assembling and exporting the non-cellulosic polysaccharides of the cell wall matrix including pectin and hemicelluloses, which are important for plant development and protection. In this review, we focus on the biosynthesis of complex polysaccharides of the primary cell wall of eudicotyledonous plants. We present and discuss the compartmental organization of the Golgi stacks with regards to complex polysaccharide assembly and secretion using immuno-electron microscopy and specific antibodies recognizing various sugar epitopes. We also discuss the significance of the recently identified Golgi-localized glycosyltransferases responsible for the biosynthesis of xyloglucan (XyG) and pectin.

Current source density correlates of cerebellar Golgi and Purkinje cell responses to tactile input

PubMed Central

Tahon, Koen; Wijnants, Mike; De Schutter, Erik

2011-01-01

The overall circuitry of the cerebellar cortex has been known for over a century, but the function of many synaptic connections remains poorly characterized in vivo. We used a one-dimensional multielectrode probe to estimate the current source density (CSD) of Crus IIa in response to perioral tactile stimuli in anesthetized rats and to correlate current sinks and sources to changes in the spike rate of corecorded Golgi and Purkinje cells. The punctate stimuli evoked two distinct early waves of excitation (at <10 and ∼20 ms) associated with current sinks in the granular layer. The second wave was putatively of corticopontine origin, and its associated sink was located higher in the granular layer than the first trigeminal sink. The distinctive patterns of granular-layer sinks correlated with the spike responses of corecorded Golgi cells. In general, Golgi cell spike responses could be linearly reconstructed from the CSD profile. A dip in simple-spike activity of coregistered Purkinje cells correlated with a current source deep in the molecular layer, probably generated by basket cell synapses, interspersed between sparse early sinks presumably generated by synapses from granule cells. The late (>30 ms) enhancement of simple-spike activity in Purkinje cells was characterized by the absence of simultaneous sinks in the granular layer and by the suppression of corecorded Golgi cell activity, pointing at inhibition of Golgi cells by Purkinje axon collaterals as a likely mechanism of late Purkinje cell excitation. PMID:21228303

Golgi targeting of human guanylate-binding protein-1 requires nucleotide binding, isoprenylation, and an IFN-γ-inducible cofactor

PubMed Central

Modiano, Nir; Lu, Yanping E.; Cresswell, Peter

2005-01-01

Human guanylate-binding protein-1 (hGBP-1) is a large GTPase, similar in structure to the dynamins. Like many smaller GTPases of the Ras/Rab family, it is farnesylated, suggesting it may dock into membranes and perhaps play a role in intracellular trafficking. To date, however, hGBP-1 has never been associated with a specific intracellular compartment. Here we present evidence that hGBP-1 can associate with the Golgi apparatus. Redistribution from the cytosol to the Golgi was observed by immunofluorescence and subcellular fractionation after aluminum fluoride treatment, suggesting that it occurs when hGBP-1 is in its GTP-bound state. Relocalization was blocked by a farnesyl transferase inhibitor. The C589S mutant of hGBP-1, which cannot be farnesylated, and the previously uncharacterized R48P mutant, which cannot bind GTP, both failed to localize to the Golgi. These two mutants had a dominant-negative effect, preventing endogenous wild-type hGBP-1 from efficiently redistributing after aluminum fluoride treatment. Furthermore, hGBP-1 requires another IFN-γ-induced factor to be targeted to the Golgi, because constitutively expressed hGBP-1 remained cytosolic in cells treated with aluminum fluoride unless the cells were preincubated with IFN-γ. Finally, two nonhydrolyzing mutants of hGBP-1, corresponding to active mutants of Ras family proteins, failed to constitutively associate with the Golgi; we propose three possible explanations for this surprising result. PMID:15937107

Golgi targeting of human guanylate-binding protein-1 requires nucleotide binding, isoprenylation, and an IFN-gamma-inducible cofactor.

PubMed

Modiano, Nir; Lu, Yanping E; Cresswell, Peter

2005-06-14

Human guanylate-binding protein-1 (hGBP-1) is a large GTPase, similar in structure to the dynamins. Like many smaller GTPases of the Ras/Rab family, it is farnesylated, suggesting it may dock into membranes and perhaps play a role in intracellular trafficking. To date, however, hGBP-1 has never been associated with a specific intracellular compartment. Here we present evidence that hGBP-1 can associate with the Golgi apparatus. Redistribution from the cytosol to the Golgi was observed by immunofluorescence and subcellular fractionation after aluminum fluoride treatment, suggesting that it occurs when hGBP-1 is in its GTP-bound state. Relocalization was blocked by a farnesyl transferase inhibitor. The C589S mutant of hGBP-1, which cannot be farnesylated, and the previously uncharacterized R48P mutant, which cannot bind GTP, both failed to localize to the Golgi. These two mutants had a dominant-negative effect, preventing endogenous wild-type hGBP-1 from efficiently redistributing after aluminum fluoride treatment. Furthermore, hGBP-1 requires another IFN-gamma-induced factor to be targeted to the Golgi, because constitutively expressed hGBP-1 remained cytosolic in cells treated with aluminum fluoride unless the cells were preincubated with IFN-gamma. Finally, two nonhydrolyzing mutants of hGBP-1, corresponding to active mutants of Ras family proteins, failed to constitutively associate with the Golgi; we propose three possible explanations for this surprising result.

Coordination of golgin tethering and SNARE assembly: GM130 binds syntaxin 5 in a p115-regulated manner.

PubMed

Diao, Aipo; Frost, Laura; Morohashi, Yuichi; Lowe, Martin

2008-03-14

During membrane traffic, transport carriers are first tethered to the target membrane prior to undergoing fusion. Mechanisms exist to connect tethering with fusion, but in most cases, the details remain poorly understood. GM130 is a member of the golgin family of coiled-coil proteins tat is involved in membrane tethering at the endoplasmic reticulum (ER) to Golgi intermediate compartment and cis-Golgi. Here, we demonstrate that GM130 interacts with syntaxin 5, a t-SNARE also localized to the early secretory pathway. Binding to syntaxin 5 is specific, direct, and mediated by the membrane-proximal region of GM130. Interestingly, interaction with syntaxin 5 is inhibited by the binding of the vesicle docking protein p115 to a distal binding site in GM130. The interaction between GM130 and the small GTPase Rab1 is also inhibited by p115 binding. Our findings suggest a mechanism for coupling membrane tethering and fusion at the ER to Golgi intermediate compartment and cis-Golgi, with GM130 playing a central role in linking these processes. Consistent with this hypothesis, we find that depletion of GM130 by RNA interference slows the rate of ER to Golgi trafficking in vivo. The interactions of GM130 with syntaxin 5 and Rab1 are also regulated by mitotic phosphorylation, which is likely to contribute to the inhibition of ER to Golgi trafficking that occurs when mammalian cells enter mitosis.

Retromer guides STxB and CD8-M6PR from early to recycling endosomes, EHD1 guides STxB from recycling endosome to Golgi

PubMed Central

McKenzie, Jenna E.; Raisley, Brent; Zhou, Xin; Naslavsky, Naava; Taguchi, Tomohiko; Caplan, Steve; Sheff, David

2012-01-01

Retrograde trafficking transports proteins, lipids and toxins from the plasma membrane to the Golgi and ER. To reach the Golgi, these cargos must transit the endosomal system, consisting of early endosomes, recycling endosomes, late endosomes and lysosomes. All cargos pass through early endosomes, but may take different routes to the Golgi. Retromer dependent cargos bypass the late endosomes to reach the Golgi. We compared how two very different retromer dependent cargos negotiate the endosomal sorting system. Shiga toxin B, bound to the external layer of the plasma membrane, and chimeric CD8-Mannose-6-Phosphate Receptor, which is anchored via a transmembrane domain. Both appear to pass through the recycling endosome. Ablation of the recycling endosome diverted both of these cargos to an aberrant compartment and prevented them from reaching the Golgi. Once in the recycling endosome, Shiga toxin required EHD1 to traffic to the TGN, while the CD8-Mannose-6-Phosphate Receptor was not significantly dependent on EHD1. Knockdown of retromer components left cargo in the early endosomes, suggesting that it is required for retrograde exit from this compartment. This work establishes the recycling endosome as a required step in retrograde traffic of at least these two retromer dependent cargos. Along this pathway, retromer is associated with EE to recycling endosome traffic, while EHD1 is associated with recycling endosome to TGN traffic of STxB. PMID:22540229

Dynamin-like protein 1 at the Golgi complex: A novel component of the sorting/targeting machinery en route to the plasma membrane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bonekamp, Nina A.; Vormund, Kerstin; Jacob, Ralf

2010-12-10

The final step in the liberation of secretory vesicles from the trans-Golgi network (TGN) involves the mechanical action of the large GTPase dynamin as well as conserved dynamin-independent fission mechanisms, e.g. mediated by Brefeldin A-dependent ADP-ribosylated substrate (BARS). Another member of the dynamin family is the mammalian dynamin-like protein 1 (DLP1/Drp1) that is known to constrict and tubulate membranes, and to divide mitochondria and peroxisomes. Here, we examined a potential role for DLP1 at the Golgi complex. DLP1 localized to the Golgi complex in some but not all cell lines tested, thus explaining controversial reports on its cellular distribution. Aftermore » silencing of DLP1, an accumulation of the apical reporter protein YFP-GL-GPI, but not the basolateral reporter VSVG-SP-GFP at the Golgi complex was observed. A reduction in the transport of YFP-GL-GPI to the plasma membrane was confirmed by surface immunoprecipitation and TGN-exit assays. In contrast, YFP-GL-GPI trafficking was not disturbed in cells silenced for BARS, which is involved in basolateral sorting and trafficking of VSVG-SP-GFP in COS-7 cells. Our data indicate a new role for DLP1 at the Golgi complex and thus a role for DLP1 as a novel component of the apical sorting machinery at the TGN is discussed.« less

Hepatitis C Virus Lipoviroparticles Assemble in the Endoplasmic Reticulum (ER) and Bud off from the ER to the Golgi Compartment in COPII Vesicles.

PubMed

Syed, Gulam H; Khan, Mohsin; Yang, Song; Siddiqui, Aleem

2017-08-01

Hepatitis C virus (HCV) exists as a lipoprotein-virus hybrid lipoviroparticle (LVP). In vitro studies have demonstrated the importance of apolipoproteins in HCV secretion and infectivity, leading to the notion that HCV coopts the secretion of very-low-density lipoprotein (VLDL) for its egress. However, the mechanisms involved in virus particle assembly and egress are still elusive. The biogenesis of VLDL particles occurs in the endoplasmic reticulum (ER), followed by subsequent lipidation in the ER and Golgi compartment. The secretion of mature VLDL particles occurs through the Golgi secretory pathway. HCV virions are believed to latch onto or fuse with the nascent VLDL particle in either the ER or the Golgi compartment, resulting in the generation of LVPs. In our attempt to unravel the collaboration between HCV and VLDL secretion, we studied HCV particles budding from the ER en route to the Golgi compartment in COPII vesicles. Biophysical characterization of COPII vesicles fractionated on an iodixanol gradient revealed that HCV RNA is enriched in the highly buoyant COPII vesicle fractions and cofractionates with apolipoprotein B (ApoB), ApoE, and the HCV core and envelope proteins. Electron microscopy of immunogold-labeled microsections revealed that the HCV envelope and core proteins colocalize with apolipoproteins and HCV RNA in Sec31-coated COPII vesicles. Ultrastructural analysis also revealed the presence of HCV structural proteins, RNA, and apolipoproteins in the Golgi stacks. These findings support the hypothesis that HCV LVPs assemble in the ER and are transported to the Golgi compartment in COPII vesicles to embark on the Golgi secretory route. IMPORTANCE HCV assembly and release accompany the formation of LVPs that circulate in the sera of HCV patients and are also produced in an in vitro culture system. The pathway of HCV morphogenesis and secretion has not been fully understood. This study investigates the exact site where the association of HCV virions with host lipoproteins occurs. Using immunoprecipitation of COPII vesicles and immunogold electron microscopy (EM), we characterize the existence of LVPs that cofractionate with lipoproteins, viral proteins, RNA, and vesicular components. Our results show that this assembly occurs in the ER, and LVPs thus formed are carried through the Golgi network by vesicular transport. This work provides a unique insight into the HCV LVP assembly process within infected cells and offers opportunities for designing antiviral therapeutic cellular targets. Copyright © 2017 American Society for Microbiology.

Hepatitis C Virus Lipoviroparticles Assemble in the Endoplasmic Reticulum (ER) and Bud off from the ER to the Golgi Compartment in COPII Vesicles

PubMed Central

Khan, Mohsin; Yang, Song

2017-01-01

ABSTRACT Hepatitis C virus (HCV) exists as a lipoprotein-virus hybrid lipoviroparticle (LVP). In vitro studies have demonstrated the importance of apolipoproteins in HCV secretion and infectivity, leading to the notion that HCV coopts the secretion of very-low-density lipoprotein (VLDL) for its egress. However, the mechanisms involved in virus particle assembly and egress are still elusive. The biogenesis of VLDL particles occurs in the endoplasmic reticulum (ER), followed by subsequent lipidation in the ER and Golgi compartment. The secretion of mature VLDL particles occurs through the Golgi secretory pathway. HCV virions are believed to latch onto or fuse with the nascent VLDL particle in either the ER or the Golgi compartment, resulting in the generation of LVPs. In our attempt to unravel the collaboration between HCV and VLDL secretion, we studied HCV particles budding from the ER en route to the Golgi compartment in COPII vesicles. Biophysical characterization of COPII vesicles fractionated on an iodixanol gradient revealed that HCV RNA is enriched in the highly buoyant COPII vesicle fractions and cofractionates with apolipoprotein B (ApoB), ApoE, and the HCV core and envelope proteins. Electron microscopy of immunogold-labeled microsections revealed that the HCV envelope and core proteins colocalize with apolipoproteins and HCV RNA in Sec31-coated COPII vesicles. Ultrastructural analysis also revealed the presence of HCV structural proteins, RNA, and apolipoproteins in the Golgi stacks. These findings support the hypothesis that HCV LVPs assemble in the ER and are transported to the Golgi compartment in COPII vesicles to embark on the Golgi secretory route. IMPORTANCE HCV assembly and release accompany the formation of LVPs that circulate in the sera of HCV patients and are also produced in an in vitro culture system. The pathway of HCV morphogenesis and secretion has not been fully understood. This study investigates the exact site where the association of HCV virions with host lipoproteins occurs. Using immunoprecipitation of COPII vesicles and immunogold electron microscopy (EM), we characterize the existence of LVPs that cofractionate with lipoproteins, viral proteins, RNA, and vesicular components. Our results show that this assembly occurs in the ER, and LVPs thus formed are carried through the Golgi network by vesicular transport. This work provides a unique insight into the HCV LVP assembly process within infected cells and offers opportunities for designing antiviral therapeutic cellular targets. PMID:28515296

Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells.

PubMed

Lajevardipour, Alireza; Chon, James W M; Chattopadhyay, Amitabha; Clayton, Andrew H A

2016-11-22

Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C 6 -NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.

Heterodimers of tyrosylprotein sulfotransferases suggest existence of a higher organization level of transferases in the membrane of the trans-Golgi apparatus.

PubMed

Hartmann-Fatu, Cristina; Trusch, Franziska; Moll, Carina N; Michin, Irina; Hassinen, Antti; Kellokumpu, Sakari; Bayer, Peter

2015-03-27

Tyrosine sulfation of proteins is an important post-translational modification shown to play a role in many membrane-associated or extracellular processes such as virus entry, blood clotting, antibody-mediated immune response, inflammation and egg fecundation. The sole two human enzymes that transfer sulfate moieties from 3'-phospho-adenosine-5'-phospho-sulfate onto tyrosine residues, TPST1 and TPST2, are anchored to the membranes of the trans-Golgi compartment with the catalytic domain oriented to the lumen. In contrast to the relatively well studied organization of medial Golgi enzymes, the organization of trans-Golgi transferases remains elusive. Although tyrosylprotein sulfotransferases are known to exist as homodimers in the Golgi membranes, this organization level may represent only a small piece of a puzzle that is linked to the entire picture. Here we report the formation of TPST1/TPST2 heterodimers and a novel interaction between either TPST1 or TPST2 and the α-2,6-sialyltransferase, indicating a higher organization level of tyrosylprotein sulfotransferases that may serve for substrate selectivity and/or effective organization of multiple post-translational modification of proteins. Copyright © 2015. Published by Elsevier Ltd.

A role for tubular networks and a COP I-independent pathway in the mitotic fragmentation of Golgi stacks in a cell-free system

PubMed Central

1995-01-01

Golgi stacks were previously shown to be converted into tubular networks when incubated in mitotic cytosol depleted of the coatomer subunit of COP I coats (Misteli and Warren, 1994). Similar, though smaller, networks are now shown to be an early intermediate on the Golgi fragmentation pathway both in vitro and in vivo. Their appearance mirrors the disappearance of Golgi cisternae and at their peak they constitute 35% of total Golgi membrane. They are consumed by two pathways, the first involving the budding of COP I-coated vesicles described previously (Misteli and Warren, 1994). The second involves a COP I-independent mechanism that leads eventually to a vesicle fraction that is larger in size and more heterogeneous than that produced by the COP I-mechanism. We suggest that both pathways operate concurrently at the onset of mitotic fragmentation. The COP I-independent pathway converts cisternae into tubular networks that then fragment. The COP I- dependent pathway partially consumes first the cisternae at the beginning of the incubation and then the tubular networks that form from them. PMID:7657690

GRASP55 Senses Glucose Deprivation through O-GlcNAcylation to Promote Autophagosome-Lysosome Fusion.

PubMed

Zhang, Xiaoyan; Wang, Leibin; Lak, Behnam; Li, Jie; Jokitalo, Eija; Wang, Yanzhuang

2018-04-23

The Golgi apparatus is the central hub for protein trafficking and glycosylation in the secretory pathway. However, how the Golgi responds to glucose deprivation is so far unknown. Here, we report that GRASP55, the Golgi stacking protein located in medial- and trans-Golgi cisternae, is O-GlcNAcylated by the O-GlcNAc transferase OGT under growth conditions. Glucose deprivation reduces GRASP55 O-GlcNAcylation. De-O-GlcNAcylated GRASP55 forms puncta outside of the Golgi area, which co-localize with autophagosomes and late endosomes/lysosomes. GRASP55 depletion reduces autophagic flux and results in autophagosome accumulation, while expression of an O-GlcNAcylation-deficient mutant of GRASP55 accelerates autophagic flux. Biochemically, GRASP55 interacts with LC3-II on the autophagosomes and LAMP2 on late endosomes/lysosomes and functions as a bridge between LC3-II and LAMP2 for autophagosome and lysosome fusion; this function is negatively regulated by GRASP55 O-GlcNAcylation. Therefore, GRASP55 senses glucose levels through O-GlcNAcylation and acts as a tether to facilitate autophagosome maturation. Copyright © 2018 Elsevier Inc. All rights reserved.

Toxoplasma gondii Syntaxin 6 Is Required for Vesicular Transport Between Endosomal-Like Compartments and the Golgi Complex

PubMed Central

Jackson, Allison J; Clucas, Caroline; Mamczur, Nicola J; Ferguson, David J; Meissner, Markus

2013-01-01

Apicomplexans are obligate intracellular parasites that invade the host cell in an active process that relies on unique secretory organelles (micronemes, rhoptries and dense granules) localized at the apical tip of these highly polarized eukaryotes. In order for the contents of these specialized organelles to reach their final destination, these proteins are sorted post-Golgi and it has been speculated that they pass through endosomal-like compartments (ELCs), where they undergo maturation. Here, we characterize a Toxoplasma gondii homologue of Syntaxin 6 (TgStx6), a well-established marker for the early endosomes and trans Golgi network (TGN) in diverse eukaryotes. Indeed, TgStx6 appears to have a role in the retrograde transport between ELCs, the TGN and the Golgi, because overexpression of TgStx6 results in the development of abnormally shaped parasites with expanded ELCs, a fragmented Golgi and a defect in inner membrane complex maturation. Interestingly, other organelles such as the micronemes, rhoptries and the apicoplast are not affected, establishing the TGN as a major sorting compartment where several transport pathways intersect. It therefore appears that Toxoplasma has retained a plant-like secretory pathway. PMID:23962112

STEM Tomography Imaging of Hypertrophied Golgi Stacks in Mucilage-Secreting Cells.

PubMed

Kang, Byung-Ho

2016-01-01

Because of the weak penetrating power of electrons, the signal-to-noise ratio of a transmission electron micrograph (TEM) worsens as section thickness increases. This problem is alleviated by the use of the scanning transmission electron microscopy (STEM). Tomography analyses using STEM of thick sections from yeast and mammalian cells are of higher quality than are bright-field (BF) images. In this study, we compared regular BF tomograms and STEM tomograms from 500-nm thick sections from hypertrophied Golgi stacks of alfalfa root cap cells. Due to their thickness and intense heavy metal staining, BF tomograms of the thick sections suffer from poor contrast and high noise levels. We were able to mitigate these drawbacks by using STEM tomography. When we performed STEM tomography of densely stained chloroplasts of Arabidopsis cotyledon, we observed similar improvements relative to BF tomograms. A longer time is required to collect a STEM tilt series than similar BF TEM images, and dynamic autofocusing required for STEM imaging often fails at high tilt angles. Despite these limitations, STEM tomography is a powerful method for analyzing structures of large or dense organelles of plant cells.

Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231.

PubMed

Tenorio, María J; Ross, Breyan H; Luchsinger, Charlotte; Rivera-Dictter, Andrés; Arriagada, Cecilia; Acuña, Diego; Aguilar, Marcelo; Cavieres, Viviana; Burgos, Patricia V; Ehrenfeld, Pamela; Mardones, Gonzalo A

2016-01-01

Golgi phosphoprotein 3 (GOLPH3) has been implicated in the development of carcinomas in many human tissues, and is currently considered a bona fide oncoprotein. Importantly, several tumor types show overexpression of GOLPH3, which is associated with tumor progress and poor prognosis. However, the underlying molecular mechanisms that connect GOLPH3 function with tumorigenicity are poorly understood. Experimental evidence shows that depletion of GOLPH3 abolishes transformation and proliferation of tumor cells in GOLPH3-overexpressing cell lines. Conversely, GOLPH3 overexpression drives transformation of primary cell lines and enhances mouse xenograft tumor growth in vivo. This evidence suggests that overexpression of GOLPH3 could result in distinct features of GOLPH3 in tumor cells compared to that of non-tumorigenic cells. GOLPH3 is a peripheral membrane protein mostly localized at the trans-Golgi network, and its association with Golgi membranes depends on binding to phosphatidylinositol-4-phosphate. GOLPH3 is also contained in a large cytosolic pool that rapidly exchanges with Golgi-associated pools. GOLPH3 has also been observed associated with vesicles and tubules arising from the Golgi, as well as other cellular compartments, and hence it has been implicated in several membrane trafficking events. Whether these and other features are typical to all different types of cells is unknown. Moreover, it remains undetermined how GOLPH3 acts as an oncoprotein at the Golgi. Therefore, to better understand the roles of GOLPH3 in cancer cells, we sought to compare some of its biochemical and cellular properties in the human breast cancer cell lines MCF7 and MDA-MB-231 with that of the non-tumorigenic breast human cell line MCF 10A. We found unexpected differences that support the notion that in different cancer cells, overexpression of GOLPH3 functions in diverse fashions, which may influence specific tumorigenic phenotypes.

Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

PubMed Central

Luchsinger, Charlotte; Rivera-Dictter, Andrés; Arriagada, Cecilia; Acuña, Diego; Aguilar, Marcelo; Cavieres, Viviana; Burgos, Patricia V.; Ehrenfeld, Pamela; Mardones, Gonzalo A.

2016-01-01

Golgi phosphoprotein 3 (GOLPH3) has been implicated in the development of carcinomas in many human tissues, and is currently considered a bona fide oncoprotein. Importantly, several tumor types show overexpression of GOLPH3, which is associated with tumor progress and poor prognosis. However, the underlying molecular mechanisms that connect GOLPH3 function with tumorigenicity are poorly understood. Experimental evidence shows that depletion of GOLPH3 abolishes transformation and proliferation of tumor cells in GOLPH3-overexpressing cell lines. Conversely, GOLPH3 overexpression drives transformation of primary cell lines and enhances mouse xenograft tumor growth in vivo. This evidence suggests that overexpression of GOLPH3 could result in distinct features of GOLPH3 in tumor cells compared to that of non-tumorigenic cells. GOLPH3 is a peripheral membrane protein mostly localized at the trans-Golgi network, and its association with Golgi membranes depends on binding to phosphatidylinositol-4-phosphate. GOLPH3 is also contained in a large cytosolic pool that rapidly exchanges with Golgi-associated pools. GOLPH3 has also been observed associated with vesicles and tubules arising from the Golgi, as well as other cellular compartments, and hence it has been implicated in several membrane trafficking events. Whether these and other features are typical to all different types of cells is unknown. Moreover, it remains undetermined how GOLPH3 acts as an oncoprotein at the Golgi. Therefore, to better understand the roles of GOLPH3 in cancer cells, we sought to compare some of its biochemical and cellular properties in the human breast cancer cell lines MCF7 and MDA-MB-231 with that of the non-tumorigenic breast human cell line MCF 10A. We found unexpected differences that support the notion that in different cancer cells, overexpression of GOLPH3 functions in diverse fashions, which may influence specific tumorigenic phenotypes. PMID:27123979

Nerve growth factor induced changes in the Golgi apparatus of PC-12 rat pheochromocytoma cells as studied by ligand endocytosis, cytochemical and morphometric methods.

PubMed

Hickey, W F; Stieber, A; Hogue-Angeletti, R; Gonatas, J; GOnatas, N K

1983-10-01

Cells of the PC-12 rat pheochromocytoma cell line respond to nerve growth factor (NGF) by sprouting neurites and biochemically differentiating into sympathetic ganglion-like cells. NGF-stimulated ('differentiated') and unstimulated ('undifferentiated') cells were studied by cytochemical techniques for the localization of the enzymes acid phosphatase (ACPase) and thiamine pyrophosphatase (TPPase), and by a morphometric analysis of the distribution of endocytosed wheat-germ agglutinin labelled with horseradish peroxidase (WGA-HRP). Both cytochemical stains showed the enzymes to be distributed in lysosomes and certain cisternae of the Golgi apparatus in both NGF stimulated and unstimulated cells. ACPase was not confined to GERL (Golgi-endoplasmic reticulum-lysosome) as in certain other cells. The morphometric studies demonstrated that the reaction product of the internalized WGA-HRP occupied 4.7% of the cytoplasmic area in unstimulated cells and 4.5% in NGF-stimulated ones. Despite this similarity, the distribution of the WGA-HRP among the studied intracellular compartments in these two cell groups varied. In the NGF-stimulated cells 3.3% of the WGA-HRP reaction product was found in the innermost Golgi cisterna(e) while in unstimulated cells only 0.3% was seen in this compartment. Similarly, 4.3% of the WGA-HRP stain was found in small vesicles at the 'trans' aspect of the Golgi apparatus in stimulated cells, when only 0.3% of the stain occupied this compartment in 'undifferentiated' cells. The morphometric analysis also revealed that when the PC-12 cells were stimulated with NGF, the Golgi apparatus increased in area by approximately 70%. These findings are consistent with the hypothesis that NGF induced differentiation of PC-12 cells is coupled with enhanced endocytosis of WGA and probably of its 'receptor' to the innermost Golgi cisterna(e) and the closely associated vesicles.

Genetics Home Reference: COG5-congenital disorder of glycosylation

MedlinePlus

... in my area? Other Names for This Condition carbohydrate deficient glycoprotein syndrome type IIi CDG IIi CDG2I ... the Golgi by the conserved oligomeric Golgi (COG) complex and the golgin TATA element modulatory factor (TMF). ...

A role for the membrane Golgi protein Ema in autophagy.

PubMed

Kim, Sungsu; DiAntonio, Aaron

2012-08-01

Autophagy is a cellular homeostatic response that involves degradation of self-components by the double-membraned autophagosome. The biogenesis of autophagosomes has been well described, but the ensuing processes after autophagosome formation are not clear. In our recent study, we proposed a model in which the Golgi complex contributes to the growth of autophagic structures, and that the Drosophila melanogaster membrane protein Ema promotes this process. In fat body cells of the D. melanogaster ema mutant, the recruitment of the Golgi complex protein Lava lamp (Lva) to autophagic structures is impaired and autophagic structures are very small. In addition, in the ema mutant autophagic turnover of SQSTM1/p62 and mitophagy are impaired. Our study not only identifies a role for Ema in autophagy, but also supports the hypothesis that the Golgi complex may be a potential membrane source for the biogenesis and development of autophagic structures.

Retention mechanisms for ER and Golgi membrane proteins.

PubMed

Gao, Caiji; Cai, Yi; Wang, Yejun; Kang, Byung-Ho; Aniento, Fernando; Robinson, David G; Jiang, Liwen

2014-08-01

Unless there are mechanisms to selectively retain membrane proteins in the endoplasmic reticulum (ER) or in the Golgi apparatus, they automatically proceed downstream to the plasma or vacuole membranes. Two types of coat protein complex I (COPI)-interacting motifs in the cytosolic tails of membrane proteins seem to facilitate membrane retention in the early secretory pathway of plants: a dilysine (KKXX) motif (which is typical of p24 proteins) for the ER and a KXE/D motif (which occurs in the Arabidopsis endomembrane protein EMP12) for the Golgi apparatus. The KXE/D motif is highly conserved in all eukaryotic EMPs and is additionally present in hundreds of other proteins of unknown subcellular localization and function. This novel signal may represent a new general mechanism for Golgi targeting and the retention of polytopic integral membrane proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

Neutral sphingomyelinase 2 (smpd3) in the control of postnatal growth and development

PubMed Central

Stoffel, Wilhelm; Jenke, Britta; Blöck, Barbara; Zumbansen, Markus; Koebke, Jürgen

2005-01-01

Neutral sphingomyelinases sphingomyelin phosphodiesterase (SMPD)2 and -3 hydrolyze sphingomyelin to phosphocholine and ceramide. smpd2 is expressed ubiquitously, and smpd3 is expressed predominantly in neurons of the CNS. Their activation and the functions of the released ceramides have been associated with signaling pathways in cell growth, differentiation, and apoptosis. However, these cellular responses remain poorly understood. Here we describe the generation and characterization of the smpd3–/– and smpd2–/–smpd3–/– double mutant mouse, which proved to be devoid of neutral sphingomyelinase activity. SMPD3 plays a pivotal role in the control of late embryonic and postnatal development: the smpd3-null mouse develops a novel form of dwarfism and delayed puberty as part of a hypothalamus-induced combined pituitary hormone deficiency. Our studies suggest that SMPD3 is segregated into detergent-resistant subdomains of Golgi membranes of hypothalamic neurosecretory neurons, where its transient activation modifies the lipid bilayer, an essential step in the Golgi secretory pathway. The smpd3–/– mouse might mimic a form of human combined pituitary hormone deficiency. PMID:15764706

Neutral sphingomyelinase 2 (smpd3) in the control of postnatal growth and development.

PubMed

Stoffel, Wilhelm; Jenke, Britta; Blöck, Barbara; Zumbansen, Markus; Koebke, Jürgen

2005-03-22

Neutral sphingomyelinases sphingomyelin phosphodiesterase (SMPD)2 and -3 hydrolyze sphingomyelin to phosphocholine and ceramide. smpd2 is expressed ubiquitously, and smpd3 is expressed predominantly in neurons of the CNS. Their activation and the functions of the released ceramides have been associated with signaling pathways in cell growth, differentiation, and apoptosis. However, these cellular responses remain poorly understood. Here we describe the generation and characterization of the smpd3(-/-) and smpd2(-/-)smpd3(-/-) double mutant mouse, which proved to be devoid of neutral sphingomyelinase activity. SMPD3 plays a pivotal role in the control of late embryonic and postnatal development: the smpd3-null mouse develops a novel form of dwarfism and delayed puberty as part of a hypothalamus-induced combined pituitary hormone deficiency. Our studies suggest that SMPD3 is segregated into detergent-resistant subdomains of Golgi membranes of hypothalamic neurosecretory neurons, where its transient activation modifies the lipid bilayer, an essential step in the Golgi secretory pathway. The smpd3(-/-) mouse might mimic a form of human combined pituitary hormone deficiency.

The Last Enzyme of the De Novo Purine Synthesis Pathway 5-aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase/IMP Cyclohydrolase (ATIC) Plays a Central Role in Insulin Signaling and the Golgi/Endosomes Protein Network*

PubMed Central

Boutchueng-Djidjou, Martial; Collard-Simard, Gabriel; Fortier, Suzanne; Hébert, Sébastien S.; Kelly, Isabelle; Landry, Christian R.; Faure, Robert L.

2015-01-01

Insulin is internalized with its cognate receptor into the endosomal apparatus rapidly after binding to hepatocytes. We performed a bioinformatic screen of Golgi/endosome hepatic protein fractions and found that ATIC, which is a rate-limiting enzyme in the de novo purine biosynthesis pathway, and PTPLAD1 are associated with insulin receptor (IR) internalization. The IR interactome (IRGEN) connects ATIC to AMPK within the Golgi/endosome protein network (GEN). Forty-five percent of the IR Golgi/endosome protein network have common heritable variants associated with type 2 diabetes, including ATIC and AMPK. We show that PTPLAD1 and AMPK are rapidly compartmentalized within the plasma membrane (PM) and Golgi/endosome fractions after insulin stimulation and that ATIC later accumulates in the Golgi/endosome fraction. Using an in vitro reconstitution system and siRNA-mediated partial knockdown of ATIC and PTPLAD1 in HEK293 cells, we show that both ATIC and PTPLAD1 affect IR tyrosine phosphorylation and endocytosis. We further show that insulin stimulation and ATIC knockdown readily increase the level of AMPK-Thr172 phosphorylation in IR complexes. We observed that IR internalization was markedly decreased after AMPKα2 knockdown, and treatment with the ATIC substrate AICAR, which is an allosteric activator of AMPK, increased IR endocytosis in cultured cells and in the liver. These results suggest the presence of a signaling mechanism that senses adenylate synthesis, ATP levels, and IR activation states and that acts in regulating IR autophosphorylation and endocytosis. PMID:25687571

Subcellular distribution of swine vesicular disease virus proteins and alterations induced in infected cells: A comparative study with foot-and-mouth disease virus and vesicular stomatitis virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin-Acebes, Miguel A.; Gonzalez-Magaldi, Monica; Rosas, Maria F.

2008-05-10

The intracellular distribution of swine vesicular disease virus (SVDV) proteins and the induced reorganization of endomembranes in IBRS-2 cells were analyzed. Fluorescence to new SVDV capsids appeared first upon infection, concentrated in perinuclear circular structures and colocalized to dsRNA. As in foot-and-mouth disease virus (FMDV)-infected cells, a vesicular pattern was predominantly found in later stages of SVDV capsid morphogenesis that colocalized with those of non-structural proteins 2C, 2BC and 3A. These results suggest that assembly of capsid proteins is associated to the replication complex. Confocal microscopy showed a decreased fluorescence to ER markers (calreticulin and protein disulfide isomerase), and disorganizationmore » of cis-Golgi gp74 and trans-Golgi caveolin-1 markers in SVDV- and FMDV-, but not in vesicular stomatitis virus (VSV)-infected cells. Electron microscopy of SVDV-infected cells at an early stage of infection revealed fragmented ER cisternae with expanded lumen and accumulation of large Golgi vesicles, suggesting alterations of vesicle traffic through Golgi compartments. At this early stage, FMDV induced different patterns of ER fragmentation and Golgi alterations. At later stages of SVDV cytopathology, cells showed a completely vacuolated cytoplasm containing vesicles of different sizes. Cell treatment with brefeldin A, which disrupts the Golgi complex, reduced SVDV ({approx} 5 log) and VSV ({approx} 4 log) titers, but did not affect FMDV growth. Thus, three viruses, which share target tissues and clinical signs in natural hosts, induce different intracellular effects in cultured cells.« less

The regulation of ER export and Golgi retention of ST3Gal5 (GM3/GM4 synthase) and B4GalNAcT1 (GM2/GD2/GA2 synthase) by arginine/lysine-based motif adjacent to the transmembrane domain.

PubMed

Uemura, Satoshi; Shishido, Fumi; Kashimura, Madoka; Inokuchi, Jin-ichi

2015-12-01

In the Golgi maturation model, the Golgi cisternae dynamically mature along a secretory pathway. In this dynamic process, glycosyltransferases are transported from the endoplasmic reticulum (ER) to the Golgi apparatus where they remain and function. The precise mechanism behind this maturation process remains unclear. We investigated two glycosyltransferases, ST3Gal5 (ST3G5) and B4GalNAcT1 (B4GN1), involved in ganglioside synthesis and examined their signal sequences for ER export and Golgi retention. Reports have suggested that the [R/K](X)[R/K] motif functions as an ER exporting signal; however, this signal sequence is insufficient in stably expressed, full-length ST3G5. Through further analysis, we have clarified that the (2)R(3)R(X)(5) (9)K(X)(3) (13)K sequence in ST3G5 is essential for ER export. We have named the sequence the R/K-based motif. On the other hand, for ER export of B4GN1, the homodimer formation in addition to the R/K-based motif is required for ER export suggesting the importance of unidentified lumenal side interaction. We found that ST3G5 R2A/R3A and K9A/K13A mutants localized not only in Golgi apparatus but also in endosomes. Furthermore, the amounts of mature type asparagine-linked (N)-glycans in ST3G5 R2A/R3A and K9A/K13A mutants were decreased compared with those in wild-type proteins, and the stability of the mutants was lower. These results suggest that the R/K-based motif is necessary for the Golgi retention of ST3G5 and that the retention is involved in the maturation of N-glycans and in stability. Thus, several basic amino acids located on the cytoplasmic tail of ST3G5 play important roles in both ER export and Golgi retention. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Function of the Golgi-located phosphate transporter PHT4;6 is critical for senescence-associated processes in Arabidopsis

PubMed Central

Hassler, Sebastian; Jung, Benjamin; Lemke, Lilia; Novák, Ondřej; Strnad, Miroslav; Martinoia, Enrico; Neuhaus, H. Ekkehard

2016-01-01

The phosphate transporter PHT4;6 locates to the trans-Golgi compartment, and its impaired activity causes altered intracellular phosphate compartmentation, leading to low cytosolic Pi levels, a blockage of Golgi-related processes such as protein glycosylation and hemicellulose biosynthesis, and a dwarf phenotype. However, it was unclear whether altered Pi homeostasis in pht4;6 mutants causes further cellular problems, typically associated with limited phosphate availability. Here we report that pht4;6 mutants exhibit a markedly increased disposition to induce dark-induced senescence. In control experiments, in which pht4;6 mutants and wild-type plants developed similarly, we confirmed that accelerated dark-induced senescence in mutants is not a ‘pleiotropic’ process associated with the dwarf phenotype. In fact, accelerated dark-induced senescence in pht4;6 mutants correlates strongly with increased levels of toxic NH4 + and higher sensitivity to ammonium, which probably contribute to the inability of pht4;6 mutants to recover from dark treatment. Experiments with modified levels of either salicylic acid (SA) or trans-zeatin (tZ) demonstrate that altered concentrations of these compounds in pht4;6 plants act as major cellular mediators for dark-induced senescence. This conclusion gained further support from the notion that the expression of the pht4;6 gene is, in contrast to genes coding for major phosphate importers, substantially induced by tZ. Taken together, our findings point to a critical function of PHT4;6 to control cellular phosphate levels, in particular the cytosolic Pi availability, required to energize plant primary metabolism for proper plant development. Phosphate and its allocation mediated by PHT4;6 is critical to prevent onset of dark-induced senescence. PMID:27325894

Human macrophage ATP7A is localized in the trans-Golgi apparatus, controls intracellular copper levels, and mediates macrophage responses to dermal wounds.

PubMed

Kim, Ha Won; Chan, Qilin; Afton, Scott E; Caruso, Joseph A; Lai, Barry; Weintraub, Neal L; Qin, Zhenyu

2012-02-01

The copper transporter ATP7A has attracted significant attention since the discovery of its gene mutation leading to human Menkes disease. We previously reported that ATP7A is highly expressed in the human vasculature and identified a novel vascular function of ATP7A in modulation of the expression and activity of extracellular superoxide dismutase. We recently identified that ATP7A expression in THP-1 cells (a monocyte/macrophage model cell line) plays a role in the oxidation of low density lipoproteins, indicating that it is necessary to further investigate its expression and function in monocytes/macrophages. In the current study, we demonstrated the protein and mRNA expression of ATP7A in human peripheral blood mononuclear cell (PBMC)-derived macrophages and alveolar macrophages. ATP7A was strongly co-localized with the trans-Golgi apparatus in PBMC-derived macrophages. Intracellular copper, detected by synchrotron X-ray fluorescence microscopy, was found to be distributed to the nucleus and cytoplasm in human THP-1 cells. To confirm the role of endogenous ATP7A in macrophage copper homeostasis, we performed inductively coupled plasma mass spectrometry in murine peritoneal macrophages, which showed markedly increased intracellular copper levels in macrophages isolated from ATP7A-deficient mice versus control mice. Moreover, the role of ATP7A in regulating macrophage responses to dermal wounds was studied by introduction of control and ATP7A-downregulated THP-1 cells into dermal wounds of nude mice. Infiltration of THP-1 cells into the wounded area (detected by expression of human macrophage markers MAC2 and CD68) was reduced in response to downregulation of ATP7A, hinting decreased macrophage accumulation subsequent to dermal wounds. In summary, alongside our previous studies, these findings indicate that human macrophage ATP7A is localized in the trans-Golgi apparatus, regulates intracellular copper levels, and mediates macrophage responses to a dermal wound.

Layer 6 cortical neurons require Reelin-Dab1 signaling for cellular orientation, Golgi deployment, and directed neurite growth into the marginal zone.

PubMed

O'Dell, Ryan S; Ustine, Candida J M; Cameron, David A; Lawless, Sean M; Williams, Rebecca M; Zipfel, Warren R; Olson, Eric C

2012-07-07

The secreted ligand Reelin is believed to regulate the translocation of prospective layer 6 (L6) neocortical neurons into the preplate, a loose layer of pioneer neurons that overlies the ventricular zone. Recent studies have also suggested that Reelin controls neuronal orientation and polarized dendritic growth during this period of early cortical development. To explicitly characterize and quantify how Reelin controls this critical aspect of neurite initiation and growth we used a new ex utero explant model of early cortical development to selectively label a subset of L6 cortical neurons for complete 3-D reconstruction. The total neurite arbor sizes of neurons in Reelin-deficient (reeler mutant) and Dab1-deficient (Reelin-non-responsive scrambler mutant) cortices were quantified and unexpectedly were not different than control arbor lengths (p = 0.51). For each mutant, however, arbor organization was markedly different: mutant neurons manifested more primary processes (neurites emitted directly from the soma) than wild type, and these neurites were longer and displayed less branching. Reeler and scrambler mutant neurites extended tangentially rather than radially, and the Golgi apparatus that normally invests the apical neurite was compact in both reeler and scrambler mutants. Mutant cortices also exhibited a neurite "exclusion zone" which was relatively devoid of L6 neuron neurites and extended at least 15 μm beneath the pial surface, an area corresponding to the marginal zone (MZ) in the wild type explants. The presence of an exclusion zone was also indicated in the orientation of mutant primary neurite and neuronal somata, which failed to adopt angles within ~20˚ of the radial line to the pial surface. Injection of recombinant Reelin to reeler, but not scrambler, mutant cortices fully rescued soma orientation, Golgi organization, and dendritic projection defects within four hrs. These findings indicate Reelin promotes directional dendritic growth into the MZ, an otherwise exclusionary zone for L6 neurites.

Golgi Compartmentation and Identity

PubMed Central

Papanikou, Effrosyni; Glick, Benjamin S.

2014-01-01

Recent work supports the idea that cisternae of the Golgi apparatus can be assigned to three classes, which correspond to discrete stages of cisternal maturation. Each stage has a unique pattern of membrane traffic. At the first stage, cisternae form in association with the ER at multifunctional membrane assembly stations. At the second stage, cisternae synthesize carbohydrates while exchanging material via COPI vesicles. At the third stage, cisternae of the trans-Golgi network segregate into domains and produce transport carriers with the aid of specific lipids and the actin cytoskeleton. These processes are coordinated by cascades of Rab and Arf/Arl GTPases. PMID:24840895

Chronic shear induces caveolae formation and alters ERK and Akt responses in endothelial cells

NASA Technical Reports Server (NTRS)

Boyd, Nolan L.; Park, Heonyong; Yi, Hong; Boo, Yong Chool; Sorescu, George P.; Sykes, Michelle; Jo, Hanjoong

2003-01-01

Caveolae are plasmalemmal domains enriched with cholesterol, caveolins, and signaling molecules. Endothelial cells in vivo are continuously exposed to shear conditions, and their caveolae density and location may be different from that of static cultured cells. Here, we show that chronic shear exposure regulates formation and localization of caveolae and caveolin-1 in bovine aortic endothelial cells (BAEC). Chronic exposure (1 or 3 days) of BAEC to laminar shear increased the total number of caveolae by 45-48% above static control. This increase was due to a rise in the luminal caveolae density without changing abluminal caveolae numbers or increasing caveolin-1 mRNA and protein levels. Whereas some caveolin-1 was found in the plasma membrane in static-cultured cells, it was predominantly localized in the Golgi. In contrast, chronic shear-exposed cells showed intense caveolin-1 staining in the luminal plasma membrane with minimum Golgi association. The preferential luminal localization of caveolae may play an important role in endothelial mechanosensing. Indeed, we found that chronic shear exposure (preconditioning) altered activation patterns of two well-known shear-sensitive signaling molecules (ERK and Akt) in response to a step increase in shear stress. ERK activation was blunted in shear preconditioned cells, whereas the Akt response was accelerated. These results suggest that chronic shear stimulates caveolae formation by translocating caveolin-1 from the Golgi to the luminal plasma membrane and alters cell signaling responses.

Gap Junction Modulation of Low-Frequency Oscillations in the Cerebellar Granule Cell Layer.

PubMed

Robinson, Jennifer Claire; Chapman, C Andrew; Courtemanche, Richard

2017-08-01

Local field potential (LFP) oscillations in the granule cell layer (GCL) of the cerebellar cortex have been identified previously in the awake rat and monkey during immobility. These low-frequency oscillations are thought to be generated through local circuit interactions between Golgi cells and granule cells within the GCL. Golgi cells display rhythmic firing and pacemaking properties, and also are electrically coupled through gap junctions within the GCL. Here, we tested if gap junctions in the rat cerebellar cortex contribute to the generation of LFP oscillations in the GCL. We recorded LFP oscillations under urethane anesthesia, and examined the effects of local infusion of gap junction blockers on 5-15 Hz oscillations. Local infusion of the gap junction blockers carbenoxolone and mefloquine resulted in significant decreases in the power of oscillations over a 30-min period, but the power of oscillations was unchanged in control experiments following vehicle injections. In addition, infusion of gap junction blockers had no significant effect on multi-unit activity, suggesting that the attenuation of low-frequency oscillations was likely due to reductions in electrical coupling rather than a decreased excitability within the granule cell layer. Our results indicate that electrical coupling among the Golgi cell networks in the cerebellar cortex contributes to the local circuit mechanisms that promote the occurrence of GCL LFP slow oscillations in the anesthetized rat.

Acid sphingomyelinase is required for cell surface presentation of Met receptor tyrosine kinase in cancer cells

PubMed Central

Zhu, Linyu; Xiong, Xiahui; Kim, Yongsoon; Okada, Naomi; Lu, Fei; Zhang, Hui

2016-01-01

ABSTRACT Receptor tyrosine kinases (RTKs) are embedded in the lipid bilayer of the plasma membrane, but the specific roles of various lipids in cell signaling remain largely uncharacterized. We have previously found that acid sphingomyelinase (ASM; also known as SMPD1) regulates the conserved DAF-2 (the ortholog IGF-1R in mammals) RTK signaling pathway in Caenorhabditis elegans. How ASM and its catalytic products, ceramides, control RTK signaling pathways remain unclear. Here, we report that ASM regulates the homeostasis of Met, an RTK that is frequently overexpressed in various cancers. Inactivation of ASM led to a rapid loss of Met from the plasma membrane, reduced Met phosphorylation and activation, and induced Met accumulation in the trans-Golgi network (TGN). However, trafficking of integrin β3 and vesicular stomatitis virus glycoprotein (VSVG) was largely unaffected. Knockdown of syntaxin 6 (STX6) also blocked the Golgi exit of Met. Depletion of either ASM or STX6 led to aberrant trafficking of Met to lysosomes, promoting its degradation. Our studies reveal that ASM and ceramides, together with STX6 and cholesterol, constitute a new regulatory mechanism for the exit of Met from the Golgi during its biosynthetic route, which is used to rapidly replenish and regulate the plasma membrane levels of Met in various cancer cells. PMID:27802163

The integrity of the plant Golgi apparatus depends on cell growth-controlled activity of GNL1.

PubMed

Du, Wenyan; Tamura, Kentaro; Stefano, Giovanni; Brandizzi, Federica

2013-05-01

Membrane traffic and organelle integrity in the plant secretory pathway depend on ARF-GTPases, which are activated by guanine-nucleotide exchange factors (ARF-GEFs). While maintenance of conserved roles, evolution of unique functions as well as tissue-specific roles have been shown for a handful of plant ARF-GEFs, a fundamental yet unanswered question concerns the extent to which their function overlaps during cell growth. To address this, we have characterized pao, a novel allele of GNOM-like 1 (GNL1), a brefeldin A (BFA)-insensitive ARF-GEF, isolated through a confocal microscopy-based forward genetics screen of the Golgi in Arabidopsis thaliana. Specifically, we have analyzed the dependence of the integrity of trafficking routes and secretory organelles on GNL1 availability during expansion stages of cotyledon epidermal cells, an exquisite model system for vegetative cell growth analyses in intact tissues. We show that Golgi traffic is influenced largely by GNL1 availability at early stages of cotyledon cell expansion but by BFA-sensitive GEFs when cell growth terminates. These data reveal an unanticipated level of complexity in the biology of GNL1 by showing that its cellular roles are correlated with cell growth. These results also indicate that the cell growth stage is an important element weighting into functional analyses of the cellular roles of ARF-GEFs.

Organization of transport from endoplasmic reticulum to Golgi in higher plants.

PubMed

Andreeva, A V; Zheng, H; Saint-Jore, C M; Kutuzov, M A; Evans, D E; Hawes, C R

2000-01-01

In plant cells, the organization of the Golgi apparatus and its interrelationships with the endoplasmic reticulum differ from those in mammalian and yeast cells. Endoplasmic reticulum and Golgi apparatus can now be visualized in plant cells in vivo with green fluorescent protein (GFP) specifically directed to these compartments. This makes it possible to study the dynamics of the membrane transport between these two organelles in the living cells. The GFP approach, in conjunction with a considerable volume of data about proteins participating in the transport between endoplasmic reticulum and Golgi in yeast and mammalian cells and the identification of their putative plant homologues, should allow the establishment of an experimental model in which to test the involvement of the candidate proteins in plants. As a first step towards the development of such a system, we are using Sar1, a small G-protein necessary for vesicle budding from the endoplasmic reticulum. This work has demonstrated that the introduction of Sar1 mutants blocks the transport from endoplasmic reticulum to Golgi in vivo in tobacco leaf epidermal cells and has therefore confirmed the feasibility of this approach to test the function of other proteins that are presumably involved in this step of endomembrane trafficking in plant cells.

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

PubMed Central

Mychasiuk, Richelle; Gibb, Robbin; Kolb, Bryan

2013-01-01

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity. PMID:24121525

A rapid method combining Golgi and Nissl staining to study neuronal morphology and cytoarchitecture.

PubMed

Pilati, Nadia; Barker, Matthew; Panteleimonitis, Sofoklis; Donga, Revers; Hamann, Martine

2008-06-01

The Golgi silver impregnation technique gives detailed information on neuronal morphology of the few neurons it labels, whereas the majority remain unstained. In contrast, the Nissl staining technique allows for consistent labeling of the whole neuronal population but gives very limited information on neuronal morphology. Most studies characterizing neuronal cell types in the context of their distribution within the tissue slice tend to use the Golgi silver impregnation technique for neuronal morphology followed by deimpregnation as a prerequisite for showing that neuron's histological location by subsequent Nissl staining. Here, we describe a rapid method combining Golgi silver impregnation with cresyl violet staining that provides a useful and simple approach to combining cellular morphology with cytoarchitecture without the need for deimpregnating the tissue. Our method allowed us to identify neurons of the facial nucleus and the supratrigeminal nucleus, as well as assessing cellular distribution within layers of the dorsal cochlear nucleus. With this method, we also have been able to directly compare morphological characteristics of neuronal somata at the dorsal cochlear nucleus when labeled with cresyl violet with those obtained with the Golgi method, and we found that cresyl violet-labeled cell bodies appear smaller at high cellular densities. Our observation suggests that cresyl violet staining is inadequate to quantify differences in soma sizes.

Isolation and characterization of multivesicular bodies from rat hepatocytes: an organelle distinct from secretory vesicles of the Golgi apparatus

PubMed Central

1985-01-01

Hepatocytes of estradiol-treated rats, which express many low density lipoprotein receptors, rapidly accumulate intravenously injected low density lipoprotein in multivesicular bodies (MVBs). We have isolated MVBs and Golgi apparatus fractions from livers of estradiol-treated rats. MVB fractions were composed mainly of large vesicles, approximately 0.55 micron diam, filled with remnantlike very low density lipoproteins, known to be taken up into hepatocytes by receptor- mediated endocytosis. MVBs also contained numerous small vesicles, 0.05- 0.07 micron in diameter, and had two types of appendages: one fingerlike and electron dense and the other saclike and electron lucent. MVBs contained little galactosyltransferase or arylsulfatase activity, and content lipoproteins were largely intact. Very low density lipoproteins from Golgi fractions, which are derived to a large extent from secretory vesicles, were larger than those of MVB fractions and contained newly synthesized triglycerides. Membranes of MVBs contained much more cholesterol and less protein than did Golgi membranes. We conclude that two distinct lipoprotein-filled organelles are located in the bile canalicular pole of hepatocytes. MVBs, a major prelysosomal organelle of low density in the endocytic pathway, contain remnants of triglyceride-rich lipoproteins, whereas secretory vesicles of the Golgi apparatus contain nascent very low density lipoproteins. PMID:3988801

Co-localization of glycine and gaba immunoreactivity in interneurons in Macaca monkey cerebellar cortex.

PubMed

Crook, J; Hendrickson, A; Robinson, F R

2006-09-15

Previous work demonstrates that the cerebellum uses glycine as a fast inhibitory neurotransmitter [Ottersen OP, Davanger S, Storm-Mathisen J (1987) Glycine-like immunoreactivity in the cerebellum of rat and Senegalese baboon, Papio papio: a comparison with the distribution of GABA-like immunoreactivity and with [3H]glycine and [3H]GABA uptake. Exp Brain Res 66(1):211-221; Ottersen OP, Storm-Mathisen J, Somogyi P (1988) Colocalization of glycine-like and GABA-like immunoreactivities in Golgi cell terminals in the rat cerebellum: a postembedding light and electron microscopic study. Brain Res 450(1-2):342-353; Dieudonne S (1995) Glycinergic synaptic currents in Golgi cells of the rat cerebellum. Proc Natl Acad Sci U S A 92:1441-1445; Dumoulin A, Triller A, Dieudonne S (2001) IPSC kinetics at identified GABAergic and mixed GABAergic and glycinergic synapses onto cerebellar Golgi cells. J Neurosci 21(16):6045-6057; Dugue GP, Dumoulin A, Triller A, Dieudonne S (2005) Target-dependent use of coreleased inhibitory transmitters at central synapses. J Neurosci 25(28):6490-6498; Zeilhofer HU, Studler B, Arabadzisz D, Schweizer C, Ahmadi S, Layh B, Bosl MR, Fritschy JM (2005) Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice. J Comp Neurol 482(2):123-141]. In the rat cerebellum glycine is not released by itself but is released together with GABA by Lugaro cells onto Golgi cells [Dumoulin A, Triller A, Dieudonne S (2001) IPSC kinetics at identified GABAergic and mixed GABAergic and glycinergic synapses onto cerebellar Golgi cells. J Neurosci 21(16):6045-6057] and by Golgi cells onto unipolar brush and granule cells [Dugue GP, Dumoulin A, Triller A, Dieudonne S (2005) Target-dependent use of coreleased inhibitory transmitters at central synapses. J Neurosci 25(28):6490-6498]. Here we report, from immunolabeling evidence in Macaca cerebellum, that interneurons in the granular cell layer are glycine+ at a density of 120 cells/linear mm. Their morphology indicates that they include Golgi and Lugaro cell types with the majority containing both glycine and GABA or glutamic acid decarboxylase. These data are consistent with the proposal that, as in the rat cerebellum, these granular cell layer interneurons corelease glycine and GABA in the primate cerebellum. The patterns of labeling for glycine and GABA within Golgi and Lugaro cells also indicate that there are biochemical sub-types which are morphologically similar. Further, we find that glycine, GABA and glutamic acid decarboxylase identified candelabrum cells adjacent to the Purkinje cells which is the first time that this interneuron has been reported in primate cerebellar cortex. We propose that candelabrum cells, like the majority of Golgi and Lugaro cells, release both glycine and GABA.

A misfolded protein conformation is not a sufficient condition for in vivo glucosylation by the UDP-Glc:glycoprotein glucosyltransferase.

PubMed

Fernández, F; D'Alessio, C; Fanchiotti, S; Parodi, A J

1998-10-15

A key element in the quality control of glycoprotein folding is the UDP-Glc:glycoprotein glucosyltransferase (GT), which in cell-free assays exclusively glucosylates misfolded glycoproteins. In order to test if such a protein conformation is a sufficient condition for in vivo glucosylation of all N-linked oligosaccharides by GT, a Schizosaccharomyces pombe double mutant (gls2/alg6) was constructed. With this mutant, Man9GlcNAc2 is transferred to proteins and no removal of glucose units added by GT occurs as it lacks glucosidase II. The same proportion of glucosylated (Glc1Man9GlcNAc2) and unglucosylated (Man9GlcNAc2 and Man8GlcNAc2) endoplasmic reticulum (ER)-specific compounds was produced when cells were pre-incubated for 10, 20 or 30 min and further incubated with [14C]glucose for 10 min at 28 degrees C with or without 5 mM dithiothreitol (DTT), thus indicating not only that DTT did not affect protein glucosylation but also that no increased glucosylation of glycoproteins occurred in the presence of the drug. Monitoring Golgi-specific modifications of oligosaccharides after pulse-chase experiments performed in the presence or absence of 5 mM DTT showed that exit of the bulk of glycoproteins synthesized from the ER and thence their proper folding had been prevented by the drug. Cells pulse-chase labeled at 37 degrees C in the absence of DTT also yielded glucosylated and unglucosylated protein-linked oligosaccharides without Golgi-specific modifications. It was concluded that a misfolded protein conformation is not a sufficient condition for in vivo glucosylation of all N-linked oligosaccharides by GT.

Blue-light-activated phototropin2 trafficking from the cytoplasm to Golgi/post-Golgi vesicles.

PubMed

Aggarwal, Chhavi; Banaś, Agnieszka Katarzyna; Kasprowicz-Maluśki, Anna; Borghetti, Carolina; Labuz, Justyna; Dobrucki, Jerzy; Gabryś, Halina

2014-07-01

Phototropins are plasma membrane-localized UVA/blue light photoreceptors which mediate phototropism, inhibition of primary hypocotyl elongation, leaf positioning, chloroplast movements, and stomatal opening. Blue light irradiation activates the C-terminal serine/threonine kinase domain of phototropin which autophosphorylates the receptor. Arabidopsis thaliana encodes two phototropins, phot1 and phot2. In response to blue light, phot1 moves from the plasma membrane into the cytosol and phot2 translocates to the Golgi complex. In this study the molecular mechanism and route of blue-light-induced phot2 trafficking are demonstrated. It is shown that Atphot2 behaves in a similar manner when expressed transiently under 35S or its native promoter. The phot2 kinase domain but not blue-light-mediated autophosphorylation is required for the receptor translocation. Using co-localization and western blotting, the receptor was shown to move from the cytoplasm to the Golgi complex, and then to the post-Golgi structures. The results were confirmed by brefeldin A (an inhibitor of the secretory pathway) which disrupted phot2 trafficking. An association was observed between phot2 and the light chain2 of clathrin via bimolecular fluorescence complementation. The fluorescence was observed at the plasma membrane. The results were confirmed using co-immunoprecipitation. However, tyrphostin23 (an inhibitor of clathrin-mediated endocytosis) and wortmannin (a suppressor of receptor endocytosis) were not able to block phot2 trafficking, indicating no involvement of receptor endocytosis in the formation of phot2 punctuate structures. Protein turnover studies indicated that the receptor was continuously degraded in both darkness and blue light. The degradation of phot2 proceeded via a transport route different from translocation to the Golgi complex. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Phospholipase D Is Involved in the Formation of Golgi Associated Clathrin Coated Vesicles in Human Parotid Duct Cells

PubMed Central

Brito de Souza, Lorena; Pinto da Silva, Luis Lamberti; Jamur, Maria Célia; Oliver, Constance

2014-01-01

Phospholipase D (PLD) has been implicated in many cellular functions, such as vesicle trafficking, exocytosis, differentiation, and proliferation. The aim of this study was to characterize the role of PLD in HSY cells, a human cell line originating from the intercalated duct of the parotid gland. As the function and intracellular localization of PLD varies according to cell type, initially, the intracellular localization of PLD1 and PLD2 was determined. By immunofluorescence, PLD1 and PLD2 both showed a punctate cytoplasmic distribution with extensive co-localization with TGN-46. PLD1 was also found in the nucleus, while PLD2 was associated with the plasma membrane. Treatment of cells with the primary alcohol 1-butanol inhibits the hydrolysis of phosphatidylcoline by PLD thereby suppressing phosphatidic acid (PA) production. In untreated HSY cells, there was only a slight co-localization of PLD with the clathrin coated vesicles. When HSY cells were incubated with 1-butanol the total number of clathrin coated vesicles increased, especially in the juxtanuclear region and the co-localization of PLD with the clathrin coated vesicles was augmented. Transmission electron microscopy confirmed that the number of Golgi-associated coated vesicles was greater. Treatment with 1-butanol also affected the Golgi apparatus, increasing the volume of the Golgi saccules. The decrease in PA levels after treatment with 1-butanol likewise resulted in an accumulation of enlarged lysosomes in the perinuclear region. Therefore, in HSY cells PLD appears to be involved in the formation of Golgi associated clathrin coated vesicles as well as in the structural maintenance of the Golgi apparatus. PMID:24618697

Identification of ER Proteins Involved in the Functional Organisation of the Early Secretory Pathway in Drosophila Cells by a Targeted RNAi Screen

PubMed Central

Kondylis, Vangelis; Tang, Yang; Fuchs, Florian; Boutros, Michael; Rabouille, Catherine

2011-01-01

Background In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation. Results To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for “more and smaller Golgi”) upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER. Conclusions This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation. PMID:21383842

Grapevine and Arabidopsis Cation-Chloride Cotransporters Localize to the Golgi and Trans-Golgi Network and Indirectly Influence Long-Distance Ion Transport and Plant Salt Tolerance1[OPEN

PubMed Central

Henderson, Sam W.; Wege, Stefanie; Qiu, Jiaen; Blackmore, Deidre H.; Walker, Amanda R.; Tyerman, Stephen D.; Walker, Rob R.; Gilliham, Matthew

2015-01-01

Plant cation-chloride cotransporters (CCCs) have been implicated in conferring salt tolerance. They are predicted to improve shoot salt exclusion by directly catalyzing the retrieval of sodium (Na+) and chloride (Cl−) ions from the root xylem. We investigated whether grapevine (Vitis vinifera [Vvi]) CCC has a role in salt tolerance by cloning and functionally characterizing the gene from the cultivar Cabernet Sauvignon. Amino acid sequence analysis revealed that VviCCC shares a high degree of similarity with other plant CCCs. A VviCCC-yellow fluorescent protein translational fusion protein localized to the Golgi and the trans-Golgi network and not the plasma membrane when expressed transiently in tobacco (Nicotiana benthamiana) leaves and Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts. AtCCC-green fluorescent protein from Arabidopsis also localized to the Golgi and the trans-Golgi network. In Xenopus laevis oocytes, VviCCC targeted to the plasma membrane, where it catalyzed bumetanide-sensitive 36Cl–, 22Na+, and 86Rb+ uptake, suggesting that VviCCC (like AtCCC) belongs to the Na+-K+-2Cl– cotransporter class of CCCs. Expression of VviCCC in an Arabidopsis ccc knockout mutant abolished the mutant’s stunted growth phenotypes and reduced shoot Cl– and Na+ content to wild-type levels after growing plants in 50 mm NaCl. In grapevine roots, VviCCC transcript abundance was not regulated by Cl– treatment and was present at similar levels in both the root stele and cortex of three Vitis spp. genotypes that exhibit differential shoot salt exclusion. Our findings indicate that CCC function is conserved between grapevine and Arabidopsis, but neither protein is likely to directly mediate ion transfer with the xylem or have a direct role in salt tolerance. PMID:26378102

The Golgi apparatus regulates cGMP-dependent protein kinase I compartmentation and proteolysis.

PubMed

Kato, Shin; Chen, Jingsi; Cornog, Katherine H; Zhang, Huili; Roberts, Jesse D

2015-06-01

cGMP-dependent protein kinase I (PKGI) is an important effector of cGMP signaling that regulates vascular smooth muscle cell (SMC) phenotype and proliferation. PKGI has been detected in the perinuclear region of cells, and recent data indicate that proprotein convertases (PCs) typically resident in the Golgi apparatus (GA) can stimulate PKGI proteolysis and generate a kinase fragment that localizes to the nucleus and regulates gene expression. However, the role of the endomembrane system in PKGI compartmentation and processing is unknown. Here, we demonstrate that PKGI colocalizes with endoplasmic reticulum (ER), ER-Golgi intermediate compartment, GA cisterna, and trans-Golgi network proteins in pulmonary artery SMC and cell lines. Moreover, PKGI localizes with furin, a trans-Golgi network-resident PC known to cleave PKGI. ER protein transport influences PKGI localization because overexpression of a constitutively inactive Sar1 transgene caused PKGI retention in the ER. Additionally, PKGI appears to reside within the GA because PKGI immunoreactivity was determined to be resistant to cytosolic proteinase K treatment in live cells. The GA appears to play a role in PKGI proteolysis because overexpression of inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate, not only tethered heterologous PKGI-β to the ER and decreased its localization to the GA, but also diminished PKGI proteolysis and nuclear translocation. Also, inhibiting intra-GA protein transport with monensin was observed to decrease PKGI cleavage. These studies detail a role for the endomembrane system in regulating PKGI compartmentation and proteolysis. Moreover, they support the investigation of mechanisms regulating PKGI-dependent nuclear cGMP signaling in the pulmonary vasculature with Golgi dysfunction. Copyright © 2015 the American Physiological Society.

Sorting of a multi-subunit ubiquitin ligase complex in the endolysosome system

PubMed Central

Yang, Xi; Arines, Felichi Mae; Zhang, Weichao

2018-01-01

The yeast Dsc E3 ligase complex has long been recognized as a Golgi-specific protein ubquitination system. It shares a striking sequence similarity to the Hrd1 complex that plays critical roles in the ER-associated degradation pathway. Using biochemical purification and mass spectrometry, we identified two novel Dsc subunits, which we named as Gld1 and Vld1. Surprisingly, Gld1 and Vld1 do not coexist in the same complex. Instead, they compete with each other to form two functionally independent Dsc subcomplexes. The Vld1 subcomplex takes the AP3 pathway to reach the vacuole membrane, whereas the Gld1 subcomplex travels through the VPS pathway and is cycled between Golgi and endosomes by the retromer. Thus, instead of being Golgi-specific, the Dsc complex can regulate protein levels at three distinct organelles, namely Golgi, endosome, and vacuole. Our study provides a novel model of achieving multi-tasking for transmembrane ubiquitin ligases with interchangeable trafficking adaptors. PMID:29355480

Structure of a C-terminal fragment of its Vps53 subunit suggests similarity of Golgi-associated retrograde protein (GARP) complex to a family of tethering complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vasan, Neil; Hutagalung, Alex; Novick, Peter

2010-08-13

The Golgi-associated retrograde protein (GARP) complex is a membrane-tethering complex that functions in traffic from endosomes to the trans-Golgi network. Here we present the structure of a C-terminal fragment of the Vps53 subunit, important for binding endosome-derived vesicles, at a resolution of 2.9 {angstrom}. We show that the C terminus consists of two {alpha}-helical bundles arranged in tandem, and we identify a highly conserved surface patch, which may play a role in vesicle recognition. Mutations of the surface result in defects in membrane traffic. The fold of the Vps53 C terminus is strongly reminiscent of proteins that belong to threemore » other tethering complexes - Dsl1, conserved oligomeric Golgi, and the exocyst - thought to share a common evolutionary origin. Thus, the structure of the Vps53 C terminus suggests that GARP belongs to this family of complexes.« less

Abnormal Glycosphingolipid Mannosylation Triggers Salicylic Acid–Mediated Responses in Arabidopsis[W][OA

PubMed Central

Mortimer, Jenny C.; Yu, Xiaolan; Albrecht, Sandra; Sicilia, Francesca; Huichalaf, Mariela; Ampuero, Diego; Michaelson, Louise V.; Murphy, Alex M.; Matsunaga, Toshiro; Kurz, Samantha; Stephens, Elaine; Baldwin, Timothy C.; Ishii, Tadashi; Napier, Johnathan A.; Weber, Andreas P.M.; Handford, Michael G.; Dupree, Paul

2013-01-01

The Arabidopsis thaliana protein GOLGI-LOCALIZED NUCLEOTIDE SUGAR TRANSPORTER (GONST1) has been previously identified as a GDP-d-mannose transporter. It has been hypothesized that GONST1 provides precursors for the synthesis of cell wall polysaccharides, such as glucomannan. Here, we show that in vitro GONST1 can transport all four plant GDP-sugars. However, gonst1 mutants have no reduction in glucomannan quantity and show no detectable alterations in other cell wall polysaccharides. By contrast, we show that a class of glycosylated sphingolipids (glycosylinositol phosphoceramides [GIPCs]) contains Man and that this mannosylation is affected in gonst1. GONST1 therefore is a Golgi GDP-sugar transporter that specifically supplies GDP-Man to the Golgi lumen for GIPC synthesis. gonst1 plants have a dwarfed phenotype and a constitutive hypersensitive response with elevated salicylic acid levels. This suggests an unexpected role for GIPC sugar decorations in sphingolipid function and plant defense signaling. Additionally, we discuss these data in the context of substrate channeling within the Golgi. PMID:23695979

Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway

PubMed Central

Goodwin, J. Shawn; Drake, Kimberly R.; Rogers, Carl; Wright, Latasha; Lippincott-Schwartz, Jennifer; Philips, Mark R.; Kenworthy, Anne K.

2005-01-01

Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes. PMID:16027222

Geldanamycin Enhances Retrograde Transport of Shiga Toxin in HEp-2 Cells

PubMed Central

Simm, Roger; Torgersen, Maria Lyngaas; Sandvig, Kirsten

2015-01-01

The heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA) has been shown to alter endosomal sorting, diverting cargo destined for the recycling pathway into the lysosomal pathway. Here we investigated whether GA also affects the sorting of cargo into the retrograde pathway from endosomes to the Golgi apparatus. As a model cargo we used the bacterial toxin Shiga toxin, which exploits the retrograde pathway as an entry route to the cytosol. Indeed, GA treatment of HEp-2 cells strongly increased the Shiga toxin transport to the Golgi apparatus. The enhanced Golgi transport was not due to increased endocytic uptake of the toxin or perturbed recycling, suggesting that GA selectively enhances endosomal sorting into the retrograde pathway. Moreover, GA activated p38 and both inhibitors of p38 or its substrate MK2 partially counteracted the GA-induced increase in Shiga toxin transport. Thus, our data suggest that GA-induced p38 and MK2 activation participate in the increased Shiga toxin transport to the Golgi apparatus. PMID:26017782

STED Imaging of Golgi Dynamics with Cer-SiR: A Two-Component, Photostable, High-Density Lipid Probe for Live Cells.

PubMed

Erdmann, Roman S; Toomre, Derek; Schepartz, Alanna

2017-01-01

Long time-lapse super-resolution imaging in live cells requires a labeling strategy that combines a bright, photostable fluorophore with a high-density localization probe. Lipids are ideal high-density localization probes, as they are >100 times more abundant than most membrane-bound proteins and simultaneously demark the boundaries of cellular organelles. Here, we describe Cer-SiR, a two-component, high-density lipid probe that is exceptionally photostable. Cer-SiR is generated in cells via a bioorthogonal reaction of two components: a ceramide lipid tagged with trans-cyclooctene (Cer-TCO) and a reactive, photostable Si-rhodamine dye (SiR-Tz). These components assemble within the Golgi apparatus of live cells to form Cer-SiR. Cer-SiR is benign to cellular function, localizes within the Golgi at a high density, and is sufficiently photostable to enable visualization of Golgi structure and dynamics by 3D confocal or long time-lapse STED microscopy.

Dynein-Dependent Transport of the Hantaan Virus Nucleocapsid Protein to the Endoplasmic Reticulum-Golgi Intermediate Compartment▿

PubMed Central

Ramanathan, Harish N.; Chung, Dong-Hoon; Plane, Steven J.; Sztul, Elizabeth; Chu, Yong-kyu; Guttieri, Mary C.; McDowell, Michael; Ali, Georgia; Jonsson, Colleen B.

2007-01-01

In contrast to most negative-stranded RNA viruses, hantaviruses and other viruses in the family Bunyaviridae mature intracellularly, deriving the virion envelope from the endoplasmic reticulum (ER) or Golgi compartment. While it is generally accepted that Old World hantaviruses assemble and bud into the Golgi compartment, some studies with New World hantaviruses have raised the possibility of maturation at the plasma membrane as well. Overall, the steps leading to virion assembly remain largely undetermined for hantaviruses. Because hantaviruses do not have matrix proteins, the nucleocapsid protein (N) has been proposed to play a key role in assembly. Herein, we examine the intracellular trafficking and morphogenesis of the prototype Old World hantavirus, Hantaan virus (HTNV). Using confocal microscopy, we show that N colocalized with the ER-Golgi intermediate compartment (ERGIC) in HTNV-infected Vero E6 cells, not with the ER, Golgi compartment, or early endosomes. Brefeldin A, which effectively disperses the ER, the ERGIC, and Golgi membranes, redistributed N with the ERGIC, implicating membrane association; however, subcellular fractionation experiments showed the majority of N in particulate fractions. Confocal microscopy revealed that N was juxtaposed to and distributed along microtubules and, over time, became surrounded by vimentin cages. To probe cytoskeletal association further, we probed trafficking of N in cells treated with nocodazole and cytochalasin D, which depolymerize microtubules and actin, respectively. We show that nocodazole, but not cytochalasin D, affected the distribution of N and reduced levels of intracellular viral RNA. These results suggested the involvement of microtubules in trafficking of N, whose movement could occur via molecular motors such as dynein. Overexpression of dynamitin, which is associated with dynein-mediated transport, creates a dominant-negative phenotype blocking transport on microtubules. Overexpression of dynamitin reduced N accumulation in the perinuclear region, which further supports microtubule components in N trafficking. The combined results of these experiments support targeting of N to the ERGIC prior to its movement to the Golgi compartment and the requirement of an intact ERGIC for viral replication and, thus, the possibility of virus factories in this region. PMID:17537852

Granules and Golgi vesicles with differential reactivity to ACTH antiserum in the corticotroph of the rat anterior pituitary.

PubMed

Weber, E; Voigt, K H; Martin, R

1978-05-01

Slices of unembedded rat anterior pituitaries, fixed with a periodate-lysine-paraformaldehyde (PLP) fixative, were incubated with guinea pig antiserum to ACTH and stained with a peroxidase-conjugated IgG fraction of anti-guinea pig gamma-globulin serum from rabbits. The fine structure of the stained cells was identical to that of the ACTH-secreting cell, as described by Siperstein and coworkers. Immunoreactive granules were mainly located at the periphery of the cell. Numerous granules of the inner cytoplasm and also the Golgi complex were nonreactive to the antiserum. The differential labeling for granules and Golgi apparatus peptide.

Deciphering the Adaptive Immune Response to Ovarian Cancer

DTIC Science & Technology

2013-10-01

Additionally, anti-cancer drugs such as 2-phenyl benzimidazole (2- PB) disrupt the Golgi apparatus, which leads to reduced cell proliferation and tumour...of Golgi processing by 2-phenyl benzimidazole analogs blocks cell proliferation and slows tumor growth. Cancer Chemother Pharmacol 2008; 61: 1045–1058

The POK/AtVPS52 protein localizes to several distinct post-Golgi compartments in sporophytic and gametophytic cells.

PubMed

Guermonprez, Hélène; Smertenko, Andrei; Crosnier, Marie-Thérèse; Durandet, Monique; Vrielynck, Nathalie; Guerche, Philippe; Hussey, Patrick J; Satiat-Jeunemaitre, Béatrice; Bonhomme, Sandrine

2008-01-01

The organization and dynamics of the plant endomembrane system require both universal and plant-specific molecules and compartments. The latter, despite the growing wealth of information, remains poorly understood. From the study of an Arabidopsis thaliana male gametophytic mutant, it was possible to isolate a gene named POKY POLLEN TUBE (POK) essential for pollen tube tip growth. The similarity between the predicted POK protein sequence and yeast Vps52p, a subunit from the GARP/VFT complex which is involved in the docking of vesicles from the prevacuolar compartment to the Golgi apparatus, suggested that the POK protein plays a role in plant membrane trafficking. Genetic analysis of Arabidopsis mutants affecting AtVPS53 or AtVPS54 genes which encode putative POK partners shows a transmission defect through the male gametophyte for all lines, which is similar to the pok mutant. Using a combination of biochemical approaches and specific antiserum it has been demonstrated that the POK protein is present in phylogenetically divergent plant species, associated with membranes and belongs to a high molecular weight complex. Combination of immunolocalization studies and pharmacological approaches in different plant cells revealed that the POK protein associates with Golgi and post-Golgi compartments. The role of POK in post-Golgi endomembrane trafficking and as a member of a putative plant GARP/VFT complex is discussed.

Blocking variant surface glycoprotein synthesis alters endoplasmic reticulum exit sites/Golgi homeostasis in Trypanosoma brucei.

PubMed

Ooi, Cher-Pheng; Smith, Terry K; Gluenz, Eva; Wand, Nadina Vasileva; Vaughan, Sue; Rudenko, Gloria

2018-06-01

The predominant secretory cargo of bloodstream form Trypanosoma brucei is variant surface glycoprotein (VSG), comprising ~10% total protein and forming a dense protective layer. Blocking VSG translation using Morpholino oligonucleotides triggered a precise pre-cytokinesis arrest. We investigated the effect of blocking VSG synthesis on the secretory pathway. The number of Golgi decreased, particularly in post-mitotic cells, from 3.5 ± 0.6 to 2.0 ± 0.04 per cell. Similarly, the number of endoplasmic reticulum exit sites (ERES) in post-mitotic cells dropped from 3.9 ± 0.6 to 2.7 ± 0.1 eight hours after blocking VSG synthesis. The secretory pathway was still functional in these stalled cells, as monitored using Cathepsin L. Rates of phospholipid and glycosylphosphatidylinositol-anchor biosynthesis remained relatively unaffected, except for the level of sphingomyelin which increased. However, both endoplasmic reticulum and Golgi morphology became distorted, with the Golgi cisternae becoming significantly dilated, particularly at the trans-face. Membrane accumulation in these structures is possibly caused by reduced budding of nascent vesicles due to the drastic reduction in the total amount of secretory cargo, that is, VSG. These data argue that the total flux of secretory cargo impacts upon the biogenesis and maintenance of secretory structures and organelles in T. brucei, including the ERES and Golgi. © 2018 The Authors. Traffic published by John Wiley & Sons Ltd.

In Vitro Reconstitution of Microtubule Plus End-directed, GTPγS-sensitive Motility of Golgi MembranesV⃞

PubMed Central

Fullerton, Aaron T.; Bau, Mu-Yeh; Conrad, Patricia A.; Bloom, George S.

1998-01-01

Purified Golgi membranes were mixed with cytosol and microtubules (MTs) and observed by video enhanced light microscopy. Initially, the membranes appeared as vesicles that moved along MTs. As time progressed, vesicles formed aggregates from which membrane tubules emerged, traveled along MTs, and eventually generated extensive reticular networks. Membrane motility required ATP, occurred mainly toward MT plus ends, and was inhibited almost completely by the H1 monoclonal antibody to kinesin heavy chain, 5′-adenylylimidodiphosphate, and 100 μM but not 20 μM vanadate. Motility was also blocked by GTPγS or AlF4− but was insensitive to AlCl3, NaF, staurosporin, or okadaic acid. The targets for GTPγS and AlF4− were evidently of cytosolic origin, did not include kinesin or MTs, and were insensitive to several probes for trimeric G proteins. Transport of Golgi membranes along MTs mediated by a kinesin has thus been reconstituted in vitro. The motility is regulated by one or more cytosolic GTPases but not by protein kinases or phosphatases that are inhibited by staurosporin or okadaic acid, respectively. The pertinent GTPases are likely to be small G proteins or possibly dynamin. The in vitro motility may correspond to Golgi-to-ER or Golgi-to-cell surface transport in vivo. PMID:9763438

A Rapid Method Combining Golgi and Nissl Staining to Study Neuronal Morphology and Cytoarchitecture

PubMed Central

Pilati, Nadia; Barker, Matthew; Panteleimonitis, Sofoklis; Donga, Revers; Hamann, Martine

2008-01-01

The Golgi silver impregnation technique gives detailed information on neuronal morphology of the few neurons it labels, whereas the majority remain unstained. In contrast, the Nissl staining technique allows for consistent labeling of the whole neuronal population but gives very limited information on neuronal morphology. Most studies characterizing neuronal cell types in the context of their distribution within the tissue slice tend to use the Golgi silver impregnation technique for neuronal morphology followed by deimpregnation as a prerequisite for showing that neuron's histological location by subsequent Nissl staining. Here, we describe a rapid method combining Golgi silver impregnation with cresyl violet staining that provides a useful and simple approach to combining cellular morphology with cytoarchitecture without the need for deimpregnating the tissue. Our method allowed us to identify neurons of the facial nucleus and the supratrigeminal nucleus, as well as assessing cellular distribution within layers of the dorsal cochlear nucleus. With this method, we also have been able to directly compare morphological characteristics of neuronal somata at the dorsal cochlear nucleus when labeled with cresyl violet with those obtained with the Golgi method, and we found that cresyl violet–labeled cell bodies appear smaller at high cellular densities. Our observation suggests that cresyl violet staining is inadequate to quantify differences in soma sizes. (J Histochem Cytochem 56:539–550, 2008) PMID:18285350

The Role of Drosophila Merlin in the Control of Mitosis Exit and Development

DTIC Science & Technology

2008-07-01

identification of new genes may not yield a clear indication of their respective functions , studies on their evolution may allow validation of their...in the elongated Nebenkern (Figure 2H). In addi- tion, Merlin was seen as a bright punctate dot in the acro- somal region, a Golgi apparatus -derived... functions as a tumor suppressor, we have confirmed that Drosophila Merlin plays important roles in the control of mitosis exit and in the

Voltage-Dependent Intrinsic Bursting in Olfactory Bulb Golgi Cells

ERIC Educational Resources Information Center

Pressler, R. Todd; Rozman, Peter A.; Strowbridge, Ben W.

2013-01-01

In the mammalian olfactory bulb (OB), local synaptic circuits modulate the evolving pattern of activity in mitral and tufted cells following olfactory sensory stimulation. GABAergic granule cells, the most numerous interneuron subtype in this brain region, have been extensively studied. However, classic studies using Golgi staining methods…

Influence of chronic ethanol consumption on toxic effects of 1,2-dichloroethane: glycolipoprotein retention and impairment of dolichol concentration in rat liver microsomes and Golgi apparatus.

PubMed

Cottalasso, Damiano; Domenicotti, Cinzia; Traverso, Nicola; Pronzato, Maria; Nanni, Giorgio

2002-09-16

Our previous investigations demonstrated that 1,2-dichloroethane (DCE) and chronic ethanol treatment separately are able to impair glycoprotein metabolism and secretion, and reduce dolichol concentration in liver membranes. The purpose of this study was to investigate whether chronic ethanol consumption can induce potentiation of rat liver damage due to DCE haloalkane used in several chemical processes and in agriculture. Rats were given 36% of their total energy as ethanol in the Lieber-DeCarli liquid diet for 8 weeks (CH group). The pair-fed control group received an isocaloric amount of dextrine-maltose (PF group). "In vitro" experiments: the DCE (6.5 mM) treatment of isolated hepatocytes from CH rats enhanced glycoprotein retention and further reduced glycoprotein secretion and 14C-glucosamine incorporation compared to the hepatocytes from CH or from PF and DCE treated rats. "In vivo" experiments: a marked decrease of dolichol concentration in microsomes (in which dolichyl phosphate is rate-limiting for the initial glycosylation of protein) and in Golgi membranes (in which total dolichol is very important for membrane permeability, fluidity and vesicle fusion) was observed in CH rats acutely treated with 628 mg/kg bw of DCE (CH+DCE) compared with CH or PF+DCE treated rats. These data suggest that chronic ethanol consumption increases DCE liver toxicity by affecting protein glycosylation processes and impairing glycolipoprotein secretion, with a concomitant retention at the level of the Golgi apparatus.

Golgi: Interactive Online Brain Mapping

PubMed Central

Brown, Ramsay A.; Swanson, Larry W.

2015-01-01

Golgi (http://www.usegolgi.com) is a prototype interactive brain map of the rat brain that helps researchers intuitively interact with neuroanatomy, connectomics, and cellular and chemical architecture. The flood of “-omic” data urges new ways to help researchers connect discrete findings to the larger context of the nervous system. Here we explore Golgi’s underlying reasoning and techniques and how our design decisions balance the constraints of building both a scientifically useful and usable tool. We demonstrate how Golgi can enhance connectomic literature searches with a case study investigating a thalamocortical circuit involving the Nucleus Accumbens and we explore Golgi’s potential and future directions for growth in systems neuroscience and connectomics. PMID:26635596

Investigating Methodological Differences in the Assessment of Dendritic Morphology of Basolateral Amygdala Principal Neurons-A Comparison of Golgi-Cox and Neurobiotin Electroporation Techniques.

PubMed

Klenowski, Paul M; Wright, Sophie E; Mu, Erica W H; Noakes, Peter G; Lavidis, Nickolas A; Bartlett, Selena E; Bellingham, Mark C; Fogarty, Matthew J

2017-12-19

Quantitative assessments of neuronal subtypes in numerous brain regions show large variations in dendritic arbor size. A critical experimental factor is the method used to visualize neurons. We chose to investigate quantitative differences in basolateral amygdala (BLA) principal neuron morphology using two of the most common visualization methods: Golgi-Cox staining and neurobiotin (NB) filling. We show in 8-week-old Wistar rats that NB-filling reveals significantly larger dendritic arbors and different spine densities, compared to Golgi-Cox-stained BLA neurons. Our results demonstrate important differences and provide methodological insights into quantitative disparities of BLA principal neuron morphology reported in the literature.

Membrane-trafficking sorting hubs: cooperation between PI4P and small GTPases at the trans-Golgi Network

PubMed Central

Santiago-Tirado, Felipe H.; Bretscher, Anthony

2011-01-01

Cell polarity in eukaryotes requires constant sorting, packaging, and transport of membrane-bound cargo within the cell. These processes occur in two sorting hubs: the recycling endosome for incoming material, and the trans-Golgi Network for outgoing. Phosphatidylinositol 3-phosphate and 4–5 phosphate are enriched at the endocytic and exocytic sorting hubs, respectively, where they act together with small GTPases to recruit factors to segregate cargo and regulate carrier formation and transport. In this review, we summarize the current understanding of how these lipids and GTPases directly regulate membrane trafficking, emphasizing the recent discoveries of phosphatidylinositol 4-phosphate functions at the trans-Golgi Network. PMID:21764313

Isolation of Hybridomas for Golgi-associated Proteins and a Plant Calmodulin

NASA Technical Reports Server (NTRS)

Kuzmanoff, K. M.; Ray, P. M.

1985-01-01

The demonstration of a role for calcium in the mechanism of the gravitropic response indicates a role for calmodulin. Localization studies indicate that plant cell walls have a high content of calmodulin which suggests a regulatory role for CaM in both gravitropic curvature and auxin-induced growth. Auxin regulation of cell wall loosening and elongation is the basis for most models of this phenomenon. Auxin treatment of pea stem tissue rapidly increases the ctivity of Golgi-localized B-1,4-glucan synthase (GS), an enzyme involved in biosynthesis of wall xyloglucan which apparently constitutes the substrate for the wall loosening process. In order to determine whether auxin stimulates GS activity either by modulation of existing enzyme or induces de novo formation of Golgi glucan synthase, a study was undertaken to isolate and quantitate glucan synthase. This enzyme appears to be an integral protein of the Golgi membrane and has resisted isolation with retention of activity. The production of monoclonal antibody for glucan synthase was undertaken due to the inability to isolate GS by standard detergent/liposome techniques.

Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells

PubMed Central

Farr, Glen A.; Hull, Michael; Mellman, Ira

2009-01-01

Newly synthesized apical and basolateral membrane proteins are sorted from one another in polarized epithelial cells. The trans-Golgi network participates in this sorting process, but some basolateral proteins travel from the Golgi to recycling endosomes (REs) before their surface delivery. Using a novel system for pulse–chase microscopy, we have visualized the postsynthetic route pursued by a newly synthesized cohort of Na,K-ATPase. We find that the basolateral delivery of newly synthesized Na,K-ATPase occurs via a pathway distinct from that pursued by the vesicular stomatitis virus G protein (VSV-G). Na,K-ATPase surface delivery occurs at a faster rate than that observed for VSV-G. The Na,K-ATPase does not pass through the RE compartment en route to the plasma membrane, and Na,K-ATPase trafficking is not regulated by the same small GTPases as other basolateral proteins. Finally, Na,K-ATPase and VSV-G travel in separate post-Golgi transport intermediates, demonstrating directly that multiple routes exist for transport from the Golgi to the basolateral membrane in polarized epithelial cells. PMID:19620635

Phosphatidic acid phospholipase A1 mediates ER–Golgi transit of a family of G protein–coupled receptors

PubMed Central

Kunduri, Govind; Yuan, Changqing; Parthibane, Velayoudame; Nyswaner, Katherine M.; Kanwar, Ritu; Nagashima, Kunio; Britt, Steven G.; Mehta, Nickita; Kotu, Varshika; Porterfield, Mindy; Tiemeyer, Michael; Dolph, Patrick J.; Acharya, Usha

2014-01-01

The coat protein II (COPII)–coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein–coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking. PMID:25002678

G-rich, a Drosophila selenoprotein, is a Golgi-resident type III membrane protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Chang Lan; Shim, Myoung Sup; Chung, Jiyeol

2006-10-06

G-rich is a Drosophila melanogaster selenoprotein, which is a homologue of human and mouse SelK. Subcellular localization analysis using GFP-tagged G-rich showed that G-rich was localized in the Golgi apparatus. The fusion protein was co-localized with the Golgi marker proteins but not with an endoplasmic reticulum (ER) marker protein in Drosophila SL2 cells. Bioinformatic analysis of G-rich suggests that this protein is either type II or type III transmembrane protein. To determine the type of transmembrane protein experimentally, GFP-G-rich in which GFP was tagged at the N-terminus of G-rich, or G-rich-GFP in which GFP was tagged at the C-terminus ofmore » G-rich, were expressed in SL2 cells. The tagged proteins were then digested with trypsin, and analyzed by Western blot analysis. The results showed that the C-terminus of the G-rich protein was exposed to the cytoplasm indicating it is a type III microsomal membrane protein. G-rich is First selenoprotein identified in the Golgi apparatus.« less

Proprioception in the extraocular muscles of mammals and man.

PubMed

Blumer, Roland; Konacki, Kadriye Zeynep; Streicher, Johannes; Hoetzenecker, Wolfram; Blumer, Michael Josef Franz; Lukas, Julius-Robert

2006-06-01

This article summarizes the authors' previous studies on proprioceptors in extraocular muscles (EOMs) of mammals and man. They report on muscle spindles in the EOMs of man, Golgi tendon organs in the EOMs of even-toed ungulates, and palisade endings in the EOMs of the cat. Muscle spindles: Muscle spindles are present in the EOMs of some mammals and in the EOMs of man. Compared with muscle spindles in other skeletal muscles, those in human EOMs exhibit structural differences. These structural differences may indicate a special function. Golgi tendon organs: Golgi tendon organs are absent in human EOMs. Golgi tendon organs exhibiting a specific morphology are present in the EOMs of even-toed ungulates. Their high number and rich innervation indicate functional importance. Palisade endings: Palisade endings are nervous end organs confined to the EOMs of mammals and man. It is assumed that these organs have a proprioceptive function. The authors show that palisade endings are immunoreactive for antibodies against choline acetyltransferase. Neuromuscular contacts, if present in palisade endings, are alpha -bungarotoxin positive as well. Taken together, these results show that palisade endings exhibit molecular characteristics of effector organs.

Nitric oxide synthase generates nitric oxide locally to regulate compartmentalized protein S-nitrosylation and protein trafficking

PubMed Central

Iwakiri, Yasuko; Satoh, Ayano; Chatterjee, Suvro; Toomre, Derek K.; Chalouni, Cecile M.; Fulton, David; Groszmann, Roberto J.; Shah, Vijay H.; Sessa, William C.

2006-01-01

Nitric oxide (NO) is a highly diffusible and short-lived physiological messenger. Despite its diffusible nature, NO modifies thiol groups of specific cysteine residues in target proteins and alters protein function via S-nitrosylation. Although intracellular S-nitrosylation is a specific posttranslational modification, the defined localization of an NO source (nitric oxide synthase, NOS) with protein S-nitrosylation has never been directly demonstrated. Endothelial NOS (eNOS) is localized mainly on the Golgi apparatus and in plasma membrane caveolae. Here, we show by using eNOS targeted to either the Golgi or the nucleus that S-nitrosylation is concentrated at the primary site of eNOS localization. Furthermore, localization of eNOS on the Golgi enhances overall Golgi protein S-nitrosylation, the specific S-nitrosylation of N-ethylmaleimide-sensitive factor and reduces the speed of protein transport from the endoplasmic reticulum to the plasma membrane in a reversible manner. These data indicate that local NOS action generates organelle-specific protein S-nitrosylation reactions that can regulate intracellular transport processes. PMID:17170139

Caspase-2 Is Localized at the Golgi Complex and Cleaves Golgin-160 during Apoptosis

PubMed Central

Mancini, Marie; Machamer, Carolyn E.; Roy, Sophie; Nicholson, Donald W.; Thornberry, Nancy A.; Casciola-Rosen, Livia A.; Rosen, Antony

2000-01-01

Caspases are an extended family of cysteine proteases that play critical roles in apoptosis. Animals deficient in caspases-2 or -3, which share very similar tetrapeptide cleavage specificities, exhibit very different phenotypes, suggesting that the unique features of individual caspases may account for distinct regulation and specialized functions. Recent studies demonstrate that unique apoptotic stimuli are transduced by distinct proteolytic pathways, with multiple components of the proteolytic machinery clustering at distinct subcellular sites. We demonstrate here that, in addition to its nuclear distribution, caspase-2 is localized to the Golgi complex, where it cleaves golgin-160 at a unique site not susceptible to cleavage by other caspases with very similar tetrapeptide specificities. Early cleavage at this site precedes cleavage at distal sites by other caspases. Prevention of cleavage at the unique caspase-2 site delays disintegration of the Golgi complex after delivery of a pro-apoptotic signal. We propose that the Golgi complex, like mitochondria, senses and integrates unique local conditions, and transduces pro-apoptotic signals through local caspases, which regulate local effectors. PMID:10791974

Visualization of the post-Golgi vesicle-mediated transportation of TGF-β receptor II by quasi-TIRFM.

PubMed

Luo, Wangxi; Xia, Tie; Xu, Li; Chen, Ye-Guang; Fang, Xiaohong

2014-10-01

Transforming growth factor β receptor II (Tβ RII) is synthesized in the cytoplasm and then transported to the plasma membrane of cells to fulfil its signalling duty. Here, we applied live-cell fluorescence imaging techniques, in particular quasi-total internal reflection fluorescence microscopy, to imaging fluorescent protein-tagged Tβ RII and monitoring its secretion process. We observed punctuate-like Tβ RII-containing post-Golgi vesicles formed in MCF7 cells. Single-particle tracking showed that these vesicles travelled along the microtubules at an average speed of 0.51 μm/s. When stimulated by TGF-β ligand, these receptor-containing vesicles intended to move towards the plasma membrane. We also identified several factors that could inhibit the formation of such post-Golgi vesicles. Although the inhibitory mechanisms still remain unknown, the observed characteristics of Tβ RII-containing vesicles provide new information on intracellular Tβ RII transportation. It also renders Tβ RII a good model system for studying post-Golgi vesicle-trafficking and protein transportation. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Guanine nucleotide dissociation inhibitor is essential for Rab1 function in budding from the endoplasmic reticulum and transport through the Golgi stack

PubMed Central

1994-01-01

The small GTPase Rab1 is required for vesicular traffic from the ER to the cis-Golgi compartment, and for transport between the cis and medial compartments of the Golgi stack. In the present study, we examine the role of guanine nucleotide dissociation inhibitor (GDI) in regulating the function of Rab1 in the transport of vesicular stomatitis virus glycoprotein (VSV-G) in vitro. Incubation in the presence of excess GDI rapidly (t1/2 < 30 s) extracted Rab1 from membranes, inhibiting vesicle budding from the ER and sequential transport between the cis-, medial-, and trans-Golgi cisternae. These results demonstrate a direct role for GDI in the recycling of Rab proteins. Analysis of rat liver cytosol by gel filtration revealed that a major pool of Rab1 fractionates with a molecular mass of approximately 80 kD in the form of a GDI-Rab1 complex. When the GDI-Rab1 complex was depleted from cytosol by use of a Rab1-specific antibody, VSV-G failed to exit the ER. However, supplementation of depleted cytosol with a GDI-Rab1 complex prepared in vitro from recombinant forms of Rab1 and GDI efficiently restored export from the ER, and transport through the Golgi stack. These results provide evidence that a cytosolic GDI-Rab1 complex is required for the formation of non-clathrin-coated vesicles mediating transport through the secretory pathway. PMID:8089173

Activation of Rab GTPase Sec4 by its GEF Sec2 is required for prospore membrane formation during sporulation in yeast Saccharomyces cerevisiae.

PubMed

Suda, Yasuyuki; Tachikawa, Hiroyuki; Inoue, Ichiro; Kurita, Tomokazu; Saito, Chieko; Kurokawa, Kazuo; Nakano, Akihiko; Irie, Kenji

2018-02-01

Sec2 activates Sec4 Rab GTPase as a guanine nucleotide exchange factor for the recruitment of downstream effectors to facilitate tethering and fusion of post-Golgi vesicles at the plasma membrane. During the meiosis and sporulation of budding yeast, post-Golgi vesicles are transported to and fused at the spindle pole body (SPB) to form a de novo membrane, called the prospore membrane. Previous studies have revealed the role of the SPB outer surface called the meiotic outer plaque (MOP) in docking and fusion of post-Golgi vesicles. However, the upstream molecular machinery for post-Golgi vesicular fusion that facilitates prospore membrane formation remains enigmatic. Here, we demonstrate that the GTP exchange factor for Sec4, Sec2, participates in the formation of the prospore membrane. A conditional mutant in which the SEC2 expression is shut off during sporulation showed sporulation defects. Inactivation of Sec2 caused Sec4 targeting defects along the prospore membranes, thereby causing insufficient targeting of downstream effectors and cargo proteins to the prospore membrane. These results suggest that the activation of Sec4 by Sec2 is required for the efficient supply of post-Golgi vesicles to the prospore membrane and thus for prospore membrane formation/extension and subsequent deposition of spore wall materials. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Quantification of apolipoprotein B-48 and B-100 in rat liver endoplasmic reticulum and Golgi fractions.

PubMed Central

Cartwright, I J; Higgins, J A

1992-01-01

We have developed a method for measurement of apolipoprotein (apo) B-48 and apo B-100 in blood and subcellular fractions of rat liver based on SDS/PAGE followed by quantitative immunoblotting using 125I-Protein A. Standard curves were prepared in each assay using apo B prepared from total rat lipoproteins by extraction with tetramethylurea. Subcellular fractions (rough and smooth endoplasmic reticulum and Golgi fractions) were prepared from rat liver and separated into membrane and cisternal-content fractions. For quantification, membrane fractions were solubilized in Triton X-100, and the apo B was immunoprecipitated before separation by SDS/PAGE and immunoblotting. Content fractions were concentrated by ultrafiltration and separated by SDS/PAGE without immunoprecipitation. Quantification of apo B in subcellular fractions and detection of apo B by immunoblotting yielded consistent results. In all fractions apo B-48 was the major form, accounting for approximately three-quarters of the total apo B. By using marker enzymes as internal standards, it was calculated that all of the apo B was recovered in the endoplasmic reticulum and Golgi fractions, with approximately 80% of each form of apo B in the endoplasmic reticulum. More than 90% of the apo B of the rough- and smooth-endoplasmic-reticulum fractions was membrane-bound, whereas approx. 33 and 15% of the apo B of the cis-enriched Golgi fractions and trans-enriched Golgi fractions respectively were membrane-bound. Images Fig. 1. Fig. 3. Fig. 4. PMID:1637294

A novel, modernized Golgi-Cox stain optimized for CLARITY cleared tissue.

PubMed

Kassem, Mustafa S; Fok, Sandra Y Y; Smith, Kristie L; Kuligowski, Michael; Balleine, Bernard W

2018-01-15

High resolution neuronal information is extraordinarily useful in understanding the brain's functionality. The development of the Golgi-Cox stain allowed observation of the neuron in its entirety with unrivalled detail. Tissue clearing techniques, e.g., CLARITY and CUBIC, provide the potential to observe entire neuronal circuits intact within tissue and without previous restrictions with regard to section thickness. Here we describe an improved Golgi-Cox stain method, optimised for use with CLARITY and CUBIC that can be used in both fresh and fixed tissue. Using this method, we were able to observe neurons in their entirety within a fraction of the time traditionally taken to clear tissue (48h). We were also able to show for the first-time that Golgi stained tissue is fluorescent when visualized using a multi-photon microscope, allowing us to image synaptic spines with a detail previously unachievable. These novel methods provide cheap and easy to use techniques to investigate the morphology of cellular processes in the brain at a new-found depth, speed, utility and detail, without previous restrictions of time, tissue type and section thickness. This is the first application of a Golgi-Cox stain to cleared brain tissue, it is investigated and discussed in detail, describing different methodologies that may be used, a comparison between the different clearing techniques and lastly the novel interaction of these techniques with this ultra-rapid stain. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of the Sterol and Phosphatidylinositol 4-Phosphate Binding Properties of Golgi-Associated OSBP-Related Protein 9 (ORP9)

PubMed Central

Liu, Xinwei; Ridgway, Neale D.

2014-01-01

Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus. PMID:25255026

Characterization of the sterol and phosphatidylinositol 4-phosphate binding properties of Golgi-associated OSBP-related protein 9 (ORP9).

PubMed

Liu, Xinwei; Ridgway, Neale D

2014-01-01

Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus.

The N-Terminus of Vps74p Is Essential for the Retention of Glycosyltransferases in the Golgi but Not for the Modulation of Apical Polarized Growth in Saccharomyces cerevisiae

PubMed Central

Huang, Chun-Fang; Lee, Fang-Jen S.

2013-01-01

Vps74p is a member of the PtdIns(4)P-binding protein family. Vps74p interacts with Golgi-resident glycosyltransferases and the coat protein COPI complex to modulate Golgi retention of glycosyltransferases and with the PtdIns(4)P phosphatase Sac1p to modulate PtdIns(4)P homeostasis at the Golgi. Genetic analysis has shown that Vps74p is required for the formation of abnormal elongated buds in cdc34-2 cells. The C-terminal region of Vps74p is required for Vps74p multimerization, Golgi localization, and glycosyltransferase interactions; however, the functional significance of the N-terminal region and three putative phosphorylation sites of Vps74p have not been well characterized. In this study, we demonstrate that Vps74p executes multiple cellular functions using different domains. We found that the N-terminal 66 amino acids of Vps74p are dispensable for its Golgi localization and modulation of cell wall integrity but are required for glycosyltransferase retention and glycoprotein processing. Deletion of the N-terminal 90 amino acids, but not the 66 amino acids, of Vps74p impaired its ability to restore the elongated bud phenotype in cdc34-2/vps74Δ cells. Deletion of Sac1p and Arf1p also specifically reduced the abnormal elongated bud phenotype in cdc34-2 cells. Furthermore, we found that three N-terminal phosphorylation sites contribute to rapamycin hypersensitivity, although these phosphorylation residues are not involved in Vps74p localization, ability to modulate glycosyltransferase retention, or elongated bud formation in cdc34-2 cells. Thus, we propose that Vps74p may use different domains to interact with specific effectors thereby differentially modulating a variety of cellular functions. PMID:24019977

Chronic alcohol exposure affects the cell components involved in membrane traffic in neuronal dendrites.

PubMed

Romero, Ana M; Renau-Piqueras, Jaime; Marín, M Pilar; Esteban-Pretel, Guillermo

2015-01-01

The specific traffic of the membrane components in neurons is a major requirement to establish and maintain neuronal domains-the axonal and the somatodendritic domains-and their polarized morphology. Unlike axons, dendrites contain membranous organelles, which are involved in the secretory pathway, including the endoplasmic reticulum, the Golgi apparatus and post-Golgi apparatus carriers, the cytoskeleton, and plasma membrane. A variety of molecules and factors are also involved in this process. Previous studies have shown that chronic alcohol exposure negatively affects several of these cell components, such as the Golgi apparatus or cytoskeleton in neurons. Yet very little information is available on the possible effects of this exposure on the remaining cell elements involved in intracellular trafficking in neurons, particularly in dendrites. By qualitative and quantitative electron microscopy, immunofluorescence and immunoblotting, we herein show that chronic exposure to moderate levels (30 mM) of ethanol in cultured neurons reduces the volume and surface density of the rough endoplasmic reticulum, and increases the levels of GRP78, a chaperone involved in endoplasmic reticulum stress. Ethanol also significantly diminishes the proportion of neurons that show an extension of Golgi into dendrites and dendritic Golgi outposts, a structure present exclusively in longer, thicker apical dendrites. Both Golgi apparatus types were also fragmented into a large number of cells. We also investigated the effect of alcohol on the levels of microtubule-based motor proteins KIF5, KIF17, KIFC2, dynein, and myosin IIb, responsible for transporting different cargoes in dendrites. Of these, alcohol differently affects several of them by lowering dynein and raising KIF5, KIFC2, and myosin IIb. These results, together with other previously published ones, suggest that practically all the protein trafficking steps in dendrites are altered to a greater or lesser extent by chronic alcohol exposure in neuronal cells, which may have negative repercussions for the development and maintenance of their polarized morphology and function.

Localization of herpes simplex virus type 1 UL37 in the Golgi complex requires UL36 but not capsid structures.

PubMed

Desai, Prashant; Sexton, Gerry L; Huang, Eugene; Person, Stanley

2008-11-01

The herpes simplex virus type 1 (HSV-1) UL37 gene encodes a 120-kDa polypeptide which resides in the tegument structure of the virion and is important for morphogenesis. The goal of this study was to use green fluorescent protein (GFP) to follow the fate of UL37 within cells during the normal course of virus replication. GFP was inserted in frame at the C terminus of UL37 to generate a fluorescent-protein-tagged UL37 polypeptide. A virus designated K37eGFP, which replicated normally on Vero cells, was isolated and was shown to express the fusion polypeptide. When cells infected with this virus were examined by confocal microscopy, the fluorescence was observed to be predominantly cytoplasmic. As the infection progressed, fluorescence began to accumulate in a juxtanuclear structure. Mannosidase II and giantin were observed to colocalize with UL37eGFP at these structures, as judged by immunofluorescence assays. Therefore, UL37 traffics to the Golgi complex during infection. A VP26mRFP marker (red fluorescent protein fused to VP26) was recombined into K37eGFP, and when cells infected with this "dual-color" virus were examined, colocalization of the red (capsid) and green (UL37) fluorescence in the Golgi structure was observed. Null mutations in VP5 (DeltaVP5), which abolished capsid assembly, and in UL36 (Delta36) were recombined into the K37eGFP virus genome. In cells infected with K37eGFP/DeltaVP5, localization of UL37eGFP to the Golgi complex was similar to that for the parental virus (K37eGFP), indicating that trafficking of UL37eGFP to the Golgi complex did not require capsid structures. Confocal analysis of cells infected with K37eGFP/Delta36 showed that, in the absence of UL36, accumulation of UL37eGFP at the Golgi complex was not evident. This indicates an interaction between these two proteins that is important for localization of UL37 in the Golgi complex and thus possibly for cytoplasmic envelopment of the capsid. This is the first demonstration of a functional role for UL36:UL37 interaction in HSV-1-infected cells.

BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication

PubMed Central

Morosky, Stefanie; Lennemann, Nicholas J.

2016-01-01

ABSTRACT Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. IMPORTANCE Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of BPIFB6 expression correlates with pronounced defects in the secretory pathway and greatly reduces the replication of CVB, PV, and EV71. Our results thus identify a novel host cell therapeutic target whose function could be targeted to alter enterovirus replication. PMID:26962226

BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication.

PubMed

Morosky, Stefanie; Lennemann, Nicholas J; Coyne, Carolyn B

2016-05-15

Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of BPIFB6 expression correlates with pronounced defects in the secretory pathway and greatly reduces the replication of CVB, PV, and EV71. Our results thus identify a novel host cell therapeutic target whose function could be targeted to alter enterovirus replication. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Direct interaction of the Golgi V-ATPase a-subunit isoform with PI(4)P drives localization of Golgi V-ATPases in yeast.

PubMed

Banerjee, Subhrajit; Kane, Patricia M

2017-09-15

Luminal pH and phosphoinositide content are fundamental features of organelle identity. Vacuolar H + -ATPases (V-ATPases) drive organelle acidification in all eukaryotes, and membrane-bound a-subunit isoforms of the V-ATPase are implicated in organelle-specific targeting and regulation. Earlier work demonstrated that the endolysosomal lipid PI(3,5)P 2 activates V-ATPases containing the vacuolar a-subunit isoform in Saccharomyces cerevisiae Here we demonstrate that PI(4)P, the predominant Golgi phosphatidylinositol (PI) species, directly interacts with the cytosolic amino terminal (NT) domain of the yeast Golgi V-ATPase a-isoform Stv1. Lysine-84 of Stv1NT is essential for interaction with PI(4)P in vitro and in vivo, and interaction with PI(4)P is required for efficient localization of Stv1-containing V-ATPases. The cytosolic NT domain of the human V-ATPase a2 isoform specifically interacts with PI(4)P in vitro, consistent with its Golgi localization and function. We propose that NT domains of V o a-subunit isoforms interact specifically with PI lipids in their organelles of residence. These interactions can transmit organelle-specific targeting or regulation information to V-ATPases. © 2017 Banerjee and Kane. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Vesicular Transport of Progeny Parvovirus Particles through ER and Golgi Regulates Maturation and Cytolysis

PubMed Central

Bär, Séverine; Rommelaere, Jean; Nüesch, Jürg P. F.

2013-01-01

Progeny particles of non-enveloped lytic parvoviruses were previously shown to be actively transported to the cell periphery through vesicles in a gelsolin-dependent manner. This process involves rearrangement and destruction of actin filaments, while microtubules become protected throughout the infection. Here the focus is on the intracellular egress pathway, as well as its impact on the properties and release of progeny virions. By colocalization with cellular marker proteins and specific modulation of the pathways through over-expression of variant effector genes transduced by recombinant adeno-associated virus vectors, we show that progeny PV particles become engulfed into COPII-vesicles in the endoplasmic reticulum (ER) and are transported through the Golgi to the plasma membrane. Besides known factors like sar1, sec24, rab1, the ERM family proteins, radixin and moesin play (an) essential role(s) in the formation/loading and targeting of virus-containing COPII-vesicles. These proteins also contribute to the transport through ER and Golgi of the well described analogue of cellular proteins, the secreted Gaussia luciferase in absence of virus infection. It is therefore likely that radixin and moesin also serve for a more general function in cellular exocytosis. Finally, parvovirus egress via ER and Golgi appears to be necessary for virions to gain full infectivity through post-assembly modifications (e.g. phosphorylation). While not being absolutely required for cytolysis and progeny virus release, vesicular transport of parvoviruses through ER and Golgi significantly accelerates these processes pointing to a regulatory role of this transport pathway. PMID:24068925

A Conserved Structural Motif Mediates Retrograde Trafficking of Shiga Toxin Types 1 and 2.

PubMed

Selyunin, Andrey S; Mukhopadhyay, Somshuvra

2015-12-01

Shiga toxin-producing Escherichia coli (STEC) produce two types of Shiga toxin (STx): STx1 and STx2. The toxin A-subunits block protein synthesis, while the B-subunits mediate retrograde trafficking. STEC infections do not have definitive treatments, and there is growing interest in generating toxin transport inhibitors for therapy. However, a comprehensive understanding of the mechanisms of toxin trafficking is essential for drug development. While STx2 is more toxic in vivo, prior studies focused on STx1 B-subunit (STx1B) trafficking. Here, we show that, compared with STx1B, trafficking of the B-subunit of STx2 (STx2B) to the Golgi occurs with slower kinetics. Despite this difference, similar to STx1B, endosome-to-Golgi transport of STx2B does not involve transit through degradative late endosomes and is dependent on dynamin II, epsinR, retromer and syntaxin5. Importantly, additional experiments show that a surface-exposed loop in STx2B (β4-β5 loop) is required for its endosome-to-Golgi trafficking. We previously demonstrated that residues in the corresponding β4-β5 loop of STx1B are required for interaction with GPP130, the STx1B-specific endosomal receptor, and for endosome-to-Golgi transport. Overall, STx1B and STx2B share a common pathway and use a similar structural motif to traffic to the Golgi, suggesting that the underlying mechanisms of endosomal sorting may be evolutionarily conserved. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Vesicular transport of progeny parvovirus particles through ER and Golgi regulates maturation and cytolysis.

PubMed

Bär, Séverine; Rommelaere, Jean; Nüesch, Jürg P F

2013-09-01

Progeny particles of non-enveloped lytic parvoviruses were previously shown to be actively transported to the cell periphery through vesicles in a gelsolin-dependent manner. This process involves rearrangement and destruction of actin filaments, while microtubules become protected throughout the infection. Here the focus is on the intracellular egress pathway, as well as its impact on the properties and release of progeny virions. By colocalization with cellular marker proteins and specific modulation of the pathways through over-expression of variant effector genes transduced by recombinant adeno-associated virus vectors, we show that progeny PV particles become engulfed into COPII-vesicles in the endoplasmic reticulum (ER) and are transported through the Golgi to the plasma membrane. Besides known factors like sar1, sec24, rab1, the ERM family proteins, radixin and moesin play (an) essential role(s) in the formation/loading and targeting of virus-containing COPII-vesicles. These proteins also contribute to the transport through ER and Golgi of the well described analogue of cellular proteins, the secreted Gaussia luciferase in absence of virus infection. It is therefore likely that radixin and moesin also serve for a more general function in cellular exocytosis. Finally, parvovirus egress via ER and Golgi appears to be necessary for virions to gain full infectivity through post-assembly modifications (e.g. phosphorylation). While not being absolutely required for cytolysis and progeny virus release, vesicular transport of parvoviruses through ER and Golgi significantly accelerates these processes pointing to a regulatory role of this transport pathway.

Polar auxin transport: controlling where and how much

NASA Technical Reports Server (NTRS)

Muday, G. K.; DeLong, A.; Brown, C. S. (Principal Investigator)

2001-01-01

Auxin is transported through plant tissues, moving from cell to cell in a unique polar manner. Polar auxin transport controls important growth and developmental processes in higher plants. Recent studies have identified several proteins that mediate polar auxin transport and have shown that some of these proteins are asymmetrically localized, paving the way for studies of the mechanisms that regulate auxin transport. New data indicate that reversible protein phosphorylation can control the amount of auxin transport, whereas protein secretion through Golgi-derived vesicles and interactions with the actin cytoskeleton might regulate the localization of auxin efflux complexes.

A role for a lithium-inhibited Golgi nucleotidase in skeletal development and sulfation

PubMed Central

Frederick, Joshua P.; Tafari, A. Tsahai; Wu, Sheue-Mei; Megosh, Louis C.; Chiou, Shean-Tai; Irving, Ryan P.; York, John D.

2008-01-01

Sulfation is an important biological process that modulates the function of numerous molecules. It is directly mediated by cytosolic and Golgi sulfotransferases, which use 3′-phosphoadenosine 5′-phosphosulfate to produce sulfated acceptors and 3′-phosphoadenosine 5′-phosphate (PAP). Here, we identify a Golgi-resident PAP 3′-phosphatase (gPAPP) and demonstrate that its activity is potently inhibited by lithium in vitro. The inactivation of gPAPP in mice led to neonatal lethality, lung abnormalities resembling atelectasis, and dwarfism characterized by aberrant cartilage morphology. The phenotypic similarities of gPAPP mutant mice to chondrodysplastic models harboring mutations within components of the sulfation pathway lead to the discovery of undersulfated chondroitin in the absence of functional enzyme. Additionally, we observed loss of gPAPP leads to perturbations in the levels of heparan sulfate species in lung tissue and whole embryos. Our data are consistent with a model that clearance of the nucleotide product of sulfotransferases within the Golgi plays an important role in glycosaminoglycan sulfation, provide a unique genetic basis for chondrodysplasia, and define a function for gPAPP in the formation of skeletal elements derived through endochondral ossification. PMID:18695242

Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering*

PubMed Central

Heinrich, Frank; Nanda, Hirsh; Goh, Haw Zan; Bachert, Collin; Lösche, Mathias; Linstedt, Adam D.

2014-01-01

The mammalian Golgi reassembly stacking protein (GRASP) proteins are Golgi-localized homotypic membrane tethers that organize Golgi stacks into a long, contiguous ribbon-like structure. It is unknown how GRASPs undergo trans pairing given that cis interactions between the proteins in the plane of the membrane are intrinsically favored. To test the hypothesis that myristoylation of the self-interacting GRASP domain restricts its orientation on the membrane to favor trans pairing, we established an in vitro assay that recapitulates GRASP-dependent membrane tethering and used neutron reflection under similar conditions to determine the orientation of the GRASP domain. In vivo, the membrane association of GRASP proteins is conferred by the simultaneous insertion of an N-terminal myristic acid and binding to a Golgi-associated binding partner. In our assay, the latter contact was replaced using a C-terminal hexa-His moiety, which bound to Ni2+-conjugated lipids incorporated into a substrate-supported bilayer lipid membrane. Nonmyristoylated protein lacked a fixed orientation on the membrane and inefficiently tethered liposomes. In contrast, myristoylated GRASP promoted tethering and exhibited a unique membrane complex. Thus, myristoylation restricts the membrane orientation of the GRASP domain favoring interactions in trans for membrane tethering. PMID:24505136

A small-molecule switch for Golgi sulfotransferases.

PubMed

de Graffenried, Christopher L; Laughlin, Scott T; Kohler, Jennifer J; Bertozzi, Carolyn R

2004-11-30

The study of glycan function is a major frontier in biology that could benefit from small molecules capable of perturbing carbohydrate structures on cells. The widespread role of sulfotransferases in modulating glycan function makes them prime targets for small-molecule modulators. Here, we report a system for conditional activation of Golgi-resident sulfotransferases using a chemical inducer of dimerization. Our approach capitalizes on two features shared by these enzymes: their requirement of Golgi localization for activity on cellular substrates and the modularity of their catalytic and localization domains. Fusion of these domains to the proteins FRB and FKBP enabled their induced assembly by the natural product rapamycin. We applied this strategy to the GlcNAc-6-sulfotransferases GlcNAc6ST-1 and GlcNAc6ST-2, which collaborate in the sulfation of L-selectin ligands. Both the activity and specificity of the inducible enzymes were indistinguishable from their WT counterparts. We further generated rapamycin-inducible chimeric enzymes comprising the localization domain of a sulfotransferase and the catalytic domain of a glycosyltransferase, demonstrating the generality of the system among other Golgi enzymes. The approach provides a means for studying sulfate-dependent processes in cellular systems and, potentially, in vivo.

Biosynthesis and Immunolocalization of Lewis a-Containing N-Glycans in the Plant Cell1

PubMed Central

Fitchette, Anne-Catherine; Cabanes-Macheteau, Marion; Marvin, Laure; Martin, Barry; Satiat-Jeunemaitre, Béatrice; Gomord, Véronique; Crooks, Kim; Lerouge, Patrice; Faye, Loïc; Hawes, Chris

1999-01-01

We recently demonstrated the presence of a new asparagine-linked complex glycan on plant glycoproteins that harbors the Lewis a (Lea), or Galβ(1-3)[Fucα(1-4)]GlcNAc, epitope, which in mammalian cells plays an important role in cell-to-cell recognition. Here we show that the monoclonal antibody JIM 84, which is widely used as a Golgi marker in light and electron microscopy of plant cells, is specific for the Lea antigen. This antigen is present on glycoproteins of a number of flowering and non-flowering plants, but is less apparent in the Cruciferae, the family that includes Arabidopsis. Lea-containing oligosaccharides are found in the Golgi apparatus, and our immunocytochemical experiments suggest that it is synthesized in the trans-most part of the Golgi apparatus. Lea epitopes are abundantly present on extracellular glycoproteins, either soluble or membrane bound, but are never observed on vacuolar glycoproteins. Double-labeling experiments suggest that vacuolar glycoproteins do not bypass the late Golgi compartments where Lea is built, and that the absence of the Lea epitope from vacuolar glycoproteins is probably the result of its degradation by glycosidases en route to or after arrival in the vacuole. PMID:10517824

The inside and outside: topological issues in plant cell wall biosynthesis and the roles of nucleotide sugar transporters.

PubMed

Temple, Henry; Saez-Aguayo, Susana; Reyes, Francisca C; Orellana, Ariel

2016-09-01

The cell wall is a complex extracellular matrix composed primarily of polysaccharides. Noncellulosic polysaccharides, glycoproteins and proteoglycans are synthesized in the Golgi apparatus by glycosyltransferases (GTs), which use nucleotide sugars as donors to glycosylate nascent glycan and glycoprotein acceptors that are subsequently exported to the extracellular space. Many nucleotide sugars are synthesized in the cytosol, leading to a topological issue because the active sites of most GTs are located in the Golgi lumen. Nucleotide sugar transporters (NSTs) overcome this problem by translocating nucleoside diphosphate sugars from the cytosol into the lumen of the organelle. The structures of the cell wall components synthesized in the Golgi are diverse and complex; therefore, transporter activities are necessary so that the nucleotide sugars can provide substrates for the GTs. In this review, we describe the topology of reactions involved in polysaccharide biosynthesis in the Golgi and focus on the roles of NSTs as well as their impacts on cell wall structure when they are altered. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Evidence that the Malaria Parasite Plasmodium falciparum Putative Rhoptry Protein 2 Localizes to the Golgi Apparatus throughout the Erythrocytic Cycle.

PubMed

Hallée, Stéphanie; Richard, Dave

2015-01-01

Invasion of a red blood cell by Plasmodium falciparum merozoites is an essential step in the malaria lifecycle. Several of the proteins involved in this process are stored in the apical complex of the merozoite, a structure containing secretory organelles that are released at specific times during invasion. The molecular players involved in erythrocyte invasion thus represent potential key targets for both therapeutic and vaccine-based strategies to block parasite development. In our quest to identify and characterize new effectors of invasion, we investigated the P. falciparum homologue of a P. berghei protein putatively localized to the rhoptries, the Putative rhoptry protein 2 (PbPRP2). We show that in P. falciparum, the protein colocalizes extensively with the Golgi apparatus across the asexual erythrocytic cycle. Furthermore, imaging of merozoites caught at different times during invasion show that PfPRP2 is not secreted during the process instead staying associated with the Golgi apparatus. Our evidence therefore suggests that PfPRP2 is a Golgi protein and that it is likely not a direct effector in the process of merozoite invasion.

In-vivo observations of a spherical aggregate of endoplasmic reticulum and of Golgi vesicles in the tip of fast-growing Chara rhizoids.

PubMed

Bartnik, E; Sievers, A

1988-11-01

In-vivo differential interference contrast microscopy was used to detect individual Golgi vesicles and a new structure in the tip of fast-growing rhizoids of Chara fragilis Desvaux. This structure is a spherical clear zone which is free of Golgi vesicles, has a diameter of 5 μm and is positioned in the center of the apical Golgi-vesicle accumulation ("Spitzenkörper"). After glutaraldehyde fixation and osmium tetroxide-potassium ferricyanide staining of the rhizoid, followed by serial sectioning and three-dimensional reconstruction, the spherical zone shows a tight accumulation of anastomosing endoplasmic reticulum (ER) membranes. The ER membranes radiate from this aggregate towards the apical plasmalemma and to the membranes of the statolith compartments. Upon gravistimulation the ER aggregate changes its position according to the new growth direction, indicating its participation in growth determination. After treatment of the rhizoid with cytochalasin B or phalloidin the ER aggregate disappears and the statoliths sediment. It is concluded that the integrity of the ER aggregate is actin-dependent and that it is related to the polar organisation of the gravitropically growing cell tip.

Transport According to GARP: Receiving Retrograde Cargo at the Trans-Golgi Network

PubMed Central

Bonifacino, Juan S.; Hierro, Aitor

2010-01-01

Tethering factors are large protein complexes that capture transport vesicles and enable their fusion with acceptor organelles at different stages of the endomembrane system. Recent studies have shed new light on the structure and function of a heterotetrameric tethering factor named Golgi-associated retrograde protein (GARP), which promotes fusion of endosome-derived, retrograde transport carriers to the trans-Golgi network (TGN). X-ray crystallography of the Vps53 and Vps54 subunits of GARP has revealed that this complex is structurally related to other tethering factors such as the exocyst, COG and Dsl1, indicating that they all might work by a similar mechanism. Loss of GARP function compromises the growth, fertility and/or viability of the defective organisms, underscoring the essential nature of GARP-mediated retrograde transport. PMID:21183348

Golgi bypass for local delivery of axonal proteins, fact or fiction?

PubMed

González, Carolina; Cornejo, Víctor Hugo; Couve, Andrés

2018-04-06

Although translation of cytosolic proteins is well described in axons, much less is known about the synthesis, processing and trafficking of transmembrane and secreted proteins. A canonical rough endoplasmic reticulum or a stacked Golgi apparatus has not been detected in axons, generating doubts about the functionality of a local route. However, axons contain mRNAs for membrane and secreted proteins, translation factors, ribosomal components, smooth endoplasmic reticulum and post-endoplasmic reticulum elements that may contribute to local biosynthesis and plasma membrane delivery. Here we consider the evidence supporting a local secretory system in axons. We discuss exocytic elements and examples of autonomous axonal trafficking that impact development and maintenance. We also examine whether unconventional post-endoplasmic reticulum pathways may replace the canonical Golgi apparatus. Copyright © 2018. Published by Elsevier Ltd.

TGN38 is maintained in the trans-Golgi network by a tyrosine-containing motif in the cytoplasmic domain.

PubMed Central

Bos, K; Wraight, C; Stanley, K K

1993-01-01

Sorting of proteins destined for different plasma membrane domains, lysosomes and secretory pathways takes place in the trans-Golgi network (TGN). TGN38 is an integral membrane protein found in this intracellular compartment. We show that TGN38 contains an autonomous targeting signal within its cytoplasmic domain which determines its intracellular location. Deletion analysis and site-directed mutagenesis of this domain demonstrate that a tyrosine motif homologous to the internalization signal of surface receptors is necessary and sufficient for correct localization. These findings suggest that TGN38 is maintained in the TGN by retrieval from the plasma membrane and employs a different mechanism for retention from that of the transferase enzymes of the trans-Golgi. Images PMID:8491209

Polarized trafficking: the palmitoylation cycle distributes cytoplasmic proteins to distinct neuronal compartments.

PubMed

Tortosa, Elena; Hoogenraad, Casper C

2018-02-01

In neurons, polarized cargo distribution occurs mainly between the soma and axonal and dendritic compartments, and requires coordinated regulation of cytoskeletal remodeling and membrane trafficking. The Golgi complex plays a critical role during neuronal polarization and secretory trafficking has been shown to differentially transport proteins to both axons and dendrites. Besides the Golgi protein sorting, recent data revealed that palmitoylation cycles are an efficient mechanism to localize cytoplasmic, non-transmembrane proteins to particular neuronal compartments, such as the newly formed axon. Palmitoylation allows substrate proteins to bind to and ride with Golgi-derived secretory vesicles to all neuronal compartments. By allowing cytoplasmic proteins to 'hitchhike' on transport carriers in a non-polarized fashion, compartmentalized depalmitoylation may act as a selective retention mechanism. Copyright © 2018 Elsevier Ltd. All rights reserved.

Analysis of thick brain sections by obverse-reverse computer microscopy: application of a new, high clarity Golgi-Nissl stain.

PubMed

Glaser, E M; Van der Loos, H

1981-08-01

Exceptionally clear Golgi-Nissl sections of 300 micron thickness have been morphometrically studied by light microscopy using oil immersion objectives. The clarity results from a new variation of a staining procedure that combines Golgi and Nissl images in one section. A viewing technique has been developed that permits a histologic preparation to be examined from its obverse (or normally viewed) side and its reverse (or under) side. The technique was designed for use with a computer microscope but can be employed with any light microscope whose stage position can be measured within 100 micron. Sections thicker than 300 micron can be studied dependent on the working distance of the objective lens, provided that the clarity of the material permits it.

Mechanisms of Organophosphorus (OP) Injury: Sarin-Induced Hippocampal Gene Expression Changes and Pathway Perturbation

DTIC Science & Technology

2012-01-01

components of the endomembrane system, including endoplasmic reticulum (ER) and Golgi apparatus were significantly down-regulated. As a result of...impairment in dopaminergic functions (Lucot JB, personal communication). Interestingly, data on sarin exposures have shown inhibition of new memory...quite unexpected that the endoplasmic reticulum (ER) and Golgi apparatus , the subcellular organelles essential for processing (e.g., folding, post

Human Papillomavirus 16 Infection Induces VAP-Dependent Endosomal Tubulation.

PubMed

Siddiqa, Abida; Massimi, Paola; Pim, David; Broniarczyk, Justyna; Banks, Lawrence

2018-03-15

Human papillomavirus (HPV) infection involves complex interactions with the endocytic transport machinery, which ultimately facilitates the entry of the incoming viral genomes into the trans -Golgi network (TGN) and their subsequent nuclear entry during mitosis. The endosomal pathway is a highly dynamic intracellular transport system, which consists of vesicular compartments and tubular extensions, although it is currently unclear whether incoming viruses specifically alter the endocytic machinery. In this study, using MICAL-L1 as a marker for tubulating endosomes, we show that incoming HPV-16 virions induce a profound alteration in global levels of endocytic tubulation. In addition, we also show a critical requirement for the endoplasmic reticulum (ER)-anchored protein VAP in this process. VAP plays an essential role in actin nucleation and endosome-to-Golgi transport. Indeed, the loss of VAP results in a dramatic decrease in the level of endosomal tubulation induced by incoming HPV-16 virions. This is also accompanied by a marked reduction in virus infectivity. In VAP knockdown cells, we see that the defect in virus trafficking occurs after capsid disassembly but prior to localization at the trans -Golgi network, with the incoming virion-transduced DNA accumulating in Vps29/TGN46-positive hybrid vesicles. Taken together, these studies demonstrate that infection with HPV-16 virions induces marked alterations of endocytic transport pathways, some of which are VAP dependent and required for the endosome-to-Golgi transport of the incoming viral L2/DNA complex. IMPORTANCE Human papillomavirus infectious entry involves multiple interactions with the endocytic transport machinery. In this study, we show that incoming HPV-16 virions induce a dramatic increase in endocytic tubulation. This tubulation requires ER-associated VAP, which plays a critical role in ensuring the delivery of cargoes from the endocytic compartments to the trans -Golgi network. Indeed, the loss of VAP blocks HPV infectious entry at a step after capsid uncoating but prior to localization at the trans -Golgi network. These results define a critical role for ER-associated VAP in endocytic tubulation and in HPV-16 infectious entry. Copyright © 2018 American Society for Microbiology.

Golgi-Associated Protein Kinase C-ε Is Delivered to Phagocytic Cups: Role of Phosphatidylinositol 4-Phosphate.

PubMed

Hanes, Cheryl M; D'Amico, Anna E; Ueyama, Takehiko; Wong, Alexander C; Zhang, Xuexin; Hynes, W Frederick; Barroso, Margarida M; Cady, Nathaniel C; Trebak, Mohamed; Saito, Naoaki; Lennartz, Michelle R

2017-07-01

Protein kinase C-ε (PKC-ε) at phagocytic cups mediates the membrane fusion necessary for efficient IgG-mediated phagocytosis. The C1B and pseudosubstrate (εPS) domains are necessary and sufficient for this concentration. C1B binds diacylglycerol; the docking partner for εPS is unknown. Liposome assays revealed that the εPS binds phosphatidylinositol 4-phosphate (PI4P) and PI(3,5)P 2 Wortmannin, but not LY294002, inhibits PKC-ε concentration at cups and significantly reduces the rate of phagocytosis. As Wortmannin inhibits PI4 kinase, we hypothesized that PI4P mediates the PKC-ε concentration at cups and the rate of phagocytosis. PKC-ε colocalizes with the trans -Golgi network (TGN) PI4P reporter, P4M, suggesting it is tethered at the TGN. Real-time imaging of GFP-PKC-ε-expressing macrophages revealed a loss of Golgi-associated PKC-ε during phagocytosis, consistent with a Golgi-to-phagosome translocation. Treatment with PIK93, a PI4 kinase inhibitor, reduces PKC-ε at both the TGN and the cup, decreases phagocytosis, and prevents the increase in capacitance that accompanies membrane fusion. Finally, expression of the Golgi-directed PI4P phosphatase, hSac1-K2A, recapitulates the PIK93 phenotype, confirming that Golgi-associated PI4P is critical for efficient phagocytosis. Together these data are consistent with a model in which PKC-ε is tethered to the TGN via an εPS-PI4P interaction. The TGN-associated pool of PKC-ε concentrates at the phagocytic cup where it mediates the membrane fusion necessary for phagocytosis. The novelty of these data lies in the demonstration that εPS binds PI4P and PI(3,5)P 2 and that PI4P is necessary for PKC-ε localization at the TGN, its translocation to the phagocytic cup, and the membrane fusion required for efficient Fc [γ] receptor-mediated phagocytosis. Copyright © 2017 by The American Association of Immunologists, Inc.

A New Approach of Short Wave Protection against Middle Cerebral Artery Occlusion/Reperfusion Injury via Attenuation of Golgi Apparatus Stress by Inhibition of Downregulation of Secretory Pathway Ca(2+)-ATPase Isoform 1 in Rats.

PubMed

Fan, Yongmei; Zhang, Changjie; Li, Ting; Peng, Wenna; Yin, Jing; Li, Xiaofao; Kong, Ying; Lan, Chunna; Wang, Rumi; Hu, Zhiping

2016-07-01

Short wave (SW), a pattern of electromagnetic therapy, achieves an oscillating electromagnetic field. It has been reported that it may have a potential effect on cerebral injury. The present study was designed to investigate the potential role and possible mechanism of SW in focal cerebral ischemia/reperfusion (I/R) injury in rats. Secretory pathway Ca(2+)/Mn(2+) ATPase isoform 1 is a major component of Golgi apparatus stress. It has been reported as representative of Golgi apparatus stress. Up to 120 minutes of middle cerebral artery occlusion (MCAO) and reperfusion injury was induced in male Sprague-Dawley rats. Different sessions of SW daily were administered over head after reperfusion from day 1 to day 7. Functional recovery scores, survival rates, infarct volume analysis, electron microscope test, and western blotting studies were used to analyze the therapy. SW protected against neuronal death and apoptosis in cornu ammon 1 region of hippocampus by reducing neuronal deficit, infarct volume, and ultrastructure. SW partly inhibited upregulation of caspase3. In addition, the expression of secretory pathway Ca(2+)-ATPase isoform 1 (SPCA1) was upregulated by SW. Our data indicate that SW can be protected against focal cerebral I/R injury, and the influence on Golgi apparatus stress might provide us a new perspective in further study. To the authors' knowledge, this is the first report using SW to increase expression of SPCA1 indicating modulate Golgi apparatus stress in MCAO and reperfusion model. Copyright © 2016 National Stroke Association. Published by Elsevier Inc. All rights reserved.

Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport.

PubMed

Hirata, Tetsuya; Fujita, Morihisa; Nakamura, Shota; Gotoh, Kazuyoshi; Motooka, Daisuke; Murakami, Yoshiko; Maeda, Yusuke; Kinoshita, Taroh

2015-09-01

The importance of endosome-to-trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51-VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport. © 2015 Hirata, Fujita, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Differential Effects of the Putative GBF1 Inhibitors Golgicide A and AG1478 on Enterovirus Replication▿

PubMed Central

van der Linden, Lonneke; van der Schaar, Hilde M.; Lanke, Kjerstin H. W.; Neyts, Johan; van Kuppeveld, Frank J. M.

2010-01-01

The genus Enterovirus, belonging to the family Picornaviridae, includes well-known pathogens, such as poliovirus, coxsackievirus, and rhinovirus. Brefeldin A (BFA) impedes replication of several enteroviruses through inhibition of Golgi-specific BFA resistance factor 1 (GBF1), a regulator of secretory pathway integrity and transport. GBF1 mediates the GTP exchange of Arf1, which in activated form recruits coatomer protein complex I (COP-I) to Golgi vesicles, a process important in transport between the endoplasmic reticulum and Golgi vesicles. Recently, the drugs AG1478 and Golgicide A (GCA) were put forward as new inhibitors of GBF1. In this study, we investigated the effects of these putative GBF1 inhibitors on secretory pathway function and enterovirus replication. We show that both drugs induced fragmentation of the Golgi vesicles and caused dissociation of Arf1 and COP-I from Golgi membranes, yet they differed in their effect on GBF1 localization. The effects of AG1478, but not those of GCA, could be countered by overexpression of Arf1, indicating a difference in their molecular mechanism of action. Consistent with this idea, we observed that GCA drastically reduced replication of coxsackievirus B3 (CVB3) and other human enterovirus species, whereas AG1478 had no effect at all on enterovirus replication. Time-of-addition studies and analysis of RNA replication using a subgenomic replicon both showed that GCA suppresses RNA replication of CVB3, which could be countered by overexpression of GBF1. These results indicate that, in contrast to AG1478, GCA inhibits CVB3 RNA replication by targeting GBF1. AG1478 and GCA may be valuable tools to further dissect enterovirus replication. PMID:20504936

Golgi-to-Endoplasmic Reticulum (ER) Retrograde Traffic in Yeast Requires Dsl1p, a Component of the ER Target Site that Interacts with a COPI Coat Subunit

PubMed Central

Reilly, Barbara A.; Kraynack, Bryan A.; VanRheenen, Susan M.; Waters, M. Gerard

2001-01-01

DSL1 was identified through its genetic interaction with SLY1, which encodes a t-SNARE-interacting protein that functions in endoplasmic reticulum (ER)-to-Golgi traffic. Conditional dsl1 mutants exhibit a block in ER-to-Golgi traffic at the restrictive temperature. Here, we show that dsl1 mutants are defective for retrograde Golgi-to-ER traffic, even under conditions where no anterograde transport block is evident. These results suggest that the primary function of Dsl1p may be in retrograde traffic, and that retrograde defects can lead to secondary defects in anterograde traffic. Dsl1p is an ER-localized peripheral membrane protein that can be extracted from the membrane in a multiprotein complex. Immunoisolation of the complex yielded Dsl1p and proteins of ∼80 and ∼55 kDa. The ∼80-kDa protein has been identified as Tip20p, a protein that others have shown to exist in a tight complex with Sec20p, which is ∼50 kDa. Both Sec20p and Tip20p function in retrograde Golgi-to-ER traffic, are ER-localized, and bind to the ER t-SNARE Ufe1p. These findings suggest that an ER-localized complex of Dsl1p, Sec20p, and Tip20p functions in retrograde traffic, perhaps upstream of a Sly1p/Ufe1p complex. Last, we show that Dsl1p interacts with the δ-subunit of the retrograde COPI coat, Ret2p, and discuss possible roles for this interaction. PMID:11739780

The Cytoplasmic Region of α-1,6-Mannosyltransferase Mnn9p Is Crucial for Retrograde Transport from the Golgi Apparatus to the Endoplasmic Reticulum in Saccharomyces cerevisiae▿ †

PubMed Central

Okamoto, Michiyo; Yoko-o, Takehiko; Miyakawa, Tokichi; Jigami, Yoshifumi

2008-01-01

In Saccharomyces cerevisiae, Och1p and Mnn9p mannosyltransferases are localized in the cis-Golgi. Attempts to live image Och1p and Mnn9p tagged with green fluorescent protein or red fluorescent protein, respectively, using a high-performance confocal laser scanning microscope system resulted in simultaneous visualization of the native proteins in a living cell. Our observations revealed that Och1p and Mnn9p are not always colocalized to the same cisternae. The difference in the dynamics of these mannosyltransferases may reflect differences in the mechanisms for their retention in the cis-Golgi, since it has been reported that Mnn9p cycles between the endoplasmic reticulum and the cis-Golgi whereas Och1p does not (Z. Todorow, A. Spang, E. Carmack, J. Yates, and R. Schekman, Proc. Natl. Acad. Sci. USA 97:13643-13648, 2000). We investigated the localization of chimeric proteins of Mnn9p and Och1p in sec12 and erd1 mutant cells. A chimeric protein, M16/O16, which consists of the N-terminal cytoplasmic region of Mnn9p and the transmembrane and luminal region of Och1p, behaved like Mnn9p, suggesting that the N-terminal cytoplasmic region is important for the intracellular dynamics of Mnn9p. This observation is supported by results from subcellular-fractionation experiments. Mutational analysis revealed that two arginine residues in the N-terminal region of Mnn9p are important for the chimeric protein to cycle between the endoplasmic reticulum and the Golgi apparatus. PMID:18083825

Prefission Constriction of Golgi Tubular Carriers Driven by Local Lipid Metabolism: A Theoretical Model

PubMed Central

Shemesh, Tom; Luini, Alberto; Malhotra, Vivek; Burger, Koert N. J.; Kozlov, Michael M.

2003-01-01

Membrane transport within mammalian cells is mediated by small vesicular as well as large pleiomorphic transport carriers (TCs). A major step in the formation of TCs is the creation and subsequent narrowing of a membrane neck connecting the emerging carrier with the initial membrane. In the case of small vesicular TCs, neck formation may be directly induced by the coat proteins that cover the emerging vesicle. However, the mechanism underlying the creation and narrowing of a membrane neck in the generation of large TCs remains unknown. We present a theoretical model for neck formation based on the elastic model of membranes. Our calculations suggest a lipid-driven mechanism with a central role for diacylglycerol (DAG). The model is applied to a well-characterized in vitro system that reconstitutes TC formation from the Golgi complex, namely the pearling and fission of Golgi tubules induced by CtBP/BARS, a protein that catalyzes the conversion of lysophosphatidic acid into phosphatidic acid. In view of the importance of a PA-DAG cycle in the formation of Golgi TCs, we assume that the newly formed phosphatidic acid undergoes rapid dephosphorylation into DAG. DAG possesses a unique molecular shape characterized by an extremely large negative spontaneous curvature, and it redistributes rapidly between the membrane monolayers and along the membrane surface. Coupling between local membrane curvature and local lipid composition results, by mutual enhancement, in constrictions of the tubule into membrane necks, and a related inhomogeneous lateral partitioning of DAG. Our theoretical model predicts the exact dimensions of the constrictions observed in the pearling Golgi tubules. Moreover, the model is able to explain membrane neck formation by physiologically relevant mole fractions of DAG. PMID:14645071

Conditional deletion of Cadherin 13 perturbs Golgi cells and disrupts social and cognitive behaviors.

PubMed

Tantra, M; Guo, L; Kim, J; Zainolabidin, N; Eulenburg, V; Augustine, G J; Chen, A I

2018-02-15

Inhibitory interneurons mediate the gating of synaptic transmission and modulate the activities of neural circuits. Disruption of the function of inhibitory networks in the forebrain is linked to impairment of social and cognitive behaviors, but the involvement of inhibitory interneurons in the cerebellum has not been assessed. We found that Cadherin 13 (Cdh13), a gene implicated in autism spectrum disorder and attention-deficit hyperactivity disorder, is specifically expressed in Golgi cells within the cerebellar cortex. To assess the function of Cdh13 and utilize the manipulation of Cdh13 expression in Golgi cells as an entry point to examine cerebellar-mediated function, we generated mice carrying Cdh13-floxed alleles and conditionally deleted Cdh13 with GlyT2::Cre mice. Loss of Cdh13 results in a decrease in the expression/localization of GAD67 and reduces spontaneous inhibitory postsynaptic current (IPSC) in cerebellar Golgi cells without disrupting spontaneous excitatory postsynaptic current (EPSC). At the behavioral level, loss of Cdh13 in the cerebellum, piriform cortex and endopiriform claustrum have no impact on gross motor coordination or general locomotor behaviors, but leads to deficits in cognitive and social abilities. Mice lacking Cdh13 exhibit reduced cognitive flexibility and loss of preference for contact region concomitant with increased reciprocal social interactions. Together, our findings show that Cdh13 is critical for inhibitory function of Golgi cells, and that GlyT2::Cre-mediated deletion of Cdh13 in non-executive centers of the brain, such as the cerebellum, may contribute to cognitive and social behavioral deficits linked to neurological disorders. © 2018 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.

In vivo intracellular pH measurements in tobacco and Arabidopsis reveal an unexpected pH gradient in the endomembrane system.

PubMed

Martinière, Alexandre; Bassil, Elias; Jublanc, Elodie; Alcon, Carine; Reguera, Maria; Sentenac, Hervé; Blumwald, Eduardo; Paris, Nadine

2013-10-01

The pH homeostasis of endomembranes is essential for cellular functions. In order to provide direct pH measurements in the endomembrane system lumen, we targeted genetically encoded ratiometric pH sensors to the cytosol, the endoplasmic reticulum, and the trans-Golgi, or the compartments labeled by the vacuolar sorting receptor (VSR), which includes the trans-Golgi network and prevacuoles. Using noninvasive live-cell imaging to measure pH, we show that a gradual acidification from the endoplasmic reticulum to the lytic vacuole exists, in both tobacco (Nicotiana tabacum) epidermal (ΔpH -1.5) and Arabidopsis thaliana root cells (ΔpH -2.1). The average pH in VSR compartments was intermediate between that of the trans-Golgi and the vacuole. Combining pH measurements with in vivo colocalization experiments, we found that the trans-Golgi network had an acidic pH of 6.1, while the prevacuole and late prevacuole were both more alkaline, with pH of 6.6 and 7.1, respectively. We also showed that endosomal pH, and subsequently vacuolar trafficking of soluble proteins, requires both vacuolar-type H(+) ATPase-dependent acidification as well as proton efflux mediated at least by the activity of endosomal sodium/proton NHX-type antiporters.

GnT1IP-L specifically inhibits MGAT1 in the Golgi via its luminal domain.

PubMed

Huang, Hung-Hsiang; Hassinen, Antti; Sundaram, Subha; Spiess, Andrej-Nikolai; Kellokumpu, Sakari; Stanley, Pamela

2015-09-15

Mouse GnT1IP-L, and membrane-bound GnT1IP-S (MGAT4D) expressed in cultured cells inhibit MGAT1, the N-acetylglucosaminyltransferase that initiates the synthesis of hybrid and complex N-glycans. However, it is not known where in the secretory pathway GnT1IP-L inhibits MGAT1, nor whether GnT1IP-L inhibits other N-glycan branching N-acetylglucosaminyltransferases of the medial Golgi. We show here that the luminal domain of GnT1IP-L contains its inhibitory activity. Retention of GnT1IP-L in the endoplasmic reticulum (ER) via the N-terminal region of human invariant chain p33, with or without C-terminal KDEL, markedly reduced inhibitory activity. Dynamic fluorescent resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) assays revealed homomeric interactions for GnT1IP-L in the ER, and heteromeric interactions with MGAT1 in the Golgi. GnT1IP-L did not generate a FRET signal with MGAT2, MGAT3, MGAT4B or MGAT5 medial Golgi GlcNAc-tranferases. GnT1IP/Mgat4d transcripts are expressed predominantly in spermatocytes and spermatids in mouse, and are reduced in men with impaired spermatogenesis.

deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster.

PubMed

Sriram, V; Krishnan, K S; Mayor, Satyajit

2003-05-12

Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.

Impaired ATP6V0A2 expression contributes to Golgi dispersion and glycosylation changes in senescent cells.

PubMed

Udono, Miyako; Fujii, Kaoru; Harada, Gakuro; Tsuzuki, Yumi; Kadooka, Keishi; Zhang, Pingbo; Fujii, Hiroshi; Amano, Maho; Nishimura, Shin-Ichiro; Tashiro, Kosuke; Kuhara, Satoru; Katakura, Yoshinori

2015-11-27

Many genes and signaling pathways have been found to be involved in cellular senescence program. In the present study, we have identified 16 senescence-associated genes by differential proteomic analysis of the normal human diploid fibroblast cell line, TIG-1, and focused on ATP6V0A2. The aim of this study is to clarify the role of ATP6V0A2, the causal gene for ARCL2, a syndrome of abnormal glycosylation and impaired Golgi trafficking, in cellular senescence program. Here we showed that ATP6V0A2 is critical for cellular senescence; impaired expression of ATP6V0A2 disperses the Golgi structure and triggers senescence, suggesting that ATP6V0A2 mediates these processes. FITC-lectin staining and glycoblotting revealed significantly different glycosylation structures in presenescent (young) and senescent (old) TIG-1 cells; reducing ATP6V0A2 expression in young TIG-1 cells yielded structures similar to those in old TIG-1 cells. Our results suggest that senescence-associated impaired expression of ATP6V0A2 triggers changes in Golgi structure and glycosylation in old TIG-1 cells, which demonstrates a role of ATP6V0A2 in cellular senescence program.

Impaired ATP6V0A2 expression contributes to Golgi dispersion and glycosylation changes in senescent cells

PubMed Central

Udono, Miyako; Fujii, Kaoru; Harada, Gakuro; Tsuzuki, Yumi; Kadooka, Keishi; Zhang, Pingbo; Fujii, Hiroshi; Amano, Maho; Nishimura, Shin-Ichiro; Tashiro, Kosuke; Kuhara, Satoru; Katakura, Yoshinori

2015-01-01

Many genes and signaling pathways have been found to be involved in cellular senescence program. In the present study, we have identified 16 senescence-associated genes by differential proteomic analysis of the normal human diploid fibroblast cell line, TIG-1, and focused on ATP6V0A2. The aim of this study is to clarify the role of ATP6V0A2, the causal gene for ARCL2, a syndrome of abnormal glycosylation and impaired Golgi trafficking, in cellular senescence program. Here we showed that ATP6V0A2 is critical for cellular senescence; impaired expression of ATP6V0A2 disperses the Golgi structure and triggers senescence, suggesting that ATP6V0A2 mediates these processes. FITC-lectin staining and glycoblotting revealed significantly different glycosylation structures in presenescent (young) and senescent (old) TIG-1 cells; reducing ATP6V0A2 expression in young TIG-1 cells yielded structures similar to those in old TIG-1 cells. Our results suggest that senescence-associated impaired expression of ATP6V0A2 triggers changes in Golgi structure and glycosylation in old TIG-1 cells, which demonstrates a role of ATP6V0A2 in cellular senescence program. PMID:26611489

Specific Rab GTPase-activating proteins define the Shiga toxin and epidermal growth factor uptake pathways.

PubMed

Fuchs, Evelyn; Haas, Alexander K; Spooner, Robert A; Yoshimura, Shin-ichiro; Lord, J Michael; Barr, Francis A

2007-06-18

Rab family guanosine triphosphatases (GTPases) together with their regulators define specific pathways of membrane traffic within eukaryotic cells. In this study, we have investigated which Rab GTPase-activating proteins (GAPs) can interfere with the trafficking of Shiga toxin from the cell surface to the Golgi apparatus and studied transport of the epidermal growth factor (EGF) from the cell surface to endosomes. This screen identifies 6 (EVI5, RN-tre/USP6NL, TBC1D10A-C, and TBC1D17) of 39 predicted human Rab GAPs as specific regulators of Shiga toxin but not EGF uptake. We show that Rab43 is the target of RN-tre and is required for Shiga toxin uptake. In contrast, RabGAP-5, a Rab5 GAP, was unique among the GAPs tested and reduced the uptake of EGF but not Shiga toxin. These results suggest that Shiga toxin trafficking to the Golgi is a multistep process controlled by several Rab GAPs and their target Rabs and that this process is discrete from ligand-induced EGF receptor trafficking.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells.

PubMed

Cheng, Chunming; Guo, Jeffrey Yunhua; Geng, Feng; Wu, Xiaoning; Cheng, Xiang; Li, Qiyue; Guo, Deliang

2016-11-08

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Complex structure of the fission yeast SREBP-SCAP binding domains reveals an oligomeric organization.

PubMed

Gong, Xin; Qian, Hongwu; Shao, Wei; Li, Jingxian; Wu, Jianping; Liu, Jun-Jie; Li, Wenqi; Wang, Hong-Wei; Espenshade, Peter; Yan, Nieng

2016-11-01

Sterol regulatory element-binding protein (SREBP) transcription factors are master regulators of cellular lipid homeostasis in mammals and oxygen-responsive regulators of hypoxic adaptation in fungi. SREBP C-terminus binds to the WD40 domain of SREBP cleavage-activating protein (SCAP), which confers sterol regulation by controlling the ER-to-Golgi transport of the SREBP-SCAP complex and access to the activating proteases in the Golgi. Here, we biochemically and structurally show that the carboxyl terminal domains (CTD) of Sre1 and Scp1, the fission yeast SREBP and SCAP, form a functional 4:4 oligomer and Sre1-CTD forms a dimer of dimers. The crystal structure of Sre1-CTD at 3.5 Å and cryo-EM structure of the complex at 5.4 Å together with in vitro biochemical evidence elucidate three distinct regions in Sre1-CTD required for Scp1 binding, Sre1-CTD dimerization and tetrameric formation. Finally, these structurally identified domains are validated in a cellular context, demonstrating that the proper 4:4 oligomeric complex formation is required for Sre1 activation.

Cosmos 1887: morphology, histochemistry, and vasculature of the growing rat tibia

NASA Technical Reports Server (NTRS)

Doty, S. B.; Morey-Holton, E. R.; Durnova, G. N.; Kaplansky, A. S.

1990-01-01

Light microscopy, electron microscopy, and enzyme histochemistry were used to study the effects of spaceflight on metaphyseal and cortical bone of the rat tibia. Cortical cross-sectional area and perimeter were not altered by a 12.5-day spaceflight in 3-month-old male rats. The endosteal osteoblast population and the vasculature near the periosteal surface in flight rats compared with ground controls showed more pronounced changes in cortical bone than in metaphyseal bone. The osteoblasts demonstrated greater numbers of transitional Golgi vesicles, possibly caused by a decreased cellular metabolic energy source, but no difference in the large Golgi saccules or the cell membrane-associated alkaline phosphatase activity. The periosteal vasculature in the diaphysis of flight rats often showed lipid accumulations within the lumen of the vessels, occasional degeneration of the vascular wall, and degeneration of osteocytes adjacent to vessels containing intraluminal deposits. These changes were not found in the metaphyseal region of flight animals. The focal vascular changes may be due to ischemia of bone or a developing fragility of the vessel walls as a result of spaceflight.

Progressive quality control of secretory proteins in the early secretory compartment by ERp44

PubMed Central

Sannino, Sara; Anelli, Tiziana; Cortini, Margherita; Masui, Shoji; Degano, Massimo; Fagioli, Claudio; Inaba, Kenji; Sitia, Roberto

2014-01-01

ERp44 is a pH-regulated chaperone of the secretory pathway. In the acidic milieu of the Golgi, its C-terminal tail changes conformation, simultaneously exposing the substrate-binding site for cargo capture and the RDEL motif for ER retrieval via interactions with cognate receptors. Protonation of cysteine 29 in the active site allows tail movements in vitro and in vivo. Here we show that also conserved histidines in the C-terminal tail regulate ERp44 in vivo. Mutants lacking these histidines are hyperactive in retaining substrates. Surprisingly, they are also O-glycosylated and partially secreted. Co-expression of client proteins prevents secretion of the histidine mutants, forcing tail opening and RDEL accessibility. Client-induced RDEL exposure allows retrieval of proteins from distinct stations along the secretory pathway, as indicated by the changes in O-glycosylation patterns upon over-expression of different partners. The ensuing gradients may help optimising folding and assembly of different cargoes. Endogenous ERp44 is O-glycosylated and secreted by human primary endometrial cells, suggesting possible pathophysiological roles of these processes. PMID:25097228

A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling.

PubMed

Bai, Zhiyong; Grant, Barth D

2015-03-24

Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1-positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42-associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling.

A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling

PubMed Central

Bai, Zhiyong; Grant, Barth D.

2015-01-01

Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1–positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42–associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling. PMID:25775511

Pompe Disease Results in a Golgi-based Glycosylation Deficit in Human Induced Pluripotent Stem Cell-derived Cardiomyocytes*

PubMed Central

Raval, Kunil K.; Tao, Ran; White, Brent E.; De Lange, Willem J.; Koonce, Chad H.; Yu, Junying; Kishnani, Priya S.; Thomson, James A.; Mosher, Deane F.; Ralphe, John C.; Kamp, Timothy J.

2015-01-01

Infantile-onset Pompe disease is an autosomal recessive disorder caused by the complete loss of lysosomal glycogen-hydrolyzing enzyme acid α-glucosidase (GAA) activity, which results in lysosomal glycogen accumulation and prominent cardiac and skeletal muscle pathology. The mechanism by which loss of GAA activity causes cardiomyopathy is poorly understood. We reprogrammed fibroblasts from patients with infantile-onset Pompe disease to generate induced pluripotent stem (iPS) cells that were differentiated to cardiomyocytes (iPSC-CM). Pompe iPSC-CMs had undetectable GAA activity and pathognomonic glycogen-filled lysosomes. Nonetheless, Pompe and control iPSC-CMs exhibited comparable contractile properties in engineered cardiac tissue. Impaired autophagy has been implicated in Pompe skeletal muscle; however, control and Pompe iPSC-CMs had comparable clearance rates of LC3-II-detected autophagosomes. Unexpectedly, the lysosome-associated membrane proteins, LAMP1 and LAMP2, from Pompe iPSC-CMs demonstrated higher electrophoretic mobility compared with control iPSC-CMs. Brefeldin A induced disruption of the Golgi in control iPSC-CMs reproduced the higher mobility forms of the LAMPs, suggesting that Pompe iPSC-CMs produce LAMPs lacking appropriate glycosylation. Isoelectric focusing studies revealed that LAMP2 has a more alkaline pI in Pompe compared with control iPSC-CMs due largely to hyposialylation. MALDI-TOF-MS analysis of N-linked glycans demonstrated reduced diversity of multiantennary structures and the major presence of a trimannose complex glycan precursor in Pompe iPSC-CMs. These data suggest that Pompe cardiomyopathy has a glycan processing abnormality and thus shares features with hypertrophic cardiomyopathies observed in the congenital disorders of glycosylation. PMID:25488666

Role of Conserved Oligomeric Golgi Complex in the Abnormalities of Glycoprotein Processing in Breast Cancer Cells

DTIC Science & Technology

2006-05-01

terminal oligosaccharide units serve as highly specific biological recognition molecules implicated in major regulatory processes of the cell...treatment or mock-treated for 9 days. To study the glycosylation process in COG complex depleted cells series of Pulse -Chase experiments have been...DAMD17-03-1-0243 TITLE: Role of the Conserved Oligomeric Golgi Complex in the Abnormalities of Glycoprotein Processing in Breast Cancer

Vacuolar protein sorting mechanisms in plants.

PubMed

Xiang, Li; Etxeberria, Ed; Van den Ende, Wim

2013-02-01

Plant vacuoles are unique, multifunctional organelles among eukaryotes. Considerable new insights in plant vacuolar protein sorting have been obtained recently. The basic machinery of protein export from the endoplasmic reticulum to the Golgi and the classical route to the lytic vacuole and the protein storage vacuole shows many similarities to vacuolar/lysosomal sorting in other eukaryotes. However, as a result of its unique functions in plant defence and as a storage compartment, some plant-specific entities and sorting determinants appear to exist. The alternative post-Golgi route, as found in animals and yeast, probably exists in plants as well. Likely, adaptor protein complex 3 fulfils a central role in this route. A Golgi-independent route involving plant-specific endoplasmic reticulum bodies appears to provide sedentary organisms such as plants with extra flexibility to cope with changing environmental conditions. © 2012 The Authors Journal compilation © 2012 FEBS.

Stacking the odds for Golgi cisternal maturation

PubMed Central

Mani, Somya; Thattai, Mukund

2016-01-01

What is the minimal set of cell-biological ingredients needed to generate a Golgi apparatus? The compositions of eukaryotic organelles arise through a process of molecular exchange via vesicle traffic. Here we statistically sample tens of thousands of homeostatic vesicle traffic networks generated by realistic molecular rules governing vesicle budding and fusion. Remarkably, the plurality of these networks contain chains of compartments that undergo creation, compositional maturation, and dissipation, coupled by molecular recycling along retrograde vesicles. This motif precisely matches the cisternal maturation model of the Golgi, which was developed to explain many observed aspects of the eukaryotic secretory pathway. In our analysis cisternal maturation is a robust consequence of vesicle traffic homeostasis, independent of the underlying details of molecular interactions or spatial stacking. This architecture may have been exapted rather than selected for its role in the secretion of large cargo. DOI: http://dx.doi.org/10.7554/eLife.16231.001 PMID:27542195

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fontana, Juan; Lopez-Iglesias, Carmen; Tzeng, Wen-Ping

Viral factories are complex structures in the infected cell where viruses compartmentalize their life cycle. Rubella virus (RUBV) assembles factories by recruitment of rough endoplasmic reticulum (RER), mitochondria and Golgi around modified lysosomes known as cytopathic vacuoles or CPVs. These organelles contain active replication complexes that transfer replicated RNA to assembly sites in Golgi membranes. We have studied the structure of RUBV factory in three dimensions by electron tomography and freeze-fracture. CPVs contain stacked membranes, rigid sheets, small vesicles and large vacuoles. These membranes are interconnected and in communication with the endocytic pathway since they incorporate endocytosed BSA-gold. RER andmore » CPVs are coupled through protein bridges and closely apposed membranes. Golgi vesicles attach to the CPVs but no tight contacts with mitochondria were detected. Immunogold labelling confirmed that the mitochondrial protein p32 is an abundant component around and inside CPVs where it could play important roles in factory activities.« less

Atkinesin-13A Modulates Cell-Wall Synthesis and Cell Expansion in Arabidopsis thaliana via the THESEUS1 Pathway

PubMed Central

Fujikura, Ushio; Elsaesser, Lore; Breuninger, Holger; Sánchez-Rodríguez, Clara; Ivakov, Alexander; Laux, Thomas; Findlay, Kim; Persson, Staffan; Lenhard, Michael

2014-01-01

Growth of plant organs relies on cell proliferation and expansion. While an increasingly detailed picture about the control of cell proliferation is emerging, our knowledge about the control of cell expansion remains more limited. We demonstrate here that the internal-motor kinesin AtKINESIN-13A (AtKIN13A) limits cell expansion and cell size in Arabidopsis thaliana, with loss-of-function atkin13a mutants forming larger petals with larger cells. The homolog, AtKINESIN-13B, also affects cell expansion and double mutants display growth, gametophytic and early embryonic defects, indicating a redundant role of the two genes. AtKIN13A is known to depolymerize microtubules and influence Golgi motility and distribution. Consistent with this function, AtKIN13A interacts genetically with ANGUSTIFOLIA, encoding a regulator of Golgi dynamics. Reduced AtKIN13A activity alters cell wall structure as assessed by Fourier-transformed infrared-spectroscopy and triggers signalling via the THESEUS1-dependent cell-wall integrity pathway, which in turn promotes the excess cell expansion in the atkin13a mutant. Thus, our results indicate that the intracellular activity of AtKIN13A regulates cell expansion and wall architecture via THESEUS1, providing a compelling case of interplay between cell wall integrity sensing and expansion. PMID:25232944

Handling Golgi-impregnated tissue for light microscopy.

PubMed

Berbel, P J; Fairén, A

1983-08-08

The use of cyanocrylic glue to fix pieces of Golgi-stained nervous tissue on a paraffin blank is proposed for obtaining thick sections of unembedded tissue with a sliding microtome. This procedure makes correct orientation of the tissue easy during sectioning and makes it possible to obtain tissue sections quickly. The sections are flat-mounted using epoxy resin, resulting in permanent preparations with excellent optical properties and enabling further thin-sectioning for light and electron microscopic studies.

Targeting Midbodies in Ovarian Cancer Stem Cells as a Therapeutic Strategy

DTIC Science & Technology

2013-10-01

functional spindle assembly. Nat. Cell Biol. 13, 1406–1414. Ori-McKenney, K.M., Jan, L.Y., and Jan, Y.-N. (2012). Golgi outposts shape dendrite morphology...1899. Yadav, S., and Linstedt, A.D. (2011). Golgi positioning. Cold Spring Harb. Perspect . Biol. 3, 3. Zhang, H., Squirrell, J.M., and White, J.G. (2008...implications for recy- cling endosome function . This new liaison has additional impli- cations for a variety of biological processes including cilia

Role of the Conserved Oligomeric Golgi Complex in the Abnormalities of Glycoprotein Processing in Breast Cancer Cells

DTIC Science & Technology

2005-05-01

AD Award Number: DAMD17-03-1-0243 TITLE: Role of the Conserved Oligomeric Golgi Complex in the Abnormalities of Glycoprotein Processing in Breast...Glycoprotein Processing in Breast Cancer 5b.GRANTNUMBER Cells DAAD17-03-1-0243 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Sergey N... processing of glycoproteins, exocytosis, protein delivery systems, gene expression, western and northern blot analysis, immunotiuorescence, gradient

In Vivo Intracellular pH Measurements in Tobacco and Arabidopsis Reveal an Unexpected pH Gradient in the Endomembrane System[W

PubMed Central

Martinière, Alexandre; Bassil, Elias; Jublanc, Elodie; Alcon, Carine; Reguera, Maria; Sentenac, Hervé; Blumwald, Eduardo; Paris, Nadine

2013-01-01

The pH homeostasis of endomembranes is essential for cellular functions. In order to provide direct pH measurements in the endomembrane system lumen, we targeted genetically encoded ratiometric pH sensors to the cytosol, the endoplasmic reticulum, and the trans-Golgi, or the compartments labeled by the vacuolar sorting receptor (VSR), which includes the trans-Golgi network and prevacuoles. Using noninvasive live-cell imaging to measure pH, we show that a gradual acidification from the endoplasmic reticulum to the lytic vacuole exists, in both tobacco (Nicotiana tabacum) epidermal (ΔpH −1.5) and Arabidopsis thaliana root cells (ΔpH −2.1). The average pH in VSR compartments was intermediate between that of the trans-Golgi and the vacuole. Combining pH measurements with in vivo colocalization experiments, we found that the trans-Golgi network had an acidic pH of 6.1, while the prevacuole and late prevacuole were both more alkaline, with pH of 6.6 and 7.1, respectively. We also showed that endosomal pH, and subsequently vacuolar trafficking of soluble proteins, requires both vacuolar-type H+ ATPase–dependent acidification as well as proton efflux mediated at least by the activity of endosomal sodium/proton NHX-type antiporters. PMID:24104564

Dual pulse-chase microscopy reveals early divergence in the biosynthetic trafficking of the Na,K-ATPase and E-cadherin

PubMed Central

Farr, Glen A.; Hull, Michael; Stoops, Emily H.; Bateson, Rosalie; Caplan, Michael J.

2015-01-01

Recent evidence indicates that newly synthesized membrane proteins that share the same distributions in the plasma membranes of polarized epithelial cells can pursue a variety of distinct trafficking routes as they travel from the Golgi complex to their common destination at the cell surface. In most polarized epithelial cells, both the Na,K-ATPase and E-cadherin are localized to the basolateral domains of the plasma membrane. To examine the itineraries pursued by newly synthesized Na,K-ATPase and E-cadherin in polarized MDCK epithelial cells, we used the SNAP and CLIP labeling systems to fluorescently tag temporally defined cohorts of these proteins and observe their behaviors simultaneously as they traverse the secretory pathway. These experiments reveal that E-cadherin is delivered to the cell surface substantially faster than is the Na,K-ATPase. Furthermore, the surface delivery of newly synthesized E-cadherin to the plasma membrane was not prevented by the 19°C temperature block that inhibits the trafficking of most proteins, including the Na,K-ATPase, out of the trans-Golgi network. Consistent with these distinct behaviors, populations of newly synthesized E-cadherin and Na,K-ATPase become separated from one another within the trans-Golgi network, suggesting that they are sorted into different carrier vesicles that mediate their post-Golgi trafficking. PMID:26424804

The BOS1 gene encodes an essential 27-kD putative membrane protein that is required for vesicular transport from the ER to the Golgi complex in yeast

PubMed Central

1991-01-01

We recently described the identification of BOS1 (Newman, A., J. Shim, and S. Ferro-Novick. 1990. Mol. Cell. Biol. 10:3405-3414.). BOS1 is a gene that in multiple copy suppresses the growth and secretion defect of bet1 and sec22, two mutants that disrupt transport from the ER to the Golgi complex in yeast. The ability of BOS1 to specifically suppress mutants blocked at a particular stage of the secretory pathway suggested that this gene encodes a protein that functions in this process. The experiments presented in this study support this hypothesis. Specifically, the BOS1 gene was found to be essential for cellular growth. Furthermore, cells depleted of the Bos1 protein fail to transport pro-alpha-factor and carboxypeptidase Y (CPY) to the Golgi apparatus. This defect in export leads to the accumulation of an extensive network of ER and small vesicles. DNA sequence analysis predicts that Bos1 is a 27-kD protein containing a putative membrane- spanning domain. This prediction is supported by differential centrifugation experiments. Thus, Bos1 appears to be a membrane protein that functions in conjunction with Bet1 and Sec22 to facilitate the transport of proteins at a step subsequent to translocation into the ER but before entry into the Golgi apparatus. PMID:2007627

Genetic, structural, and chemical insights into the dual function of GRASP55 in germ cell Golgi remodeling and JAM-C polarized localization during spermatogenesis.

PubMed

Cartier-Michaud, Amandine; Bailly, Anne-Laure; Betzi, Stéphane; Shi, Xiaoli; Lissitzky, Jean-Claude; Zarubica, Ana; Sergé, Arnauld; Roche, Philippe; Lugari, Adrien; Hamon, Véronique; Bardin, Florence; Derviaux, Carine; Lembo, Frédérique; Audebert, Stéphane; Marchetto, Sylvie; Durand, Bénédicte; Borg, Jean-Paul; Shi, Ning; Morelli, Xavier; Aurrand-Lions, Michel

2017-06-01

Spermatogenesis is a dynamic process that is regulated by adhesive interactions between germ and Sertoli cells. Germ cells express the Junctional Adhesion Molecule-C (JAM-C, encoded by Jam3), which localizes to germ/Sertoli cell contacts. JAM-C is involved in germ cell polarity and acrosome formation. Using a proteomic approach, we demonstrated that JAM-C interacted with the Golgi reassembly stacking protein of 55 kDa (GRASP55, encoded by Gorasp2) in developing germ cells. Generation and study of Gorasp2-/- mice revealed that knock-out mice suffered from spermatogenesis defects. Acrosome formation and polarized localization of JAM-C in spermatids were altered in Gorasp2-/- mice. In addition, Golgi morphology of spermatocytes was disturbed in Gorasp2-/- mice. Crystal structures of GRASP55 in complex with JAM-C or JAM-B revealed that GRASP55 interacted via PDZ-mediated interactions with JAMs and induced a conformational change in GRASP55 with respect of its free conformation. An in silico pharmacophore approach identified a chemical compound called Graspin that inhibited PDZ-mediated interactions of GRASP55 with JAMs. Treatment of mice with Graspin hampered the polarized localization of JAM-C in spermatids, induced the premature release of spermatids and affected the Golgi morphology of meiotic spermatocytes.

Involvement of the Golgi apparatus in the synthesis and secretion of hydroxyproline-rich cell wall glycoproteins. [carrot

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardiner, M.; Chrispeels, M.J.

1975-01-01

Pulse labeling of carrot root phloem parenchyma (Daucus carota L. ev. Nantes) tissue with /sup 14/C-proline followed by fractionation of the cytoplasmic organelles on sucrose gradients was used to determine the identiy of the membranous organelles involved in the secretion of the hydroxyproline-rich glycoproteins of the cell wall. Identification of the organelles was done through electron-microscopical observations and through the localization of marker enzymes on the sucrose gradients. Enrichment of the organelles involved in secretion was determined by measuring the percentage of the incorporated radioactivity present as /sup 14/C-hydroxyproline. The Golgi apparatus (dictyosome) was found to be a major sitemore » of glycoprotein transport. This identification was based on the observed enrichment of dictyosomes paralleling the purification of newly synthesized cell-wall glycoproteins. A marker enzyme for the Golgi apparatus, inosinediphosphatase, banded with the newly synthesized cell wall glycoproteins on sequential isopycnic and rate zonal sucrose gradients. Marker enzymes for the endoplasmic reticulum and the plasma memebrane were clearly separated from the dictyosome-rich fraction. UDP-arabinose arabinosyl transferase, an enzyme involved in the glycosylation of the peptide moiety of this glycoprotein, also banded with the dictyosomes on both kinds of gradients. The results suggest an important role of the Golgi apparatus in the biosynthesis and the secretion of the cell wall glycoproteins of higher plants. (auth)« less

Neutral sphingomyelinase (SMPD3) deficiency disrupts the Golgi secretory pathway and causes growth inhibition

PubMed Central

Stoffel, Wilhelm; Hammels, Ina; Jenke, Bitta; Binczek, Erika; Schmidt-Soltau, Inga; Brodesser, Susanne; Schauss, Astrid; Etich, Julia; Heilig, Juliane; Zaucke, Frank

2016-01-01

Systemic loss of neutral sphingomyelinase (SMPD3) in mice leads to a novel form of systemic, juvenile hypoplasia (dwarfism). SMPD3 deficiency in mainly two growth regulating cell types contributes to the phenotype, in chondrocytes of skeletal growth zones to skeletal malformation and chondrodysplasia, and in hypothalamic neurosecretory neurons to systemic hypothalamus–pituitary–somatotropic hypoplasia. The unbiased smpd3−/− mouse mutant and derived smpd3−/− primary chondrocytes were instrumental in defining the enigmatic role underlying the systemic and cell autonomous role of SMPD3 in the Golgi compartment. Here we describe the unprecedented role of SMPD3. SMPD3 deficiency disrupts homeostasis of sphingomyelin (SM), ceramide (Cer) and diacylglycerol (DAG) in the Golgi SMPD3-SMS1 (SM-synthase1) cycle. Cer and DAG, two fusogenic intermediates, modify the membrane lipid bilayer for the initiation of vesicle formation and transport. Dysproteostasis, unfolded protein response, endoplasmic reticulum stress and apoptosis perturb the Golgi secretory pathway in the smpd3−/− mouse. Secretion of extracellular matrix proteins is arrested in chondrocytes and causes skeletal malformation and chondrodysplasia. Similarly, retarded secretion of proteo-hormones in hypothalamic neurosecretory neurons leads to hypothalamus induced combined pituitary hormone deficiency. SMPD3 in the regulation of the protein vesicular secretory pathway may become a diagnostic target in the etiology of unknown forms of juvenile growth and developmental inhibition. PMID:27882938

Golgi Traffic and Integrity Depend on N-Myristoyl Transferase-1 in Arabidopsis[W

PubMed Central

Renna, Luciana; Stefano, Giovanni; Majeran, Wojciech; Micalella, Chiara; Meinnel, Thierry; Giglione, Carmela; Brandizzi, Federica

2013-01-01

N-myristoylation is a crucial irreversible eukaryotic lipid modification allowing a key subset of proteins to be targeted at the periphery of specific membrane compartments. Eukaryotes have conserved N-myristoylation enzymes, involving one or two N-myristoyltransferases (NMT1 and NMT2), among which NMT1 is the major enzyme. In the postembryonic developmental stages, defects in NMT1 lead to aberrant cell polarity, flower differentiation, fruit maturation, and innate immunity; however, no specific NMT1 target responsible for such deficiencies has hitherto been identified. Using a confocal microscopy forward genetics screen for the identification of Arabidopsis thaliana secretory mutants, we isolated STINGY, a recessive mutant with defective Golgi traffic and integrity. We mapped STINGY to a substitution at position 160 of Arabidopsis NMT1 (NMT1A160T). In vitro kinetic studies with purified NMT1A160T enzyme revealed a significant reduction in its activity due to a remarkable decrease in affinity for both myristoyl-CoA and peptide substrates. We show here that this recessive mutation is responsible for the alteration of Golgi traffic and integrity by predominantly affecting the Golgi membrane/cytosol partitioning of ADP-ribosylation factor proteins. Our results provide important functional insight into N-myristoylation in plants by ascribing postembryonic functions of Arabidopsis NMT1 that involve regulation of the functional and morphological integrity of the plant endomembranes. PMID:23673980

Involvement of complex sphingolipids and phosphatidylserine in endosomal trafficking in yeast Saccharomyces cerevisiae.

PubMed

Tani, Motohiro; Kuge, Osamu

2012-12-01

Sphingolipids play critical roles in many physiologically important events in the yeast Saccharomyces cerevisiae. In this study, we found that csg2Δ mutant cells defective in the synthesis of mannosylinositol phosphorylceramide exhibited abnormal intracellular accumulation of an exocytic v-SNARE, Snc1, under phosphatidylserine synthase gene (PSS1)-repressive conditions, although in wild-type cells, Snc1 was known to cycle between plasma membranes and the late Golgi via post-Golgi endosomes. The mislocalized Snc1 was co-localized with an endocytic marker dye, FM4-64, upon labelling for a short time. The abnormal distribution of Snc1 was suppressed by deletion of GYP2 encoding a GTPase-activating protein that negatively regulates endosomal vesicular trafficking, or expression of GTP-restricted form of Ypt32 GTPase. Furthermore, an endocytosis-deficient mutant of Snc1 was localized to plasma membranes in PSS1-repressed csg2Δ mutant cells as well as wild-type cells. Thus, the PSS1-repressed csg2Δ mutant cells were indicated to be defective in the trafficking of Snc1 from post-Golgi endosomes to the late Golgi. In contrast, the vesicular trafficking pathways via pre-vacuolar endosomes in the PSS1-repressed csg2Δ mutant cells seemed to be normal. These results suggested that specific complex sphingolipids and phosphatidylserine are co-ordinately involved in specific vesicular trafficking pathway. © 2012 Blackwell Publishing Ltd.

Neutral sphingomyelinase (SMPD3) deficiency disrupts the Golgi secretory pathway and causes growth inhibition.

PubMed

Stoffel, Wilhelm; Hammels, Ina; Jenke, Bitta; Binczek, Erika; Schmidt-Soltau, Inga; Brodesser, Susanne; Schauss, Astrid; Etich, Julia; Heilig, Juliane; Zaucke, Frank

2016-11-24

Systemic loss of neutral sphingomyelinase (SMPD3) in mice leads to a novel form of systemic, juvenile hypoplasia (dwarfism). SMPD3 deficiency in mainly two growth regulating cell types contributes to the phenotype, in chondrocytes of skeletal growth zones to skeletal malformation and chondrodysplasia, and in hypothalamic neurosecretory neurons to systemic hypothalamus-pituitary-somatotropic hypoplasia. The unbiased smpd3-/- mouse mutant and derived smpd3-/- primary chondrocytes were instrumental in defining the enigmatic role underlying the systemic and cell autonomous role of SMPD3 in the Golgi compartment. Here we describe the unprecedented role of SMPD3. SMPD3 deficiency disrupts homeostasis of sphingomyelin (SM), ceramide (Cer) and diacylglycerol (DAG) in the Golgi SMPD3-SMS1 (SM-synthase1) cycle. Cer and DAG, two fusogenic intermediates, modify the membrane lipid bilayer for the initiation of vesicle formation and transport. Dysproteostasis, unfolded protein response, endoplasmic reticulum stress and apoptosis perturb the Golgi secretory pathway in the smpd3-/- mouse. Secretion of extracellular matrix proteins is arrested in chondrocytes and causes skeletal malformation and chondrodysplasia. Similarly, retarded secretion of proteo-hormones in hypothalamic neurosecretory neurons leads to hypothalamus induced combined pituitary hormone deficiency. SMPD3 in the regulation of the protein vesicular secretory pathway may become a diagnostic target in the etiology of unknown forms of juvenile growth and developmental inhibition.

Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

PubMed Central

Hoang, Huy-Dung; Maruyama, Jun-ichi

2014-01-01

Filamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, using Aspergillus oryzae as a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors, A. oryzae Vip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme α-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of α-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi. PMID:25362068

Control systems and coordination protocols of the secretory pathway.

PubMed

Luini, Alberto; Mavelli, Gabriella; Jung, Juan; Cancino, Jorge

2014-01-01

Like other cellular modules, the secretory pathway and the Golgi complex are likely to be supervised by control systems that support homeostasis and optimal functionality under all conditions, including external and internal perturbations. Moreover, the secretory apparatus must be functionally connected with other cellular modules, such as energy metabolism and protein degradation, via specific rules of interaction, or "coordination protocols". These regulatory devices are of fundamental importance for optimal function; however, they are generally "hidden" at steady state. The molecular components and the architecture of the control systems and coordination protocols of the secretory pathway are beginning to emerge through studies based on the use of controlled transport-specific perturbations aimed specifically at the detection and analysis of these internal regulatory devices.

Role of Autism Susceptibility Gene, CNTNAP2, in Neural Circuitry for Vocal Communication

DTIC Science & Technology

2013-10-01

Grimbert et al., 2003), and ATP2C2 is part of a pathway responsible for shuttling divalent ions to the Golgi apparatus (Faddy et al., 2008...L., & Wuytack, F. (2007). Calcium in the Golgi apparatus . Cell Calcium, 41(5), 405–416. doi:10.1016/j. ceca.2006.11.001 Mizutani, A., Matsuzaki, A...determination. Nature Structural & Molecular Biology, 16(4), 365–371. doi:10.1038/nsmb.1576 This is a contribution from New Perspectives on the Origins

Lower numbers of mechanoreceptors in the posterior cruciate ligament and anterior capsule of the osteoarthritic knees.

PubMed

Çabuk, Haluk; Kuşku Çabuk, Fatmagül; Tekin, Ali Çağrı; Dedeoğlu, Süleyman Semih; Çakar, Murat; Büyükkurt, Cem Dinçay

2017-10-01

Impaired proprioception accuracy of the knee has been proposed as a local factor in the onset and progression of knee osteoarthritis. Patients with decreased numbers of mechanoreceptors could be more likely to develop arthrosis due to a loss in proprioception of the joint. We aimed to identify and quantify the mechanoreceptors of the posterior cruciate ligament (PCL), the anterior capsule (AC) and the medial meniscocapsular junction (MCJ) in knee arthrosis. PCLs, ACs and MCJs were harvested from 30 patients with Kellgren and Lawrence grades 3 and 4 osteoarthritis (OA), and ten knees taken from five cadavers without OA were used as a control group. PCL degeneration was evaluated with haematoxylin & eosin, and the types and numbers of mechanoreceptors were evaluated using S100 immunostaining. The patient ages in the OA and control groups (n.s.) did not differ. PCL degeneration was more severe in the gonarthrosis group than in the control group (p = 0.04). The numbers of Golgi corpuscles, Ruffini corpuscles, free nerve endings, total nerve endings and small vessels of the PCL were low in the OA group, as were the numbers of Golgi corpuscles, free nerve endings and total nerve endings of the AC. No significant correlation was found regarding the mechanoreceptors of the MCJ between the two groups. The numbers of mechanoreceptors in patients with OA were low in the PCLs and ACs. A loss in proprioception could be a local risk factor in OA. The proprioceptive impact of preserving PCL while performing total knee arthroplasty may not be exaggerated as its thought. Prognostic study, Level I.

SAP97-mediated ADAM10 trafficking from Golgi outposts depends on PKC phosphorylation

PubMed Central

Saraceno, C; Marcello, E; Di Marino, D; Borroni, B; Claeysen, S; Perroy, J; Padovani, A; Tramontano, A; Gardoni, F; Di Luca, M

2014-01-01

A disintegrin and metalloproteinase 10 (ADAM10) is the major α-secretase that catalyzes the amyloid precursor protein (APP) ectodomain shedding in the brain and prevents amyloid formation. Its activity depends on correct intracellular trafficking and on synaptic membrane insertion. Here, we describe that in hippocampal neurons the synapse-associated protein-97 (SAP97), an excitatory synapse scaffolding element, governs ADAM10 trafficking from dendritic Golgi outposts to synaptic membranes. This process is mediated by a previously uncharacterized protein kinase C phosphosite in SAP97 SRC homology 3 domain that modulates SAP97 association with ADAM10. Such mechanism is essential for ADAM10 trafficking from the Golgi outposts to the synapse, but does not affect ADAM10 transport from the endoplasmic reticulum. Notably, this process is altered in Alzheimer's disease brains. These results help in understanding the mechanism responsible for the modulation of ADAM10 intracellular path, and can constitute an innovative therapeutic strategy to finely tune ADAM10 shedding activity towards APP. PMID:25429624

Role of tetanus neurotoxin insensitive vesicle-associated membrane protein in membrane domains transport and homeostasis

PubMed Central

Molino, Diana; Nola, Sébastien; Lam, Sin Man; Verraes, Agathe; Proux-Gillardeaux, Véronique; Boncompain, Gaëlle; Perez, Franck; Wenk, Markus; Shui, Guanghou; Danglot, Lydia; Galli, Thierry

2015-01-01

Biological membranes in eukaryotes contain a large variety of proteins and lipids often distributed in domains in plasma membrane and endomembranes. Molecular mechanisms responsible for the transport and the organization of these membrane domains along the secretory pathway still remain elusive. Here we show that vesicular SNARE TI-VAMP/VAMP7 plays a major role in membrane domains composition and transport. We found that the transport of exogenous and endogenous GPI-anchored proteins was altered in fibroblasts isolated from VAMP7-knockout mice. Furthermore, disassembly and reformation of the Golgi apparatus induced by Brefeldin A treatment and washout were impaired in VAMP7-depleted cells, suggesting that loss of VAMP7 expression alters biochemical properties and dynamics of the Golgi apparatus. In addition, lipid profiles from these knockout cells indicated a defect in glycosphingolipids homeostasis. We conclude that VAMP7 is required for effective transport of GPI–anchored proteins to cell surface and that VAMP7-dependent transport contributes to both sphingolipids and Golgi homeostasis. PMID:26196023

System-wide organization of actin cytoskeleton determines organelle transport in hypocotyl plant cells

PubMed Central

Nowak, Jacqueline; Ivakov, Alexander; Somssich, Marc; Persson, Staffan; Nikoloski, Zoran

2017-01-01

The actin cytoskeleton is an essential intracellular filamentous structure that underpins cellular transport and cytoplasmic streaming in plant cells. However, the system-level properties of actin-based cellular trafficking remain tenuous, largely due to the inability to quantify key features of the actin cytoskeleton. Here, we developed an automated image-based, network-driven framework to accurately segment and quantify actin cytoskeletal structures and Golgi transport. We show that the actin cytoskeleton in both growing and elongated hypocotyl cells has structural properties facilitating efficient transport. Our findings suggest that the erratic movement of Golgi is a stable cellular phenomenon that might optimize distribution efficiency of cell material. Moreover, we demonstrate that Golgi transport in hypocotyl cells can be accurately predicted from the actin network topology alone. Thus, our framework provides quantitative evidence for system-wide coordination of cellular transport in plant cells and can be readily applied to investigate cytoskeletal organization and transport in other organisms. PMID:28655850

3D Printing of Plant Golgi Stacks from Their Electron Tomographic Models.

PubMed

Mai, Keith Ka Ki; Kang, Madison J; Kang, Byung-Ho

2017-01-01

Three-dimensional (3D) printing is an effective tool for preparing tangible 3D models from computer visualizations to assist in scientific research and education. With the recent popularization of 3D printing processes, it is now possible for individual laboratories to convert their scientific data into a physical form suitable for presentation or teaching purposes. Electron tomography is an electron microscopy method by which 3D structures of subcellular organelles or macromolecular complexes are determined at nanometer-level resolutions. Electron tomography analyses have revealed the convoluted membrane architectures of Golgi stacks, chloroplasts, and mitochondria. But the intricacy of their 3D organizations is difficult to grasp from tomographic models illustrated on computer screens. Despite the rapid development of 3D printing technologies, production of organelle models based on experimental data with 3D printing has rarely been documented. In this chapter, we present a simple guide to creating 3D prints of electron tomographic models of plant Golgi stacks using the two most accessible 3D printing technologies.

Structural Basis for the Interaction of the Golgi-Associated Retrograde Protein (GARP) Complex with the t-SNARE Syntaxin 6

PubMed Central

Abascal-Palacios, Guillermo; Schindler, Christina; Rojas, Adriana L; Bonifacino, Juan S.; Hierro, Aitor

2016-01-01

Summary The Golgi-Associated Retrograde Protein (GARP) is a tethering complex involved in the fusion of endosome-derived transport vesicles to the trans-Golgi network through interaction with components of the Syntaxin 6/Syntaxin 16/Vti1a/VAMP4 SNARE complex. The mechanisms by which GARP and other tethering factors engage the SNARE fusion machinery are poorly understood. Herein we report the structural basis for the interaction of the human Ang2 subunit of GARP with Syntaxin 6 and the closely related Syntaxin 10. The crystal structure of Syntaxin 6 Habc domain in complex with a peptide from the N terminus of Ang2 shows a novel binding mode in which a di-tyrosine motif of Ang2 interacts with a highly conserved groove in Syntaxin 6. Structure-based mutational analyses validate the crystal structure and support the phylogenetic conservation of this interaction. The same binding determinants are found in other tethering proteins and syntaxins, suggesting a general interaction mechanism. PMID:23932592

Herpes simplex virus 1 induces de novo phospholipid synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sutter, Esther; Oliveira, Anna Paula de; Tobler, Kurt

2012-08-01

Herpes simplex virus type 1 capsids bud at nuclear membranes and Golgi membranes acquiring an envelope composed of phospholipids. Hence, we measured incorporation of phospholipid precursors into these membranes, and quantified changes in size of cellular compartments by morphometric analysis. Incorporation of [{sup 3}H]-choline into both nuclear and cytoplasmic membranes was significantly enhanced upon infection. [{sup 3}H]-choline was also part of isolated virions even grown in the presence of brefeldin A. Nuclei expanded early in infection. The Golgi complex and vacuoles increased substantially whereas the endoplasmic reticulum enlarged only temporarily. The data suggest that HSV-1 stimulates phospholipid synthesis, and thatmore » de novo synthesized phospholipids are inserted into nuclear and cytoplasmic membranes to i) maintain membrane integrity in the course of nuclear and cellular expansion, ii) to supply membrane constituents for envelopment of capsids by budding at nuclear membranes and Golgi membranes, and iii) to provide membranes for formation of transport vacuoles.« less

A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface.

PubMed

Buser, Dominik P; Schleicher, Kai D; Prescianotto-Baschong, Cristina; Spiess, Martin

2018-06-18

Retrograde transport of membranes and proteins from the cell surface to the Golgi and beyond is essential to maintain homeostasis, compartment identity, and physiological functions. To study retrograde traffic biochemically, by live-cell imaging or by electron microscopy, we engineered functionalized anti-GFP nanobodies (camelid VHH antibody domains) to be bacterially expressed and purified. Tyrosine sulfation consensus sequences were fused to the nanobody for biochemical detection of trans -Golgi arrival, fluorophores for fluorescence microscopy and live imaging, and APEX2 (ascorbate peroxidase 2) for electron microscopy and compartment ablation. These functionalized nanobodies are specifically captured by GFP-modified reporter proteins at the cell surface and transported piggyback to the reporters' homing compartments. As an application of this tool, we have used it to determine the contribution of adaptor protein-1/clathrin in retrograde transport kinetics of the mannose-6-phosphate receptors from endosomes back to the trans -Golgi network. Our experiments establish functionalized nanobodies as a powerful tool to demonstrate and quantify retrograde transport pathways.

Unconventional secretory processing diversifies neuronal ion channel properties

PubMed Central

Hanus, Cyril; Geptin, Helene; Tushev, Georgi; Garg, Sakshi; Alvarez-Castelao, Beatriz; Sambandan, Sivakumar; Kochen, Lisa; Hafner, Anne-Sophie; Langer, Julian D; Schuman, Erin M

2016-01-01

N-glycosylation – the sequential addition of complex sugars to adhesion proteins, neurotransmitter receptors, ion channels and secreted trophic factors as they progress through the endoplasmic reticulum and the Golgi apparatus – is one of the most frequent protein modifications. In mammals, most organ-specific N-glycosylation events occur in the brain. Yet, little is known about the nature, function and regulation of N-glycosylation in neurons. Using imaging, quantitative immunoblotting and mass spectrometry, we show that hundreds of neuronal surface membrane proteins are core-glycosylated, resulting in the neuronal membrane displaying surprisingly high levels of glycosylation profiles that are classically associated with immature intracellular proteins. We report that while N-glycosylation is generally required for dendritic development and glutamate receptor surface expression, core-glycosylated proteins are sufficient to sustain these processes, and are thus functional. This atypical glycosylation of surface neuronal proteins can be attributed to a bypass or a hypo-function of the Golgi apparatus. Core-glycosylation is regulated by synaptic activity, modulates synaptic signaling and accelerates the turnover of GluA2-containing glutamate receptors, revealing a novel mechanism that controls the composition and sensing properties of the neuronal membrane. DOI: http://dx.doi.org/10.7554/eLife.20609.001 PMID:27677849

Functional significance of differential eNOS translocation

PubMed Central

Sánchez, Fabiola A.; Savalia, Nirav B.; Durán, Ricardo G.; Lal, Brajesh K.; Boric, Mauricio P.; Durán, Walter N.

2006-01-01

Nitric oxide (NO) regulates flow and permeability. ACh and platelet-activating factor (PAF) lead to endothelial NO synthase (eNOS) phosphorylation and NO release. While ACh causes only vasodilation, PAF induces vasoconstriction and hyperpermeability. The key differential signaling mechanisms for discriminating between vasodilation and hyperpermeability are unknown. We tested the hypothesis that differential translocation may serve as a regulatory mechanism of eNOS to determine specific vascular responses. We used ECV-304 cells permanently transfected with eNOS-green fluorescent protein (ECVeNOS-GFP) and demonstrated that the agonists activate eNOS and reproduce their characteristic endothelial permeability effects in these cells. We evaluated eNOS localization by lipid raft analysis and immunofluorescence microscopy. After PAF and ACh, eNOS moves away from caveolae. eNOS distributes both in the plasma membrane and Golgi in control cells. ACh (10−5 M, 10−4 M) translocated eNOS preferentially to the trans-Golgi network (TGN) and PAF (10−7 M) preferentially to the cytosol. We suggest that PAF-induced eNOS translocation preferentially to cytosol reflects a differential signaling mechanism related to changes in permeability, whereas ACh-induced eNOS translocation to the TGN is related to vasodilation. PMID:16679407

G protein βγ complex translocation from plasma membrane to Golgi complex is influenced by receptor γ subunit interaction

PubMed Central

Akgoz, Muslum; Kalyanaraman, Vani; Gautam, N.

2008-01-01

On activation of a receptor the G protein βγ complex translocates away from the receptor on the plasma membrane to the Golgi complex. The rate of translocation is influenced by the type of γ subunit associated with the G protein. Complementary approaches — imaging living cells expressing fluorescent protein tagged G proteins and assaying reconstituted receptors and G proteins in vitro — were used to identify mechanisms at the basis of the translocation process. Translocation of Gβγ containing mutant γ subunits with altered prenyl moieties showed that the differences in the prenyl moieties were not sufficient to explain the differential effects of geranylgeranylated γ5 and farnesylated γ11 on the translocation process. The translocation properties of Gβγ were altered dramatically by mutating the C terminal tail region of the γ subunit. The translocation characteristics of these mutants suggest that after receptor activation, Gβγ retains contact with a receptor through the γ subunit C terminal domain and that differential interaction of the activated receptor with this domain controls Gβγ translocation from the plasma membrane. PMID:16517125

Functional analysis of the Arabidopsis PHT4 family of intracellular phosphate transporters.

PubMed

Guo, B; Jin, Y; Wussler, C; Blancaflor, E B; Motes, C M; Versaw, W K

2008-01-01

The transport of phosphate (Pi) between subcellular compartments is central to metabolic regulation. Although some of the transporters involved in controlling the intracellular distribution of Pi have been identified in plants, others are predicted from genetic, biochemical and bioinformatics studies. Heterologous expression in yeast, and gene expression and localization in plants were used to characterize all six members of an Arabidopsis thaliana membrane transporter family designated here as PHT4. PHT4 proteins share similarity with SLC17/type I Pi transporters, a diverse group of animal proteins involved in the transport of Pi, organic anions and chloride. All of the PHT4 proteins mediate Pi transport in yeast with high specificity. Bioinformatic analysis and localization of PHT4-GFP fusion proteins indicate that five of the proteins are targeted to the plastid envelope, and the sixth resides in the Golgi apparatus. PHT4 genes are expressed in both roots and leaves, although two of the genes are expressed predominantly in leaves and one mostly in roots. These expression patterns, together with Pi transport activities and subcellular locations, suggest roles for PHT4 proteins in the transport of Pi between the cytosol and chloroplasts, heterotrophic plastids and the Golgi apparatus.

A Novel Pulse-Chase Paradigm to Visualize the Trafficking of Transport Vesicles in Neurons

NASA Astrophysics Data System (ADS)

Al-Bassam, Sarmad

In neurons transmembrane proteins are targeted to dendrites in vesicles that traffic solely within the somatodendritic compartment. How these vesicles are retained within the somatodendritic domain is unknown. Here we adapt a novel pulse chase system that allows synchronous release of exogenous transmembrane proteins from the endoplasmic reticulum using FKBP12 and Rapamycin. We demonstrate proof-of-concept and establish protein trafficking controls in incremental steps. We demonstrate the utility of this approach in studying protein trafficking and establish parameters for analysis of time-lapse images. We implement this novel pulse-chase strategy to track the movements of post-Golgi transport vesicles. Surprisingly, we found that post-Golgi vesicles carrying dendritic proteins were equally likely to enter axons and dendrites. However, once such vesicles entered the axon they very rarely moved beyond the axon initial segment, but instead either halted or reversed direction in an actin and Myosin Va-dependent manner. In contrast, vesicles carrying either an axonal or a nonspecifically localized protein only rarely halted or reversed and instead generally proceeded to the distal axon. Thus, our results are consistent with the axon initial segment behaving as a vesicle filter that mediates the differential trafficking of transport vesicles.

E-cadherin transport from the trans-Golgi network in tubulovesicular carriers is selectively regulated by golgin-97.

PubMed

Lock, John G; Hammond, Luke A; Houghton, Fiona; Gleeson, Paul A; Stow, Jennifer L

2005-12-01

E-cadherin is a cell-cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membrane-bound carriers for the post-Golgi exocytosis of E-cadherin have not been characterized. Green fluorescent protein (GFP)-tagged E-cadherin (Ecad-GFP) is transported from the trans-Golgi network (TGN) to the recycling endosome on its way to the cell surface in tubulovesicular carriers that resemble TGN tubules labeled by members of the golgin family of tethering proteins. Here, we examine the association of golgins with tubular carriers containing E-cadherin as cargo. Fluorescent GRIP domains from golgin proteins replicate the membrane binding of the full-length proteins and were coexpressed with Ecad-GFP. The GRIP domains of p230/golgin-245 and golgin-97 had overlapping but nonidentical distributions on the TGN; both domains were on TGN-derived tubules but only the golgin-97 GRIP domain coincided with Ecad-GFP tubules in live cells. When the Arl1-binding endogenous golgins, p230/golgin-245 and golgin-97 were displaced from Golgi membranes by overexpression of the p230 GRIP domain, trafficking of Ecad-GFP was inhibited. siRNA knockdown of golgin-97 also inhibited trafficking of Ecad-GFP. Thus, the GRIP domains of p230/golgin-245 and golgin-97 bind discriminately to distinct membrane subdomains of the TGN. Golgin-97 is identified as a selective and essential component of the tubulovesicular carriers transporting E-cadherin out of the TGN.

UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage

DOE PAGES

Saez-Aguayo, Susana; Rautengarten, Carsten; Temple, Henry; ...

2017-01-01

UDP-glucuronic acid (UDP-GlcA) is the precursor of many plant cell wall polysaccharides and is required for production of seed mucilage. Following synthesis in the cytosol, it is transported into the lumen of the Golgi apparatus, where it is converted to UDP-galacturonic acid (UDP-GalA), UDP-arabinose, and UDP-xylose. To identify the Golgi-localized UDP-GlcA transporter, we screened Arabidopsis thaliana mutants in genes coding for putative nucleotide sugar transporters for altered seed mucilage, a structure rich in the GalA-containing polysaccharide rhamnogalacturonan I. As a result, we identified UUAT1, which encodes a Golgi-localized protein that transports UDP-GlcA and UDP-GalA in vitro. The seed coat ofmore » uuat1 mutants had less GalA, rhamnose, and xylose in the soluble mucilage, and the distal cell walls had decreased arabinan content. Cell walls of other organs and cells had lower arabinose levels in roots and pollen tubes, but no differences were observed in GalA or xylose contents. Furthermore, the GlcA content of glucuronoxylan in the stem was not affected in the mutant. Interestingly, the degree of homogalacturonan methylation increased in uuat1. These results suggest that this UDP-GlcA transporter plays a key role defining the seed mucilage sugar composition and that its absence produces pleiotropic effects in this component of the plant extracellular matrix.« less

UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saez-Aguayo, Susana; Rautengarten, Carsten; Temple, Henry

UDP-glucuronic acid (UDP-GlcA) is the precursor of many plant cell wall polysaccharides and is required for production of seed mucilage. Following synthesis in the cytosol, it is transported into the lumen of the Golgi apparatus, where it is converted to UDP-galacturonic acid (UDP-GalA), UDP-arabinose, and UDP-xylose. To identify the Golgi-localized UDP-GlcA transporter, we screened Arabidopsis thaliana mutants in genes coding for putative nucleotide sugar transporters for altered seed mucilage, a structure rich in the GalA-containing polysaccharide rhamnogalacturonan I. As a result, we identified UUAT1, which encodes a Golgi-localized protein that transports UDP-GlcA and UDP-GalA in vitro. The seed coat ofmore » uuat1 mutants had less GalA, rhamnose, and xylose in the soluble mucilage, and the distal cell walls had decreased arabinan content. Cell walls of other organs and cells had lower arabinose levels in roots and pollen tubes, but no differences were observed in GalA or xylose contents. Furthermore, the GlcA content of glucuronoxylan in the stem was not affected in the mutant. Interestingly, the degree of homogalacturonan methylation increased in uuat1. These results suggest that this UDP-GlcA transporter plays a key role defining the seed mucilage sugar composition and that its absence produces pleiotropic effects in this component of the plant extracellular matrix.« less

Golgi, electron-microscopic and combined Golgi-electron-microscopic studies of the mitral cells in the goldfish olfactory bulb.

PubMed

Oka, Y

1983-04-01

The local neuronal circuitry of goldfish olfactory bulb was analyzed in Golgi preparations combining light- and electron-microscopy, as well as in routinely prepared ultrastructural preparations. Mitral cells were identified with the light-microscope in Golgi-impregnated thick sections according to the following criteria: (1) cell bodies were distributed irregularly in a wide layer between 100 and 200 micrometer from the surface, (2) cell bodies were larger than other neurons (10-20 micrometer in diameter), and (3) the dendrites were directed toward the superficially-located olfactory nerve layer where they ended as highly branched glomerular tufts. These impregnated cells were examined by electron-microscopy in serial section. The results demonstrate synaptic organization in relation to the mitral cells. (1) Glomerular tufts received afferent input from primary olfactory axons which made Gray's Type I synaptic contacts. These dendrites also had reciprocal dendrodendritic synapses with dendrites of certain non-mitral cells. (2) Dendritic shafts of mitral cells made reciprocal dendritic synapses with dendrites of certain non-mitral cells. (3) Cell bodies and their initial axon segments had reciprocal synapses with certain dendrites but occurred infrequently. In reciprocal synapses, the direction of the Gray Type I (asymmetrical) is away from the mitral cell while those with Gray Type II synapses (symmetrical) are toward the mitral cell. Assuming that the type I synapse is excitatory and Type II is inhibitory, these findings explain the electrophysiological demonstration of self-inhibition discharge found in mitral cells.

Neuroanatomy: Cajal and after Cajal.

PubMed

Jones, Edward G

2007-10-01

This essay commences with a consideration of the relative contributions of Cajal and Golgi to the study of the anatomy of the nervous system. It demonstrates the extent to which Cajal depended upon Golgi's work and how his modifications of the Golgi technique permitted a remarkable series of investigations in which the foundations of the neuron doctrine were laid and in which the intrinsic connectivity of virtually every part of the central nervous system was charted. Cajal's readiness to seize on and develop new techniques was one of the many keys to his success. After him, neuroanatomical studies tended to be focused more on long tract connectivity, using techniques such as those of Nissl and Marchi that had been in place before Cajal commenced his studies. Development of degeneration-based techniques of tracing connections in the late 1950s spearheaded a revolution in neuroanatomy while introduction of mixed aldehyde fixation made possible similarly intensive studies of the fine structure of the nervous system. At this time, the Golgi technique experienced a brief resurgence as neuroanatomists made efforts to bridge the gap between light and electron microscopy. Later developments in techniques for tracing connections included anterograde tracing by autoradiography and retrograde tracing by horseradish peroxidase. These were soon superseded by tracing techniques of increasing sensitivity and specificity that rely upon the cellular and molecular biology of neurons. Although neuroanatomy in its traditional form is perhaps no longer fashionable as a discipline, the techniques of neuroanatomy remain preeminent in many, perhaps all areas of neuroscience.

Ethanol-induced disruption of Golgi apparatus morphology, primary neurite number and cellular orientation in developing cortical neurons

PubMed Central

Powrozek, Teresa A.; Olson, Eric C.

2012-01-01

Prenatal ethanol exposure disrupts cortical neurite initiation and outgrowth, but prior studies have reported both ethanol-dependent growth promotion and inhibition. To resolve this ambiguity and better approximate in vivo conditions, we quantitatively analyzed neuronal morphology using a new, whole hemisphere explant model. In this model, Layer 6 (L6) cortical neurons migrate, laminate and extend neurites in an organotypic fashion. To selectively label L6 neurons we performed ex utero electroporation of a GFP expression construct at embryonic day 13 and allowed the explants to develop for 2 days in vitro. Explants were exposed to (400mg/dL) ethanol for either 4 or 24 hrs prior to fixation. Complete 3-D reconstructions were made of >80 GFP-positive neurons in each experimental condition. Acute responses to ethanol exposure included compaction of the Golgi apparatus accompanied by elaboration of supernumerary primary apical neurites, as well as a modest (~15%) increase in higher order apical neurite length. With longer exposure time, ethanol exposure leads to a consistent, significant disorientation of the cell (cell body, primary apical neurite, and Golgi) with respect to the pial surface. The effects on cellular orientation were accompanied by decreased expression of cytoskeletal elements, microtubule associated protein 2 and F-actin. These findings indicate that upon exposure to ethanol, developing L6 neurons manifest disruptions in Golgi apparatus and cytoskeletal elements which may in turn trigger selective and significant perturbations to primary neurite formation and neuronal polarity. PMID:22840816

Neoplasia Driven by Mutant c-KIT Is Mediated by Intracellular, Not Plasma Membrane, Receptor Signaling▿

PubMed Central

Xiang, Zhifu; Kreisel, Frederike; Cain, Jennifer; Colson, AnnaLynn; Tomasson, Michael H.

2007-01-01

Activating mutations in c-KIT are associated with gastrointestinal stromal tumors, mastocytosis, and acute myeloid leukemia. In attempting to establish a murine model of human KITD816V (hKITD816V)-mediated leukemia, we uncovered an unexpected relationship between cellular transformation and intracellular trafficking. We found that transport of hKITD816V protein was blocked at the endoplasmic reticulum in a species-specific fashion. We exploited these species-specific trafficking differences and a set of localization domain-tagged KIT mutants to explore the relationship between subcellular localization of mutant KIT and cellular transformation. The protein products of fully transforming KIT mutants localized to the Golgi apparatus and to a lesser extent the plasma membrane. Domain-tagged KITD816V targeted to the Golgi apparatus remained constitutively active and transforming. Chemical inhibition of intracellular transport demonstrated that Golgi localization is sufficient, but plasma membrane localization is dispensable, for downstream signaling mediated by KIT mutation. When expressed in murine bone marrow, endoplasmic reticulum-localized hKITD816V failed to induce disease in mice, while expression of either Golgi-localized HyKITD816V or cytosol-localized, ectodomain-deleted KITD816V uniformly caused fatal myeloproliferative diseases. Taken together, these data demonstrate that intracellular, non-plasma membrane receptor signaling is sufficient to drive neoplasia caused by mutant c-KIT and provide the first animal model of myelomonocytic neoplasia initiated by human KITD816V. PMID:17060458

Role of the AP-5 adaptor protein complex in late endosome-to-Golgi retrieval

PubMed Central

Hirst, Jennifer; Itzhak, Daniel N.; Antrobus, Robin; Borner, Georg H. H.

2018-01-01

The AP-5 adaptor protein complex is presumed to function in membrane traffic, but so far nothing is known about its pathway or its cargo. We have used CRISPR-Cas9 to knock out the AP-5 ζ subunit gene, AP5Z1, in HeLa cells, and then analysed the phenotype by subcellular fractionation profiling and quantitative mass spectrometry. The retromer complex had an altered steady-state distribution in the knockout cells, and several Golgi proteins, including GOLIM4 and GOLM1, were depleted from vesicle-enriched fractions. Immunolocalisation showed that loss of AP-5 led to impaired retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR), GOLIM4, and GOLM1 from endosomes back to the Golgi region. Knocking down the retromer complex exacerbated this phenotype. Both the CIMPR and sortilin interacted with the AP-5–associated protein SPG15 in pull-down assays, and we propose that sortilin may act as a link between Golgi proteins and the AP-5/SPG11/SPG15 complex. Together, our findings suggest that AP-5 functions in a novel sorting step out of late endosomes, acting as a backup pathway for retromer. This provides a mechanistic explanation for why mutations in AP-5/SPG11/SPG15 cause cells to accumulate aberrant endolysosomes, and highlights the role of endosome/lysosome dysfunction in the pathology of hereditary spastic paraplegia and other neurodegenerative disorders. PMID:29381698

Small molecule schweinfurthins selectively inhibit cancer cell proliferation and mTOR/AKT signaling by interfering with trans-Golgi-network trafficking

PubMed Central

Bao, Xingfeng; Zheng, Wanjun; Sugi, Naoko Hata; Agarwala, Kishan L; Xu, Qunli; Wang, Zichun; Tendyke, Karen; Lee, Winnie; Parent, Lana; Li, Wei; Cheng, Hongsheng; Shen, Yongchun; Taylor, Noel; Dezso, Zoltan; Du, Hong; Kotake, Yoshihiko; Zhao, Nanding; Wang, John; Postema, Maarten; Woodall-Jappe, Mary; Takase, Yasutaka; Uenaka, Toshimitsu; Kingston, David G I; Nomoto, Kenichi

2015-01-01

Natural compound schweinfurthins are of considerable interest for novel therapy development because of their selective anti-proliferative activity against human cancer cells. We previously reported the isolation of highly active schweinfurthins E-H, and in the present study, mechanisms of the potent and selective anti-proliferation were investigated. We found that schweinfurthins preferentially inhibited the proliferation of PTEN deficient cancer cells by indirect inhibition of AKT phosphorylation. Mechanistically, schweinfurthins and their analogs arrested trans-Golgi-network trafficking, an intracellular vesicular trafficking system, resulting in the induction of endoplasmic reticulum stress and the suppression of both lipid raft-mediated PI3K activation and mTOR/RheB complex formation, which collectively led to an effective inhibition of mTOR/AKT signaling. The trans-Golgi-network traffic arresting effect of schweinfurthins was associated with their in vitro binding activity to oxysterol-binding proteins that are known to regulate intracellular vesicular trafficking. Moreover, schweinfurthins were found to be highly toxic toward PTEN-deficient B cell lymphoma cells, and displayed 2 orders of magnitude lower activity toward normal human peripheral blood mononuclear cells and primary fibroblasts in vitro. These results revealed a previously unrecognized role of schweinfurthins in regulating trans-Golgi-network trafficking, and linked mechanistically this cellular effect with mTOR/AKT signaling and with cancer cell survival and growth. Our findings suggest the schweinfurthin class of compounds as a novel approach to modulate oncogenic mTOR/AKT signaling for cancer treatment. PMID:25729885

Intracellular zinc distribution in mitochondria, ER and the Golgi apparatus

PubMed Central

Lu, Qiping; Haragopal, Hariprakash; Slepchenko, Kira G; Stork, Christian; Li, Yang V

2016-01-01

Zinc (Zn2+) is required for numerous cellular functions. As such, the homeostasis and distribution of intracellular zinc can influence cellular metabolism and signaling. However, the exact distribution of free zinc within live cells remains elusive. Previously we showed the release of zinc from thapsigargin/IP3-sensitive endoplasmic reticulum (ER) storage in cortical neurons. In the present study, we investigated if other cellular organelles also contain free chelatable zinc and function as organelle storage for zinc. To identify free zinc within the organelles, live cells were co-stained with Zinpyr-1, a zinc fluorescent dye, and organelle-specific fluorescent dyes (MitoFluor Red 589: mitochondria; ER Tracker Red: endoplasmic reticulum; BODIPY TR ceramide: Golgi apparatus; Syto Red 64: nucleus). We examined organelles that represent potential storing sites for intracellular zinc. We showed that zinc fluorescence staining was co-localized with MitoFluor Red 589, ER Tracker Red, and BODIPY TR ceramide respectively, suggesting the presence of free zinc in mitochondria, endoplasmic reticulum, and the Golgi apparatus. On the other hand, cytosol and nucleus had nearly no detectable zinc fluorescence. It is known that nucleus contains high amount of zinc binding proteins that have high zinc binding affinity. The absence of zinc fluorescence suggests that there is little free zinc in these two regions. It also indicates that the zinc fluorescence detected in mitochondria, ER and Golgi apparatus represents free chelatable zinc. Taken together, our results support that these organelles are potential zinc storing organelles during cellular zinc homeostasis. PMID:27186321

Mutations in proteins of the Conserved Oligomeric Golgi Complex affect polarity, cell wall structure, and glycosylation in the filamentous fungus Aspergillus nidulans.

PubMed

Gremillion, S K; Harris, S D; Jackson-Hayes, L; Kaminskyj, S G W; Loprete, D M; Gauthier, A C; Mercer, S; Ravita, A J; Hill, T W

2014-12-01

We have described two Aspergillus nidulans gene mutations, designated podB1 (polarity defective) and swoP1 (swollen cell), which cause temperature-sensitive defects during polarization. Mutant strains also displayed unevenness and abnormal thickness of cell walls. Un-polarized or poorly-polarized mutant cells were capable of establishing normal polarity after a shift to a permissive temperature, and mutant hyphae shifted from permissive to restrictive temperature show wall and polarity abnormalities in subsequent growth. The mutated genes (podB=AN8226.3; swoP=AN7462.3) were identified as homologues of COG2 and COG4, respectively, each predicted to encode a subunit of the multi-protein COG (Conserved Oligomeric Golgi) Complex involved in retrograde vesicle trafficking in the Golgi apparatus. Down-regulation of COG2 or COG4 resulted in abnormal polarization and cell wall staining. The GFP-tagged COG2 and COG4 homologues displayed punctate, Golgi-like localization. Lectin-blotting indicated that protein glycosylation was altered in the mutant strains compared to the wild type. A multicopy expression experiment showed evidence for functional interactions between the homologues COG2 and COG4 as well as between COG2 and COG3. To date, this work is the first regarding a functional role of the COG proteins in the development of a filamentous fungus. Copyright © 2014 Elsevier Inc. All rights reserved.

TBC-8, a putative RAB-2 GAP, regulates dense core vesicle maturation in Caenorhabditis elegans.

PubMed

Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M; Koenig, Sabine; Eimer, Stefan

2012-01-01

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.

Determinants of [Cl−] in recycling and late endosomes and Golgi complex measured using fluorescent ligands

PubMed Central

Sonawane, N.D.; Verkman, A.S.

2003-01-01

Chloride concentration ([Cl−]) was measured in defined organellar compartments using fluorescently labeled transferrin, α2-macroglobulin, and cholera toxin B-subunit conjugated with Cl−-sensitive and -insensitive dyes. In pulse-chase experiments, [Cl−] in Tf-labeled early/recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over ∼10 min in parallel to a drop in pH from 6.91 to 6.05. The low [Cl−] just after internalization (compared with 137 mM solution [Cl−]) was prevented by reducing the interior-negative Donnan potential. [Cl−] in α2-macroglobulin–labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1–45 min after internalization, whereas pH decreased from 6.85 to 5.20. Cl− accumulation was prevented by bafilomycin but restored by valinomycin. A Cl− channel inhibitor slowed endosomal acidification and Cl− accumulation by ∼2.5-fold. [Cl−] was 49 mM and pH was 6.42 in cholera toxin B subunit–labeled Golgi complex in Vero cells; Golgi compartment Cl− accumulation and acidification were reversed by bafilomycin. Our experiments provide evidence that Cl− is the principal counter ion accompanying endosomal and Golgi compartment acidification, and that an interior-negative Donnan potential is responsible for low endosomal [Cl−] early after internalization. We propose that reduced [Cl−] and volume in early endosomes permits endosomal acidification and [Cl−] accumulation without lysis. PMID:12668661

Computational Reconstruction of Pacemaking and Intrinsic Electroresponsiveness in Cerebellar Golgi Cells

PubMed Central

Solinas, Sergio; Forti, Lia; Cesana, Elisabetta; Mapelli, Jonathan; De Schutter, Erik; D'Angelo, Egidio

2007-01-01

The Golgi cells have been recently shown to beat regularly in vitro (Forti et al., 2006. J. Physiol. 574, 711–729). Four main currents were shown to be involved, namely a persistent sodium current (I Na-p), an h current (I h), an SK-type calcium-dependent potassium current (I K-AHP), and a slow M-like potassium current (I K-slow). These ionic currents could take part, together with others, also to different aspects of neuronal excitability like responses to depolarizing and hyperpolarizing current injection. However, the ionic mechanisms and their interactions remained largely hypothetical. In this work, we have investigated the mechanisms of Golgi cell excitability by developing a computational model. The model predicts that pacemaking is sustained by subthreshold oscillations tightly coupled to spikes. I Na-p and I K-slow emerged as the critical determinants of oscillations. I h also played a role by setting the oscillatory mechanism into the appropriate membrane potential range. I K-AHP, though taking part to the oscillation, appeared primarily involved in regulating the ISI following spikes. The combination with other currents, in particular a resurgent sodium current (I Na-r) and an A-current (I K-A), allowed a precise regulation of response frequency and delay. These results provide a coherent reconstruction of the ionic mechanisms determining Golgi cell intrinsic electroresponsiveness and suggests important implications for cerebellar signal processing, which will be fully developed in a companion paper (Solinas et al., 2008. Front. Neurosci. 2:4). PMID:18946520

UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage[OPEN

PubMed Central

Saez-Aguayo, Susana; Rautengarten, Carsten; Temple, Henry; Sanhueza, Dayan; Ejsmentewicz, Troy; Sandoval-Ibañez, Omar; Parra-Rojas, Juan Pablo; Ebert, Berit; Reyes, Francisca C.

2017-01-01

UDP-glucuronic acid (UDP-GlcA) is the precursor of many plant cell wall polysaccharides and is required for production of seed mucilage. Following synthesis in the cytosol, it is transported into the lumen of the Golgi apparatus, where it is converted to UDP-galacturonic acid (UDP-GalA), UDP-arabinose, and UDP-xylose. To identify the Golgi-localized UDP-GlcA transporter, we screened Arabidopsis thaliana mutants in genes coding for putative nucleotide sugar transporters for altered seed mucilage, a structure rich in the GalA-containing polysaccharide rhamnogalacturonan I. As a result, we identified UUAT1, which encodes a Golgi-localized protein that transports UDP-GlcA and UDP-GalA in vitro. The seed coat of uuat1 mutants had less GalA, rhamnose, and xylose in the soluble mucilage, and the distal cell walls had decreased arabinan content. Cell walls of other organs and cells had lower arabinose levels in roots and pollen tubes, but no differences were observed in GalA or xylose contents. Furthermore, the GlcA content of glucuronoxylan in the stem was not affected in the mutant. Interestingly, the degree of homogalacturonan methylation increased in uuat1. These results suggest that this UDP-GlcA transporter plays a key role defining the seed mucilage sugar composition and that its absence produces pleiotropic effects in this component of the plant extracellular matrix. PMID:28062750

In Vitro Intracellular Trafficking of Virulence Antigen during Infection by Yersinia pestis

DTIC Science & Technology

2009-07-17

endosomes after 10 min, late endosomes after 30–45 min, lysosomes after 1–2 h, mitochondria after 2.5–3 h, and the Golgi apparatus after 4 h. Scale bar...of infection, cell viability decreased significantly, as detected by IFM, and V was not tracked beyond the Golgi apparatus . Thus, flow cytometry was...involve the T3SS. This finding as reported by Fields and Straley [34] was evidenced by infections with strains lacking a functional T3SS apparatus or

Detecting the golgi protein 73 of liver cancer with micro cantilever

NASA Astrophysics Data System (ADS)

Thanh Tuyen Le, Thi; Pham, Van Tho; Nhat Khoa Phan, Thanh; Binh Pham, Van; Thao Le, Van; Hien Tong, Duy

2014-12-01

Golgi protein 73 (GP73) is a potential serum biomarker used in diagnosing human hepatocellular carcinoma (HCC). Compared to alpha-fetoprotein, detection of GP73 is expected to give better sensitivity and specificity and thus offers a better method for diagnosis of HCC at an early stage. In this paper, silicon nitride microcantilever was used to detect GP73. The cantilever was modified through many steps to contain antibody of GP73. The result shows that the cantilever can be used as a label-free sensor to detect this kind of biomarker.

Recurrent Muscle Weakness with Rhabdomyolysis, Metabolic Crises, and Cardiac Arrhythmia Due to Bi-allelic TANGO2 Mutations

PubMed Central

Lalani, Seema R.; Liu, Pengfei; Rosenfeld, Jill A.; Watkin, Levi B.; Chiang, Theodore; Leduc, Magalie S.; Zhu, Wenmiao; Ding, Yan; Pan, Shujuan; Vetrini, Francesco; Miyake, Christina Y.; Shinawi, Marwan; Gambin, Tomasz; Eldomery, Mohammad K.; Akdemir, Zeynep Hande Coban; Emrick, Lisa; Wilnai, Yael; Schelley, Susan; Koenig, Mary Kay; Memon, Nada; Farach, Laura S.; Coe, Bradley P.; Azamian, Mahshid; Hernandez, Patricia; Zapata, Gladys; Jhangiani, Shalini N.; Muzny, Donna M.; Lotze, Timothy; Clark, Gary; Wilfong, Angus; Northrup, Hope; Adesina, Adekunle; Bacino, Carlos A.; Scaglia, Fernando; Bonnen, Penelope E.; Crosson, Jane; Duis, Jessica; Maegawa, Gustavo H.B.; Coman, David; Inwood, Anita; McGill, Jim; Boerwinkle, Eric; Graham, Brett; Beaudet, Art; Eng, Christine M.; Hanchard, Neil A.; Xia, Fan; Orange, Jordan S.; Gibbs, Richard A.; Lupski, James R.; Yang, Yaping

2016-01-01

The underlying genetic etiology of rhabdomyolysis remains elusive in a significant fraction of individuals presenting with recurrent metabolic crises and muscle weakness. Using exome sequencing, we identified bi-allelic mutations in TANGO2 encoding transport and Golgi organization 2 homolog (Drosophila) in 12 subjects with episodic rhabdomyolysis, hypoglycemia, hyperammonemia, and susceptibility to life-threatening cardiac tachyarrhythmias. A recurrent homozygous c.460G>A (p.Gly154Arg) mutation was found in four unrelated individuals of Hispanic/Latino origin, and a homozygous ∼34 kb deletion affecting exons 3–9 was observed in two families of European ancestry. One individual of mixed Hispanic/European descent was found to be compound heterozygous for c.460G>A (p.Gly154Arg) and the deletion of exons 3–9. Additionally, a homozygous exons 4–6 deletion was identified in a consanguineous Middle Eastern Arab family. No homozygotes have been reported for these changes in control databases. Fibroblasts derived from a subject with the recurrent c.460G>A (p.Gly154Arg) mutation showed evidence of increased endoplasmic reticulum stress and a reduction in Golgi volume density in comparison to control. Our results show that the c.460G>A (p.Gly154Arg) mutation and the exons 3–9 heterozygous deletion in TANGO2 are recurrent pathogenic alleles present in the Latino/Hispanic and European populations, respectively, causing considerable morbidity in the homozygotes in these populations. PMID:26805781

Progressive quality control of secretory proteins in the early secretory compartment by ERp44.

PubMed

Sannino, Sara; Anelli, Tiziana; Cortini, Margherita; Masui, Shoji; Degano, Massimo; Fagioli, Claudio; Inaba, Kenji; Sitia, Roberto

2014-10-01

ERp44 is a pH-regulated chaperone of the secretory pathway. In the acidic milieu of the Golgi, its C-terminal tail changes conformation, simultaneously exposing the substrate-binding site for cargo capture and the RDEL motif for ER retrieval through interactions with cognate receptors. Protonation of cysteine 29 in the active site allows tail movements in vitro and in vivo. Here, we show that conserved histidine residues in the C-terminal tail also regulate ERp44 in vivo. Mutants lacking these histidine residues retain substrates more efficiently. Surprisingly, they are also O-glycosylated and partially secreted. Co-expression of client proteins prevents secretion of the histidine mutants, forcing tail opening and RDEL accessibility. Client-induced RDEL exposure allows retrieval of proteins from distinct stations along the secretory pathway, as indicated by the changes in O-glycosylation patterns upon overexpression of different partners. The ensuing gradients might help to optimize folding and assembly of different cargoes. Endogenous ERp44 is O-glycosylated and secreted by human primary endometrial cells, suggesting possible pathophysiological roles of these processes. © 2014. Published by The Company of Biologists Ltd.

Non-genomic STAT5-dependent effects at the endoplasmic reticulum and Golgi apparatus and STAT6-GFP in mitochondria

PubMed Central

Sehgal, Pravin B

2013-01-01

STAT protein species are well-known as transcription factors that regulate nuclear gene expression. Recent novel lines of research suggest new non-genomic functions of STAT5A/B and STAT6. It was discovered in human pulmonary arterial endothelial cells that STAT5A, including STAT5A-GFP, constitutively associated with the Golgi apparatus, and both STAT5A and B with the endoplasmic reticulum. Acute siRNA-mediated knockdown of STAT5A/B led to the rapid development of a dramatic cystic change in the endoplasmic reticulum (ER) characterized by deposition of the ER structural protein reticulon-4 (RTN4; also called Nogo-B) and the ER-resident GTPase atlastin-3 (ATL3) along cyst membranes and cyst-zone boundaries, accompanied by Golgi fragmentation. Functional consequences included reduced anterograde trafficking, an ER stress response (increased GRP78/BiP) and eventual mitochondrial fragmentation. This phenotype was “non-genomic” in that it was elicited in enucleated cytoplasts. In cross-immunopanning assays STAT5A and B species associated with ATL3, and the ER-lumen spacer CLIMP63 (also called cytoskeleton-associated protein 4, CKAP4) but not RTN4. From a disease significance perspective we posit that STAT5, which is known to be affected by estradiol-17β and prolactin, represents the gender-sensitive determinant in the pathogenesis of idiopathic pulmonary hypertension (IPAH), a disease which includes ER/Golgi dysfunctions but with a 2- to 4-fold higher prevalence in postpubertal women. A separate line of recent research produced evidence for the association of STAT6-GFP, but not STAT3-GFP, STAT3-DsRed, or STAT3-Flag, with mitochondria in live-cell, immunofluorescence, and immunoelectron microscopy. An N-terminal truncation of STAT6-GFP (1–459), which lacked the SH2 domain and Tyr-phosphorylation site, constitutively associated with mitochondria. Thus, the emergent new of biology STAT proteins includes non-genomic roles—structurally and functionally—in the three closely related membrane organelles consisting of the endoplasmic reticulum, Golgi apparatus, and mitochondria. PMID:24470974

In silico analysis of the fucosylation-associated genome of the human blood fluke Schistosoma mansoni: cloning and characterization of the enzymes involved in GDP-L-fucose synthesis and Golgi import

PubMed Central

2013-01-01

Background Carbohydrate structures of surface-expressed and secreted/excreted glycoconjugates of the human blood fluke Schistosoma mansoni are key determinants that mediate host-parasite interactions in both snail and mammalian hosts. Fucose is a major constituent of these immunologically important glycans, and recent studies have sought to characterize fucosylation-associated enzymes, including the Golgi-localized fucosyltransferases that catalyze the transfer of L-fucose from a GDP-L-fucose donor to an oligosaccharide acceptor. Importantly, GDP-L-fucose is the only nucleotide-sugar donor used by fucosyltransferases and its availability represents a bottleneck in fucosyl-glycotope expression. Methods A homology-based genome-wide bioinformatics approach was used to identify and molecularly characterize the enzymes that contribute to GDP-L-fucose synthesis and Golgi import in S. mansoni. Putative functions were further investigated through molecular phylogenetic and immunocytochemical analyses. Results We identified homologs of GDP-D-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (GMER), which constitute a de novo pathway for GDP-L-fucose synthesis, in addition to a GDP-L-fucose transporter (GFT) that putatively imports cytosolic GDP-L-fucose into the Golgi. In silico primary sequence analyses identified characteristic Rossman loop and short-chain dehydrogenase/reductase motifs in GMD and GMER as well as 10 transmembrane domains in GFT. All genes are alternatively spliced, generating variants of unknown function. Observed quantitative differences in steady-state transcript levels between miracidia and primary sporocysts may contribute to differential glycotope expression in early larval development. Additionally, analyses of protein expression suggest the occurrence of cytosolic GMD and GMER in the ciliated epidermal plates and tegument of miracidia and primary sporocysts, respectively, which is consistent with previous localization of highly fucosylated glycotopes. Conclusions This study is the first to identify and characterize three key genes that are putatively involved in the synthesis and Golgi import of GDP-L-fucose in S. mansoni and provides fundamental information regarding their genomic organization, genetic variation, molecular phylogenetics, and developmental expression in intramolluscan larval stages. PMID:23835114

In silico analysis of the fucosylation-associated genome of the human blood fluke Schistosoma mansoni: cloning and characterization of the enzymes involved in GDP-L-fucose synthesis and Golgi import.

PubMed

Peterson, Nathan A; Anderson, Tavis K; Wu, Xiao-Jun; Yoshino, Timothy P

2013-07-09

Carbohydrate structures of surface-expressed and secreted/excreted glycoconjugates of the human blood fluke Schistosoma mansoni are key determinants that mediate host-parasite interactions in both snail and mammalian hosts. Fucose is a major constituent of these immunologically important glycans, and recent studies have sought to characterize fucosylation-associated enzymes, including the Golgi-localized fucosyltransferases that catalyze the transfer of L-fucose from a GDP-L-fucose donor to an oligosaccharide acceptor. Importantly, GDP-L-fucose is the only nucleotide-sugar donor used by fucosyltransferases and its availability represents a bottleneck in fucosyl-glycotope expression. A homology-based genome-wide bioinformatics approach was used to identify and molecularly characterize the enzymes that contribute to GDP-L-fucose synthesis and Golgi import in S. mansoni. Putative functions were further investigated through molecular phylogenetic and immunocytochemical analyses. We identified homologs of GDP-D-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (GMER), which constitute a de novo pathway for GDP-L-fucose synthesis, in addition to a GDP-L-fucose transporter (GFT) that putatively imports cytosolic GDP-L-fucose into the Golgi. In silico primary sequence analyses identified characteristic Rossman loop and short-chain dehydrogenase/reductase motifs in GMD and GMER as well as 10 transmembrane domains in GFT. All genes are alternatively spliced, generating variants of unknown function. Observed quantitative differences in steady-state transcript levels between miracidia and primary sporocysts may contribute to differential glycotope expression in early larval development. Additionally, analyses of protein expression suggest the occurrence of cytosolic GMD and GMER in the ciliated epidermal plates and tegument of miracidia and primary sporocysts, respectively, which is consistent with previous localization of highly fucosylated glycotopes. This study is the first to identify and characterize three key genes that are putatively involved in the synthesis and Golgi import of GDP-L-fucose in S. mansoni and provides fundamental information regarding their genomic organization, genetic variation, molecular phylogenetics, and developmental expression in intramolluscan larval stages.

Development of a modified straw method for vitrification of in vitro-produced bovine blastocysts and various genes expression in between the methods.

PubMed

Ha, A-Na; Lee, Sang-Ryeul; Jeon, Jeong-Seon; Park, Han-Seul; Lee, Sang-Ho; Jin, Jong-In; Sessions, Benjamin R; Wang, Zhongde; White, Kenneth L; Kong, Il-Keun

2014-02-01

This study evaluated a modified plastic straw loading method for vitrification of in vitro-produced bovine blastocysts. A modified straw was used with a depressed area on its inner surface to which embryos attach. In vitro-produced blastocysts were randomly assigned into three groups: (i) blastocysts attached to the inner surface of a plastic straw (aV), (ii) blastocysts attached to the inner surface of a modified plastic straw (maV), and (iii) non-vitrified blastocysts (control). The recovery rates were not significantly different between aV and maV groups (95.8% vs. 94.3%). The post-thaw survival rate did not significantly differ between aV and maV groups (86.4% vs. 88.2%). The total cell numbers of blastocyst was higher in control than in aV and maV groups (142 ± 21.8 vs. 117 ± 29.7 and 120 ± 25.2; P < 0.05), but not significantly differ between aV and maV groups. The mRNA levels of pro-apoptosis related genes Bax and Caspase-3 were higher in aV and maV than in control (P < 0.05). By contrast, the mRNA levels of anti-apoptotic genes Bcl-2 and Mcl-1 and of antioxidant-related genes MnSOD and Prdx5 were lower in aV and maV than in control (P < 0.05). Confocal microscopy analysis of Golgi apparatus and mitochondria showed that the fluorescence intensity of Golgi apparatus and mitochondria was higher in control than in aV and maV groups. In conclusion, both aV and maV methods can be used to successfully vitrify IVP blastocysts, with maV method to be preferable because of its easiness in embryo loading. Copyright © 2013 Elsevier Inc. All rights reserved.

Different oligosaccharide processing of the membrane-integrated and the secretory form of gp 80 in rat liver.

PubMed

Tauber, R; Schenck, I; Josić, D; Gross, V; Heinrich, P C; Gerok, W; Reutter, W

1986-09-01

Rat liver synthesizes a glycoprotein with Mr of 80.000 (gp 80) which is partly inserted into the plasma membrane and partly secreted into the serum. The membrane-integrated and the secretory form of this glycoprotein have an identical peptide pattern, but different N-linked glycans. Whereas gp 80 from the serum is glycosylated with complex-type oligosaccharides, gp 80 from the plasma membrane has high mannose glycans. Phase separation with Triton X-114 showed that membrane-integrated gp 80 contains hydrophobic portions, whereas secretory gp 80 has hydrophilic properties. Intracellular transport and oligosaccharide processing of gp 80 were studied in vivo in the endoplasmic reticulum, the Golgi apparatus and plasma membranes of rat liver and in serum using pulse-chase labeling with L-[35S]methionine and immunoprecipitation. Peak labeling of gp 80 was reached in the endoplasmic reticulum 10 min after the pulse, in the Golgi apparatus 20 min later, and in the plasma membrane after 2 h; in the serum the specific radioactivity was steadily increasing during the experiment. Gp 80 of the endoplasmic reticulum was completely sensitive to endo-beta-N-glucosaminidase H (endo H), but simultaneously occurred in the Golgi apparatus in an endo H-sensitive and endo H-resistant form. The endo H-sensitive form was transported to the plasma membrane, the endo H-resistant species secreted into the serum. Conversion from the endo H-sensitive to the endo H-resistant form was completed within 10 min after transfer of gp 80 to the Golgi apparatus.(ABSTRACT TRUNCATED AT 250 WORDS)

A membrane-anchored E-type endo-1,4-beta-glucanase is localized on Golgi and plasma membranes of higher plants.

PubMed

Brummell, D A; Catala, C; Lashbrook, C C; Bennett, A B

1997-04-29

Endo-1,4-beta-D-glucanases (EGases, EC 3.2.1.4) are enzymes produced in bacteria, fungi, and plants that hydrolyze polysaccharides possessing a 1,4-beta-D-glucan backbone. All previously identified plant EGases are E-type endoglucanases that possess signal sequences for endoplasmic reticulum entry and are secreted to the cell wall. Here we report the characterization of a novel E-type plant EGase (tomato Cel3) with a hydrophobic transmembrane domain and structure typical of type II integral membrane proteins. The predicted protein is composed of 617 amino acids and possesses seven potential sites for N-glycosylation. Cel3 mRNA accumulates in young vegetative tissues with highest abundance during periods of rapid cell expansion, but is not hormonally regulated. Antibodies raised to a recombinant Cel3 protein specifically recognized three proteins, with apparent molecular masses of 93, 88, and 53 kDa, in tomato root microsomal membranes separated by sucrose density centrifugation. The 53-kDa protein comigrated in the gradient with plasma membrane markers, the 88-kDa protein with Golgi membrane markers, and the 93-kDa protein with markers for both Golgi and plasma membranes. EGase enzyme activity was also found in regions of the density gradient corresponding to both Golgi and plasma membranes, suggesting that Cel3 EGase resides in both membrane systems, the sites of cell wall polymer biosynthesis. The in vivo function of Cel3 is not known, but the only other known membrane-anchored EGase is present in Agrobacterium tumefaciens where it is required for cellulose biosynthesis.

Hygromycin B hypersensitive (hhy) mutants implicate an intact trans-Golgi and late endosome interface in efficient Tor1 vacuolar localization and TORC1 function.

PubMed

Ejzykowicz, Daniele E; Locken, Kristopher M; Ruiz, Fiona J; Manandhar, Surya P; Olson, Daniel K; Gharakhanian, Editte

2017-06-01

Saccharomyces cerevisiae vacuoles are functionally analogous to mammalian lysosomes. Both also serve as physical platforms for Tor Complex 1 (TORC1) signal transduction, the master regulator of cellular growth and proliferation. Hygromycin B is a eukaryotic translation inhibitor. We recently reported on hygromycin B hypersensitive (hhy) mutants that fail to grow at subtranslation inhibitory concentrations of the drug and exhibit vacuolar defects (Banuelos et al. in Curr Genet 56:121-137, 2010). Here, we show that hhy phenotype is not due to increased sensitivity to translation inhibition and establish a super HHY (s-HHY) subgroup of genes comprised of ARF1, CHC1, DRS2, SAC1, VPS1, VPS34, VPS45, VPS52, and VPS54 that function exclusively or inclusively at trans-Golgi and late endosome interface. Live cell imaging of s-hhy mutants revealed that hygromycin B treatment disrupts vacuolar morphology and the localization of late endosome marker Pep12, but not that of late endosome-independent vacuolar SNARE Vam3. This, along with normal post-late endosome trafficking of the vital dye FM4-64, establishes that severe hypersensitivity to hygromycin B correlates specifically with compromised trans-Golgi and late endosome interface. We also show that Tor1p vacuolar localization and TORC1 anabolic functions, including growth promotion and phosphorylation of its direct substrate Sch9, are compromised in s-hhy mutants. Thus, an intact trans-Golgi and late endosome interface is a requisite for efficient Tor1 vacuolar localization and TORC1 function.

TBC-8, a Putative RAB-2 GAP, Regulates Dense Core Vesicle Maturation in Caenorhabditis elegans

PubMed Central

Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M.; Koenig, Sabine; Eimer, Stefan

2012-01-01

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2–specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation. PMID:22654674

Interaction between the PH and START domains of ceramide transfer protein competes with phosphatidylinositol 4-phosphate binding by the PH domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prashek, Jennifer; Bouyain, Samuel; Fu, Mingui

De novo synthesis of the sphingolipid sphingomyelin requires non-vesicular transport of ceramide from the endoplasmic reticulum to the Golgi by the multidomain protein ceramide transfer protein (CERT). CERT's N-terminal pleckstrin homology (PH) domain targets it to the Golgi by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P) in the Golgi membrane, whereas its C-terminal StAR-related lipid transfer domain (START) carries out ceramide transfer. Hyperphosphorylation of a serine-rich motif immediately after the PH domain decreases both PtdIns(4)P binding and ceramide transfer by CERT. This down-regulation requires both the PH and START domains, suggesting a possible inhibitory interaction between the two domains. In this studymore » we show that isolated PH and START domains interact with each other. The crystal structure of a PH–START complex revealed that the START domain binds to the PH domain at the same site for PtdIns(4)P-binding, suggesting that the START domain competes with PtdIns(4)P for association with the PH domain. We further report that mutations disrupting the PH–START interaction increase both PtdIns(4)P-binding affinity and ceramide transfer activity of a CERT-serine–rich phosphorylation mimic. We also found that these mutations increase the Golgi localization of CERT inside the cell, consistent with enhanced PtdIns(4)P binding of the mutant. Collectively, our structural, biochemical, and cellular investigations provide important structural insight into the regulation of CERT function and localization.« less

Loss of a membrane trafficking protein αSNAP induces non-canonical autophagy in human epithelia

PubMed Central

Naydenov, Nayden G.; Harris, Gianni; Morales, Victor; Ivanov, Andrei I.

2012-01-01

Autophagy is a catabolic process that sequesters intracellular proteins and organelles within membrane vesicles called autophagosomes with their subsequent delivery to lyzosomes for degradation. This process involves multiple fusions of autophagosomal membranes with different vesicular compartments; however, the role of vesicle fusion in autophagosomal biogenesis remains poorly understood. This study addresses the role of a key vesicle fusion regulator, soluble N-ethylmaleimide-sensitive factor attachment protein α (αSNAP), in autophagy. Small interfering RNA-mediated downregulation of αSNAP expression in cultured epithelial cells stimulated the autophagic flux, which was manifested by increased conjugation of microtubule-associated protein light chain 3 (LC3-II) and accumulation of LC3-positive autophagosomes. This enhanced autophagy developed via a non-canonical mechanism that did not require beclin1-p150-dependent nucleation, but involved Atg5 and Atg7-mediated elongation of autophagosomal membranes. Induction of autophagy in αSNAP-depleted cells was accompanied by decreased mTOR signaling but appeared to be independent of αSNAP-binding partners, N-ethylmaleimide-sensitive factor and BNIP1. Loss of αSNAP caused fragmentation of the Golgi and downregulation of the Golgi-specific GTP exchange factors, GBF1, BIG1 and BIG2. Pharmacological disruption of the Golgi and genetic inhibition of GBF1 recreated the effects of αSNAP depletion on the autophagic flux. Our study revealed a novel role for αSNAP as a negative regulator of autophagy that acts by enhancing mTOR signaling and regulating the integrity of the Golgi complex. PMID:23187805

Endoplasmic reticulum-Golgi intermediate compartment protein 3 knockdown suppresses lung cancer through endoplasmic reticulum stress-induced autophagy.

PubMed

Hong, Seong-Ho; Chang, Seung-Hee; Cho, Kyung-Cho; Kim, Sanghwa; Park, Sungjin; Lee, Ah Young; Jiang, Hu-Lin; Kim, Hyeon-Jeong; Lee, Somin; Yu, Kyeong-Nam; Seo, Hwi Won; Chae, Chanhee; Kim, Kwang Pyo; Park, Jongsun; Cho, Myung-Haing

2016-10-04

Trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus is elevated in cancer cells. Therefore, proteins of the ER-Golgi intermediate compartment (ERGIC) attract significant attention as targets for cancer treatment. Enhanced cancer cell growth and epithelial-mesenchymal transition by ERGICs correlates with poor-prognosis of lung cancer. This prompted us to assess whether knockdown of ERGIC3 may decrease lung cancer growth. To test the hypothesis, the effects of ERGIC3 short hairpin RNA (shERGIC3) on ER stress-induced cell death and lung tumorigenesis were investigated both in vitro and in vivo. Knockdown of ERGIC3 led to ER stress-induced autophagic cell death and suppression of proliferation in the A549 human lung cancer cell-line. Moreover, non-invasive aerosol-delivery of shERGIC3 using the biocompatible carrier glycerol propoxylate triacrylate and spermine (GPT-SPE) inhibited lung tumorigenesis in the K-rasLA1 murine model of lung cancer. Our data suggest that suppression of ERGIC3 could provide a framework for the development of effective lung cancer therapies.

Ubiquitin-specific protease 8 deubiquitinates Sec31A and decreases large COPII carriers and collagen IV secretion.

PubMed

Kawaguchi, Kohei; Endo, Akinori; Fukushima, Toshiaki; Madoka, Yuka; Tanaka, Toshiaki; Komada, Masayuki

2018-05-15

Nascent cargo proteins in the endoplasmic reticulum are transported to the Golgi by COPII carriers. Typical COPII vesicles are 60-70 nm in diameter, and much larger macromolecules, such as procollagen, are transported by atypical large COPII carriers in mammalian cells. The formation of large COPII carriers is enhanced by Cul3 ubiquitin ligase, which mono-ubiquitinates Sec31A, a COPII coat protein. However, the deubiquitinating enzyme for Sec31A was unclear. Here, we show that the deubiquitinating enzyme USP8 interacts with and deubiquitinates Sec31A. The interaction was mediated by the adaptor protein STAM1. USP8 overexpression inhibited the formation of large COPII carriers. By contrast, USP8 knockdown caused the accumulation of COPII coat proteins around the cis-Golgi, promoted the intracellular trafficking of procollagen IV from the endoplasmic reticulum to the Golgi, and increased collagen IV secretion. We concluded that USP8 deubiquitinates Sec31A and inhibits the formation of large COPII carriers, thereby suppressing collagen IV secretion. Copyright © 2018 Elsevier Inc. All rights reserved.

RORγt+ cells selectively express redundant cation channels linked to the Golgi apparatus

PubMed Central

Drujont, Lucile; Lemoine, Aurélie; Moreau, Aurélie; Bienvenu, Géraldine; Lancien, Mélanie; Cens, Thierry; Guillot, Flora; Bériou, Gaëlle; Bouchet-Delbos, Laurence; Fehling, Hans Jörg; Chiffoleau, Elise; Nicot, Arnaud B.; Charnet, Pierre; Martin, Jérôme C.; Josien, Régis; Cuturi, Maria Cristina; Louvet, Cédric

2016-01-01

Retinoid-related orphan receptor gamma t (RORγt) is a master transcription factor central to type 17 immunity involving cells such as T helper 17, group 3 innate lymphoid cells or IL-17-producing γδ T cells. Here we show that the intracellular ion channel TMEM176B and its homologue TMEM176A are strongly expressed in these RORγt+ cells. We demonstrate that TMEM176A and B exhibit a similar cation channel activity and mainly colocalise in close proximity to the trans-Golgi network. Strikingly, in the mouse, the loss of Tmem176b is systematically associated with a strong upregulation of Tmem176a. While Tmem176b single-deficiency has no effect on the course of experimental autoimmune encephalomyelitis, T cell or DSS-induced colitis, it significantly reduces imiquimod-induced psoriasis-like skin inflammation. These findings shed light on a potentially novel specific process linked to post-Golgi trafficking for modulating the function of RORγt+ cells and indicate that both homologues should be simultaneously targeted to clearly elucidate the role of this intracellular ion flow. PMID:27009467

Soluble Glucan Is Internalized and Trafficked to the Golgi Apparatus in Macrophages via a Clathrin-Mediated, Lipid Raft-Regulated Mechanism

PubMed Central

Goldman, Matthew P.; Kalbfleisch, John H.; Williams, David L.

2012-01-01

Glucans are natural product carbohydrates that stimulate immunity. Glucans are internalized by the pattern recognition receptor, Dectin-1. Glucans were thought to be trafficked to phagolysosomes, but this is unproven. We examined the internalization and trafficking of soluble glucans in macrophages. Incubation of macrophages with glucan resulted in internalization of Dectin-1 and glucan. Inhibition of clathrin blocked internalization of the Dectin-1/glucan complex. Lipid raft depletion resulted in decreased Dectin levels and glucan uptake. Once internalized, glucans colocalized with early endosomes at 0 to 15 min, with the Golgi apparatus at 15 min to 24 h, and with Dectin-1 immediately (0 h) and again later (15 min-24 h). Glucans did not colocalize with lysosomes at any time interval examined. We conclude that the internalization of Dectin-1/glucan complexes in macrophages is mediated by clathrin and negatively regulated by lipid rafts and/or caveolin-1. Upon internalization, soluble glucans are trafficked via endosomes to the Golgi apparatus, not lysosomes. PMID:22700434

Illuminating cellular structure and function in the early secretory pathway by multispectral 3D imaging in living cells

NASA Astrophysics Data System (ADS)

Rietdorf, Jens; Stephens, David J.; Squire, Anthony; Simpson, Jeremy; Shima, David T.; Paccaud, Jean-Pierre; Bastiaens, Philippe I.; Pepperkok, Rainer

2000-04-01

Membrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in selective packaging of anterograde cargo into coated transport vesicles budding from the ER. COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER. We have used multi spectral 3D imaging to visualize COPI and COPII behavior simultaneously with various GFP-tagged secretory markers in living cells. This shows that COPII and COPI act sequentially whereby COPI association with anterograde transport complexes is involved in microtubule-based transport and the en route segregation of ER recycling molecules from secretory cargo within TCS in transit to the Golgi complex. We have also investigated the possibility to discriminate spectrally GFP fusion proteins by fluorescence lifetime imaging. This shows that at least two, and possibly up to three GFP fusion proteins can be discriminated and localized in living cells using a single excitation wavelength and a single broad band emission filter.

A Structure-Based Mechanism for Arf1-Dependent Recruitment of Coatomer to Membranes

PubMed Central

Yu, Xinchao; Breitman, Marianna; Goldberg, Jonathan

2012-01-01

Summary Budding of COPI-coated vesicles from Golgi membranes requires an Arf-family G protein and the coatomer complex recruited from cytosol. Arf is also required with coatomer-related clathrin adaptor complexes to bud vesicles from the trans-Golgi network and endosomal compartments. To understand the structural basis for Arf-dependent recruitment of a vesicular coat to the membrane, we determined the structure of Arf1 bound to the γζ-COP subcomplex of coatomer. Structure-guided biochemical analysis reveals that a second Arf1-GTP molecule binds to βδ-COP at a site common to the γ- and β-COP subunits. The Arf1-binding sites on coatomer are spatially related to PtdIns4,5P2-binding sites on the endocytic AP2 complex, providing evidence that the orientation of membrane binding is general for this class of vesicular coat proteins. A bivalent GTP-dependent binding mode has implications for the dynamics of coatomer interaction with the Golgi and for the selection of cargo molecules. PMID:22304919

Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus

PubMed Central

Lipovsky, Alex; Popa, Andreea; Pimienta, Genaro; Wyler, Michael; Bhan, Ashima; Kuruvilla, Leena; Guie, Marie-Aude; Poffenberger, Adrian C.; Nelson, Christian D. S.; Atwood, Walter J.; DiMaio, Daniel

2013-01-01

Despite major advances in our understanding of many aspects of human papillomavirus (HPV) biology, HPV entry is poorly understood. To identify cellular genes required for HPV entry, we conducted a genome-wide screen for siRNAs that inhibited infection of HeLa cells by HPV16 pseudovirus. Many retrograde transport factors were required for efficient infection, including multiple subunits of the retromer, which initiates retrograde transport from the endosome to the trans-Golgi network (TGN). The retromer has not been previously implicated in virus entry. Furthermore, HPV16 capsid proteins arrive in the TGN/Golgi in a retromer-dependent fashion during entry, and incoming HPV proteins form a stable complex with retromer subunits. We propose that HPV16 directly engages the retromer at the early or late endosome and traffics to the TGN/Golgi via the retrograde pathway during cell entry. These results provide important insights into HPV entry, identify numerous potential antiviral targets, and suggest that the role of the retromer in infection by other viruses should be assessed. PMID:23569269

INTRACELLULAR CHOLESTEROL HOMEOSTASIS AND AMYLOID PRECURSOR PROTEIN PROCESSING

PubMed Central

Burns, Mark; Rebeck, G. William

2010-01-01

Many preclinical and clinical studies have implied a role for cholesterol in the pathogenesis of Alzheimer's disease (AD). In this review we will discuss the movement of intracellular cholesterol and how normal distribution, transport, and export of cholesterol is vital for regulation of the AD related protein, Aβ. We focus on cholesterol distribution in the plasma membrane, transport through the endosomal/lysosomal system, control of cholesterol intracellular signaling at the endoplasmic reticulum and Golgi, the HMG-CoA reductase pathway and finally export of cholesterol from the cell. PMID:20304094

The shape of things to come: regulation of shape changes in endoplasmic reticulum.

PubMed

Paiement, J; Bergeron, J

2001-01-01

Shape changes in the endoplasmic reticulum control fundamental cell processes including nuclear envelope assembly in mitotic cells, calcium homeostasis in cytoplasmic domains of secreting and motile cells, and membrane traffic in the early secretion apparatus between the endoplasmic reticulum and Golgi. Opposing forces of assembly (membrane fusion) and disassembly (membrane fragmentation) ultimately determine the size and shape of this organelle. This review examines some of the regulatory mechanisms involved in these processes and how they occur at specific sites or subcompartments of the endoplasmic reticulum.

Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38).

PubMed Central

Luzio, J P; Brake, B; Banting, G; Howell, K E; Braghetta, P; Stanley, K K

1990-01-01

Organelle-specific integral membrane proteins were identified by a novel strategy which gives rise to monospecific antibodies to these proteins as well as to the cDNA clones encoding them. A cDNA expression library was screened with a polyclonal antiserum raised against Triton X-114-extracted organelle proteins and clones were then grouped using antibodies affinity-purified on individual fusion proteins. The identification, molecular cloning and sequencing are described of a type 1 membrane protein (TGN38) which is located specifically in the trans-Golgi network. Images Fig. 1. Fig. 3. PMID:2204342

Sorting by COP I-coated vesicles under interphase and mitotic conditions

PubMed Central

1996-01-01

COP I-coated vesicles were analyzed for their content of resident Golgi enzymes (N-acetylgalactosaminyltransferase; N- acetylglucosaminyltransferase I; mannosidase II; galactosyltransferase), cargo (rat serum albumin; polyimmunoglobulin receptor), and recycling proteins (-KDEL receptor; ERGIC-53/p58) using biochemical and morphological techniques. The levels of these proteins were similar when the vesicles were prepared under interphase or mitotic conditions showing that sorting was unaffected. The average density relative to starting membranes for resident enzymes (14-30%), cargo (16-23%), and recycling proteins (81-125%) provides clues to the function of COP I vesicles in transport through the Golgi apparatus. PMID:8830771

Adaptor protein 2–mediated endocytosis of the β-secretase BACE1 is dispensable for amyloid precursor protein processing

PubMed Central

Prabhu, Yogikala; Burgos, Patricia V.; Schindler, Christina; Farías, Ginny G.; Magadár, Javier G.; Bonifacino, Juan S.

2012-01-01

The β-site amyloid precursor protein (APP)–cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER–Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway. PMID:22553349

Experiment K-6-03. Gravity and skeletal growth, part 1. Part 2: Morphology and histochemistry of bone cells and vasculature of the tibia; Part 3: Nuclear volume analysis of osteoblast histogenesis in periodontal ligament cells; Part 4: Intervertebral disc swelling pressure associated with microgravity

NASA Technical Reports Server (NTRS)

Holton, E.; Hargens, A.; Gonsalves, M.; Berretta, D.; Doty, S.; Roberts, W.; Garetto, L.; Kaplansky, A.; Durnova, G.; Gott, S.

1990-01-01

Bone area, bone electrophysiology, bone vascularity, osteoblast morphology, and osteoblast histogenesis were studied in rats associated with Cosmos 1887. The results suggest that the synchronous animals were the only group with a significantly larger bone area than the basal group, that the bone electrical potential was more negative in flight than in the synchronous rats, that the endosteal osteoblasts from flight rats had greater numbers of transitional Golgi vesicles but no difference in the large Golgi saccules or the alkaline phosphatase activity, that the perioteal vasculature in the shaft of flight rats often showed very dense intraluminal deposits with adjacent degenerating osteocytes as well as lipid accumulations within the lumen of the vessels and sometimes degeneration of the vascular wall (this change was not present in the metaphyseal region of flight animals), and that the progenitor cells decreased in flight rats while the preosteoblasts increased compared to controls. Many of the results suggest that the animals were beginning to recover from the effects of spaceflight during the two day interval between landing and euthanasia; flight effects, such as the vascular changes, did not appear to recover.

Phosphatidylinositol transfer protein beta displays minimal sphingomyelin transfer activity and is not required for biosynthesis and trafficking of sphingomyelin.

PubMed

Ségui, Bruno; Allen-Baume, Victoria; Cockcroft, Shamshad

2002-08-15

Mammalian phosphatidylinositol transfer proteins (PITPs) alpha and beta, which share 77% identity, have been shown to exhibit distinct lipid-transfer activities. In addition to transferring phosphatidylinositol (PI) and phosphatidylcholine (PC), PITPbeta has been shown to transfer sphingomyelin (SM), and this has led to the suggestion that PITPbeta is important for the regulation of SM metabolism. In the present study, we have analysed the ability of human PITPbeta to transfer and regulate the metabolism of cellular SM. We report that, in vitro, the two PITP isoforms were comparable in mediating PI, PC or SM transfer. Using permeabilized HL-60 cells as the donor compartment, both PITP isoforms efficiently transferred PI and PC, and were slightly active towards SM, with the activity of PITPbeta being slightly greater. To identify which cellular lipids were selected by PITPs, PITPalpha and PITPbeta were exposed to permeabilized HL-60 cells, and subsequently repurified and analysed for their bound lipids. Both PITPs were able to select only PI and PC, but not SM. SM synthesis takes place at the Golgi, and PITPbeta was shown to localize in that compartment. To examine the role of PITPbeta in SM biosynthesis, Golgi membranes were used. Purified Golgi membranes had lost their endogenous PITPbeta, but were able to recruit PITPbeta when added exogenously. However, PITPbeta did not enhance the activities of either SM synthase or glucosylceramide synthase. Further analysis in COS-7 cells overexpressing PITPbeta showed no effects on (a) SM and glucosylceramide biosynthesis, (b) diacylglycerol or ceramide levels, (c) SM transport from the Golgi to the plasma membrane, or (d) resynthesis of SM after exogenous sphingomyelinase treatment. Altogether, these observations do not support the suggestion that PITPbeta participates in the transfer of SM, the regulation of SM biosynthesis or its intracellular trafficking.

Integrating glycomics and genomics uncovers SLC10A7 as essential factor for bone mineralization by regulating post-Golgi protein transport and glycosylation.

PubMed

Ashikov, Angel; Abu Bakar, Nurulamin; Wen, Xiao-Yan; Niemeijer, Marco; Rodrigues Pinto Osorio, Glentino; Brand-Arzamendi, Koroboshka; Hasadsri, Linda; Hansikova, Hana; Raymond, Kimiyo; Vicogne, Dorothée; Simon, Marleen E H; Pfundt, Rolph; Timal, Sharita; Beumers, Roel; Biot, Christophe; Smeets, Roel; Kersten, Marjan; Huijben, Karin; Linders, Peter T A; van den Bogaart, Geert; van Hijum, Sacha A F T; Rodenburg, Richard; van den Heuvel, Lambertus P; van Spronsen, Francjan; Honzik, Tomas; Foulquier, Francois; van Scherpenzeel, Monique; Lefeber, Dirk J

2018-06-05

Genomics methodologies have significantly improved elucidation of Mendelian disorders. The combination with high-throughput functional-omics technologies potentiates the identification and confirmation of causative genetic variants, especially in singleton families of recessive inheritance. In a cohort of 99 individuals with abnormal Golgi glycosylation, 47 of which being unsolved, glycomics profiling was performed of total plasma glycoproteins. Combination with whole-exome sequencing in 31 cases revealed a known genetic defect in 15 individuals. To identify additional genetic factors, hierarchical clustering of the plasma glycomics data was done, which indicated a subgroup of four patients that shared a unique glycomics signature of hybrid type N-glycans. In two siblings, compound heterozygous mutations were found in SLC10A7, a gene of unknown function in human. These included a missense mutation that disrupted transmembrane domain 4 and a mutation in a splice acceptor site resulting in skipping of exon 9. The two other individuals showed a complete loss of SLC10A7 mRNA. The patients' phenotype consisted of amelogenesis imperfecta, skeletal dysplasia, and decreased bone mineral density compatible with osteoporosis. The patients' phenotype was mirrored in SLC10A7 deficient zebrafish. Furthermore, alizarin red staining of calcium deposits in zebrafish morphants showed a strong reduction in bone mineralization. Cell biology studies in fibroblasts of affected individuals showed intracellular mislocalization of glycoproteins and a defect in post-Golgi transport of glycoproteins to the cell membrane. In contrast to yeast, human SLC10A7 localized to the Golgi.Our combined data indicate an important role for SLC10A7 in bone mineralization and transport of glycoproteins to the extracellular matrix.

The Kringle-like Domain Facilitates Post-endoplasmic Reticulum Changes to Premelanosome Protein (PMEL) Oligomerization and Disulfide Bond Configuration and Promotes Amyloid Formation*

PubMed Central

Ho, Tina; Watt, Brenda; Spruce, Lynn A.; Seeholzer, Steven H.; Marks, Michael S.

2016-01-01

The formation of functional amyloid must be carefully regulated to prevent the accumulation of potentially toxic products. Premelanosome protein (PMEL) forms non-toxic functional amyloid fibrils that assemble into sheets upon which melanins ultimately are deposited within the melanosomes of pigment cells. PMEL is synthesized in the endoplasmic reticulum but forms amyloid only within post-Golgi melanosome precursors; thus, PMEL must traverse the secretory pathway in a non-amyloid form. Here, we identified two pre-amyloid PMEL intermediates that likely regulate the timing of fibril formation. Analyses by non-reducing SDS-PAGE, size exclusion chromatography, and sedimentation velocity revealed two native high Mr disulfide-bonded species that contain Golgi-modified forms of PMEL. These species correspond to disulfide bond-containing dimeric and monomeric PMEL isoforms that contain no other proteins as judged by two-dimensional PAGE of metabolically labeled/immunoprecipitated PMEL and by mass spectrometry of affinity-purified complexes. Metabolic pulse-chase analyses, small molecule inhibitor treatments, and evaluation of site-directed mutants suggest that the PMEL dimer forms around the time of endoplasmic reticulum exit and is resolved by disulfide bond rearrangement into a monomeric form within the late Golgi or a post-Golgi compartment. Mutagenesis of individual cysteine residues within the non-amyloid cysteine-rich Kringle-like domain stabilizes the disulfide-bonded dimer and impairs fibril formation as determined by electron microscopy. Our data show that the Kringle-like domain facilitates the resolution of disulfide-bonded PMEL dimers and promotes PMEL functional amyloid formation, thereby suggesting that PMEL dimers must be resolved to monomers to generate functional amyloid fibrils. PMID:26694611

Wide-range high-resolution transmission electron microscopy reveals morphological and distributional changes of endomembrane compartments during log to stationary transition of growth phase in tobacco BY-2 cells.

PubMed

Toyooka, Kiminori; Sato, Mayuko; Kutsuna, Natsumaro; Higaki, Takumi; Sawaki, Fumie; Wakazaki, Mayumi; Goto, Yumi; Hasezawa, Seiichiro; Nagata, Noriko; Matsuoka, Ken

2014-09-01

Rapid growth of plant cells by cell division and expansion requires an endomembrane trafficking system. The endomembrane compartments, such as the Golgi stacks, endosome and vesicles, are important in the synthesis and trafficking of cell wall materials during cell elongation. However, changes in the morphology, distribution and number of these compartments during the different stages of cell proliferation and differentiation have not yet been clarified. In this study, we examined these changes at the ultrastructural level in tobacco Bright yellow 2 (BY-2) cells during the log and stationary phases of growth. We analyzed images of the BY-2 cells prepared by the high-pressure freezing/freeze substitution technique with the aid of an auto-acquisition transmission electron microscope system. We quantified the distribution of secretory and endosomal compartments in longitudinal sections of whole cells by using wide-range gigapixel-class images obtained by merging thousands of transmission electron micrographs. During the log phase, all Golgi stacks were composed of several thick cisternae. Approximately 20 vesicle clusters (VCs), including the trans-Golgi network and secretory vesicle cluster, were observed throughout the cell. In the stationary-phase cells, Golgi stacks were thin with small cisternae, and only a few VCs were observed. Nearly the same number of multivesicular body and small high-density vesicles were observed in both the stationary and log phases. Results from electron microscopy and live fluorescence imaging indicate that the morphology and distribution of secretory-related compartments dramatically change when cells transition from log to stationary phases of growth. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Determinants in the β and δ subunit cytoplasmic loop regulate Golgi trafficking and surface expression of the muscle acetylcholine receptor.

PubMed

Rudell, Jolene Chang; Borges, Lucia S; Rudell, John B; Beck, Kenneth A; Ferns, Michael J

2014-01-03

The molecular determinants that govern nicotinic acetylcholine receptor (AChR) assembly and trafficking are poorly defined, and those identified operate largely during initial receptor biogenesis in the endoplasmic reticulum. To identify determinants that regulate later trafficking steps, we performed an unbiased screen using chimeric proteins consisting of CD4 fused to the muscle AChR subunit cytoplasmic loops. In C2 mouse muscle cells, we found that CD4-β and δ subunit loops were expressed at very low levels on the cell surface, whereas the other subunit loops were robustly expressed on the plasma membrane. The low surface expression of CD4-β and δ loops was due to their pronounced retention in the Golgi apparatus and also to their rapid internalization from the plasma membrane. Both retention and recovery were mediated by the proximal 25-28 amino acids in each loop and were dependent on an ordered sequence of charged and hydrophobic residues. Indeed, βK353L and δK351L mutations increased surface trafficking of the CD4-subunit loops by >6-fold and also decreased their internalization from the plasma membrane. Similarly, combined βK353L and δK351L mutations increased the surface levels of assembled AChR expressed in HEK cells to 138% of wild-type levels. This was due to increased trafficking to the plasma membrane and not decreased AChR turnover. These findings identify novel Golgi retention signals in the β and δ subunit loops that regulate surface trafficking of assembled AChR and may help prevent surface expression of unassembled subunits. Together, these results define molecular determinants that govern a Golgi-based regulatory step in nicotinic AChR trafficking.

Molecular Determinants and Dynamics of Hepatitis C Virus Secretion

PubMed Central

Coller, Kelly E.; Heaton, Nicholas S.; Berger, Kristi L.; Cooper, Jacob D.; Saunders, Jessica L.; Randall, Glenn

2012-01-01

The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells. PMID:22241992

In Contrast to Chlamydia trachomatis, Waddlia chondrophila Grows in Human Cells without Inhibiting Apoptosis, Fragmenting the Golgi Apparatus, or Diverting Post-Golgi Sphingomyelin Transport

PubMed Central

Dille, Stephanie; Kleinschnitz, Eva-Maria; Kontchou, Collins Waguia; Nölke, Thilo

2015-01-01

The Chlamydiales are an order of obligate intracellular bacteria sharing a developmental cycle inside a cytosolic vacuole, with very diverse natural hosts, from amoebae to mammals. The clinically most important species is Chlamydia trachomatis. Many uncertainties remain as to how Chlamydia organizes its intracellular development and replication. The discovery of new Chlamydiales species from other families permits the comparative analysis of cell-biological events and may indicate events that are common to all or peculiar to some species and more or less tightly linked to “chlamydial” development. We used this approach in the infection of human cells with Waddlia chondrophila, a species from the family Waddliaceae whose natural host is uncertain. Compared to C. trachomatis, W. chondrophila had slightly different growth characteristics, including faster cytotoxicity. The embedding in cytoskeletal structures was not as pronounced as for the C. trachomatis inclusion. C. trachomatis infection generates proteolytic activity by the protease Chlamydia protease-like activity factor (CPAF), which degrades host substrates upon extraction; these substrates were not cleaved in the case of W. chondrophila. Unlike Chlamydia, W. chondrophila did not protect against staurosporine-induced apoptosis. C. trachomatis infection causes Golgi apparatus fragmentation and redirects post-Golgi sphingomyelin transport to the inclusion; both were absent from W. chondrophila-infected cells. When host cells were infected with both species, growth of both species was reduced. This study highlights differences between bacterial species that both depend on obligate intracellular replication inside an inclusion. Some features seem principally dispensable for intracellular development of Chlamydiales in vitro but may be linked to host adaptation of Chlamydia and the higher virulence of C. trachomatis. PMID:26056386

Function of OPG as a traffic regulator for RANKL is crucial for controlled osteoclastogenesis.

PubMed

Aoki, Shigeki; Honma, Masashi; Kariya, Yoshiaki; Nakamichi, Yuko; Ninomiya, Tadashi; Takahashi, Naoyuki; Udagawa, Nobuyuki; Suzuki, Hiroshi

2010-09-01

The amount of the receptor activator of NF-κB ligand (RANKL) on the osteoblastic cell surface is considered to determine the magnitude of the signal input to osteoclast precursors and the degree of osteoclastogenesis. Previously, we have shown that RANKL is localized predominantly in lysosomal organelles, but little is found on the osteoblastic cell surface, and consequently, the regulated subcellular trafficking of RANKL in osteoblastic cells is important for controlled osteoclastogenesis. Here we have examined the involvement of osteoprotegerin (OPG), which is currently recognized as a decoy receptor for RANKL, in the regulation of RANKL behavior. It was suggested that OPG already makes a complex with RANKL in the Golgi apparatus and that the complex formation is necessary for RANKL sorting to the secretory lysosomes. It was also shown that each structural domain of OPG is indispensable for exerting OPG function as a traffic regulator. In particular, the latter domains of OPG, whose physiologic functions have been unclear, were indicated to sort RANKL molecules to lysosomes from the Golgi apparatus. In addition, the overexpression of RANK-OPG chimeric protein, which retained OPG function as a decoy receptor but lost the function as a traffic regulator, inhibited endogenous OPG function as a traffic regulator selectively in osteoblastic cells and resulted in the upregulation of osteoclastogenic ability despite the increased number of decoy receptor molecules. Conclusively, OPG function as a traffic regulator for RANKL is crucial for regulating osteoclastogenesis at least as well as that as a decoy receptor. © 2010 American Society for Bone and Mineral Research.

Autophagy in Saccharomyces cerevisiae requires the monomeric GTP-binding proteins, Arl1 and Ypt6.

PubMed

Yang, Shu; Rosenwald, Anne G

2016-10-02

Macroautophagy/autophagy is a cellular degradation process that sequesters organelles or proteins into a double-membrane structure called the phagophore; this transient compartment matures into an autophagosome, which then fuses with the lysosome or vacuole to allow hydrolysis of the cargo. Factors that control membrane traffic are also essential for each step of autophagy. Here we demonstrate that 2 monomeric GTP-binding proteins in Saccharomyces cerevisiae, Arl1 and Ypt6, which belong to the Arf/Arl/Sar protein family and the Rab family, respectively, and control endosome-trans-Golgi traffic, are also necessary for starvation-induced autophagy under high temperature stress. Using established autophagy-specific assays we found that cells lacking either ARL1 or YPT6, which exhibit synthetic lethality with one another, were unable to undergo autophagy at an elevated temperature, although autophagy proceeds normally at normal growth temperature; specifically, strains lacking one or the other of these genes are unable to construct the autophagosome because these 2 proteins are required for proper traffic of Atg9 to the phagophore assembly site (PAS) at the restrictive temperature. Using degron technology to construct an inducible arl1Δ ypt6Δ double mutant, we demonstrated that cells lacking both genes show defects in starvation-inducted autophagy at the permissive temperature. We also found Arl1 and Ypt6 participate in autophagy by targeting the Golgi-associated retrograde protein (GARP) complex to the PAS to regulate the anterograde trafficking of Atg9. Our data show that these 2 membrane traffic regulators have novel roles in autophagy.

The impact of multiple memory formation on dendritic complexity in the hippocampus and anterior cingulate cortex assessed at recent and remote time points

PubMed Central

Wartman, Brianne C.; Holahan, Matthew R.

2014-01-01

Consolidation processes, involving synaptic and systems level changes, are suggested to stabilize memories once they are formed. At the synaptic level, dendritic structural changes are associated with long-term memory storage. At the systems level, memory storage dynamics between the hippocampus and anterior cingulate cortex (ACC) may be influenced by the number of sequentially encoded memories. The present experiment utilized Golgi-Cox staining and neuron reconstruction to examine recent and remote structural changes in the hippocampus and ACC following training on three different behavioral procedures. Rats were trained on one hippocampal-dependent task only (a water maze task), two hippocampal-dependent tasks (a water maze task followed by a radial arm maze task), or one hippocampal-dependent and one non-hippocampal-dependent task (a water maze task followed by an operant conditioning task). Rats were euthanized recently or remotely. Brains underwent Golgi-Cox processing and neurons were reconstructed using Neurolucida software (MicroBrightField, Williston, VT, USA). Rats trained on two hippocampal-dependent tasks displayed increased dendritic complexity compared to control rats, in neurons examined in both the ACC and hippocampus at recent and remote time points. Importantly, this behavioral group showed consistent, significant structural differences in the ACC compared to the control group at the recent time point. These findings suggest that taxing the demand placed upon the hippocampus, by training rats on two hippocampal-dependent tasks, engages synaptic and systems consolidation processes in the ACC at an accelerated rate for recent and remote storage of spatial memories. PMID:24795581

A theory of cerebellar cortex and adaptive motor control based on two types of universal function approximation capability.

PubMed

Fujita, Masahiko

2016-03-01

Lesions of the cerebellum result in large errors in movements. The cerebellum adaptively controls the strength and timing of motor command signals depending on the internal and external environments of movements. The present theory describes how the cerebellar cortex can control signals for accurate and timed movements. A model network of the cerebellar Golgi and granule cells is shown to be equivalent to a multiple-input (from mossy fibers) hierarchical neural network with a single hidden layer of threshold units (granule cells) that receive a common recurrent inhibition (from a Golgi cell). The weighted sum of the hidden unit signals (Purkinje cell output) is theoretically analyzed regarding the capability of the network to perform two types of universal function approximation. The hidden units begin firing as the excitatory inputs exceed the recurrent inhibition. This simple threshold feature leads to the first approximation theory, and the network final output can be any continuous function of the multiple inputs. When the input is constant, this output becomes stationary. However, when the recurrent unit activity is triggered to decrease or the recurrent inhibition is triggered to increase through a certain mechanism (metabotropic modulation or extrasynaptic spillover), the network can generate any continuous signals for a prolonged period of change in the activity of recurrent signals, as the second approximation theory shows. By incorporating the cerebellar capability of two such types of approximations to a motor system, in which learning proceeds through repeated movement trials with accompanying corrections, accurate and timed responses for reaching the target can be adaptively acquired. Simple models of motor control can solve the motor error vs. sensory error problem, as well as the structural aspects of credit (or error) assignment problem. Two physiological experiments are proposed for examining the delay and trace conditioning of eyelid responses, as well as saccade adaptation, to investigate this novel idea of cerebellar processing. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ultrastructural pathology of cortical capillary pericytes in human traumatic brain oedema.

PubMed

Castejón, Orlando J

2011-01-01

In human traumatic brain oedema pericytes exhibit remarkable oedematous changes, increased vacuolar and vesicular transport, transient transpericytal channels, and tubular structures demonstrating pericyte brain barrier dysfunction. They show nuclear invaginations, actin and myosin-like filaments, and coupled interaction with endothelial cells through the macula occludens. Some pericytes display hypertrophic and necrotic changes, and phagocytic capacity. Hypertrophic pericytes induce basement membrane splitting. Degenerated pericytes exhibit lacunar enlargement of endoplasmic reticulum, dense osmiophilic bodies, glycogen granules, vacuolization, oedematous Golgi apparatus, and pleomorphic mitochondria. Certain micropinocytotic vesicles are orientated to the Golgi complex and multivesicular bodies, suggesting that pericytes play some role in oedema resolution.

SCRG1, a potential marker of autophagy in transmissible spongiform encephalopathies.

PubMed

Dron, Michel; Bailly, Yannick; Beringue, Vincent; Haeberlé, Anne-Marie; Griffond, Bernadette; Risold, Pierre-Yves; Tovey, Michael G; Laude, Hubert; Dandoy-Dron, Françoise

2006-01-01

The Scrg1 gene was initially discovered as one of the genes upregulated in transmissible spongiform encephalopathies (TSE). Scrg1 encodes a highly conserved, cysteine-rich protein expressed principally in the central nervous system. The protein is targeted to the Golgi apparatus and large dense-core vesicles/secretory granules in neurons. We have recently shown that the Scrg1 protein is widely induced in neurons of scrapie-infected mice, suggesting that Scrg1 is involved in the host response to stress and/or the death of neurons. At the ultrastructural level, Scrg1 is associated with dictyosomes of the Golgi apparatus and autophagic vacuoles of degenerative neurons. It is well known that apoptosis plays a major role in the events leading to neuronal cell death in TSE. However, autophagy was identified in experimentally induced scrapie a long time ago and was recently reevaluated as a possible cell death program in prion diseases. The consistent association of Scrg1 with autophagic structures typical of scrapie is in agreement with the recruitment of Golgi-specific proteins in this degradation process and we suggest that Scrg1 might be used as a specific probe to identify neuronal autophagy in TSE.

Disturbed vesicular trafficking of membrane proteins in prion disease.

PubMed

Uchiyama, Keiji; Miyata, Hironori; Sakaguchi, Suehiro

2013-01-01

The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.

Connectingthe puzzle pieces between cytoskeleton andsecretory pathway

PubMed Central

Gurel, Pinar S.; Hatch, Anna L.; Higgs, Henry N.

2014-01-01

A tendency in cell biology is to divide and conquer. For example, decades of painstaking work have led to an understanding of endoplasmic reticulum (ER) and Golgi structure, dynamics, and transport. In parallel, cytoskeletal researchers have revealed a fantastic diversity of structure and cellular function in both actin and microtubules. Increasingly, these areas overlap, necessitating an understanding of both organelle and cytoskeletal biology. This review addressesconnections between the actin/microtubule cytoskeletons and organelles in animal cells, focusing on threetopics: ER structure/function, ER-to-Golgi transport; and Golgi structure/function. Making these connections has been challenging, due to 1) the small sizes and dynamic characteristics of some components, 2) the fact that organelle-specific cytoskeleton can easily be obscured by more abundant cytoskeletal structures, and 3) the difficulties in imaging membranes and cytoskeleton simultaneously, especially at the ultra-structural level. One major concept is that the cytoskeleton is frequently used to generate force for membrane movement, with two potential consequences: translocation of the organelle, or deformation of the organelle membrane. While initially discussing issues common to metazoan cells in general, we subsequently highlight specific features of neurons, since these highly polarized cells present unique challenges for organellar distribution and dynamics. PMID:25050967

Ceramide Phosphoethanolamine Biosynthesis in Drosophila Is Mediated by a Unique Ethanolamine Phosphotransferase in the Golgi Lumen♦

PubMed Central

Vacaru, Ana M.; van den Dikkenberg, Joep; Ternes, Philipp; Holthuis, Joost C. M.

2013-01-01

Sphingomyelin (SM) is a vital component of mammalian membranes, providing mechanical stability and a structural framework for plasma membrane organization. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase in the Golgi lumen. Drosophila lacks SM and instead synthesizes the SM analogue ceramide phosphoethanolamine (CPE) as the principal membrane sphingolipid. The corresponding CPE synthase shares mechanistic features with enzymes mediating phospholipid biosynthesis via the Kennedy pathway. Using a functional cloning strategy, we here identified a CDP-ethanolamine:ceramide ethanolamine phosphotransferase as the enzyme responsible for CPE production in Drosophila. CPE synthase constitutes a new branch within the CDP-alcohol phosphotransferase superfamily with homologues in Arthropoda (insects, spiders, mites, scorpions), Cnidaria (Hydra, sea anemones), and Mollusca (oysters) but not in most other animal phyla. The enzyme resides in the Golgi complex with its active site facing the lumen, contrary to the membrane topology of other CDP-alcohol phosphotransferases. Our findings open up an important new avenue to address the biological role of CPE, an enigmatic membrane constituent of a wide variety of invertebrate and marine organisms. PMID:23449981

α-Synuclein-induced lysosomal dysfunction occurs through disruptions in protein trafficking in human midbrain synucleinopathy models.

PubMed

Mazzulli, Joseph R; Zunke, Friederike; Isacson, Ole; Studer, Lorenz; Krainc, Dimitri

2016-02-16

Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by the accumulation of protein aggregates comprised of α-synuclein (α-syn). A major barrier in treatment discovery for PD is the lack of identifiable therapeutic pathways capable of reducing aggregates in human neuronal model systems. Mutations in key components of protein trafficking and cellular degradation machinery represent important risk factors for PD; however, their precise role in disease progression and interaction with α-syn remains unclear. Here, we find that α-syn accumulation reduced lysosomal degradation capacity in human midbrain dopamine models of synucleinopathies through disrupting hydrolase trafficking. Accumulation of α-syn at the cell body resulted in aberrant association with cis-Golgi-tethering factor GM130 and disrupted the endoplasmic reticulum-Golgi localization of rab1a, a key mediator of vesicular transport. Overexpression of rab1a restored Golgi structure, improved hydrolase trafficking and activity, and reduced pathological α-syn in patient neurons. Our work suggests that enhancement of lysosomal hydrolase trafficking may prove beneficial in synucleinopathies and indicates that human midbrain disease models may be useful for identifying critical therapeutic pathways in PD and related disorders.

Retention of secretory proteins in an intermediate compartment and disappearance of the Golgi complex in an END4 mutant of Chinese hamster ovary cells

PubMed Central

1992-01-01

Mutant V.24.1, a member of the End4 complementation group of temperature-sensitive CHO cells, is defective in secretion at the restrictive temperature (Wang, R.-H., P. A. Colbaugh, C.-Y. Kao, E. A. Rutledge, and R. K. Draper. 1990. J. Biol. Chem. 265:20179-20187; Presley, J. F., R. K. Draper, and D. T. Brown. 1991. J. Virol. 65:1332- 1339). We have further investigated the secretory lesion and report three main findings. First, the block in secretion is not due to aberrant folding or oligomerization of secretory proteins in the endoplasmic reticulum because the hemagglutinin of influenza virus folded and oligomerized at the same rate in mutant and parental cells at the restrictive temperature. Second, secretory proteins accumulated in a compartment intermediate between the ER and the Golgi. Several lines of evidence support this conclusion, the most direct being the colocalization by immunofluorescence microscopy of influenza virus hemagglutinin with a 58-kD protein that is known to reside in an intermediate compartment. Third, at the resolution of fluorescence microscopy, the Golgi complex in the mutant cells vanished at the restrictive temperature. PMID:1577851

Changes in the Golgi Apparatus of Neocortical and Hippocampal Neurons in the Hibernating Hamster.

PubMed

Antón-Fernández, Alejandro; León-Espinosa, Gonzalo; DeFelipe, Javier; Muñoz, Alberto

2015-01-01

Hibernating animals have been used as models to study several aspects of the plastic changes that occur in the metabolism and physiology of neurons. These models are also of interest in the study of Alzheimer's disease because the microtubule-associated protein tau is hyperphosphorylated during the hibernation state known as torpor, similar to the pretangle stage of Alzheimer's disease. Hibernating animals undergo torpor periods with drops in body temperature and metabolic rate, and a virtual cessation of neural activity. These processes are accompanied by morphological and neurochemical changes in neurons, which reverse a few hours after coming out of the torpor state. Since tau has been implicated in the structural regulation of the neuronal Golgi apparatus (GA) we have used Western Blot and immunocytochemistry to analyze whether the GA is modified in cortical neurons of the Syrian hamster at different hibernation stages. The results show that, during the hibernation cycle, the GA undergo important structural changes along with differential modifications in expression levels and distribution patterns of Golgi structural proteins. These changes were accompanied by significant transitory reductions in the volume and surface area of the GA elements during torpor and arousal stages as compared with euthermic animals.

Changes in the Golgi Apparatus of Neocortical and Hippocampal Neurons in the Hibernating Hamster

PubMed Central

Antón-Fernández, Alejandro; León-Espinosa, Gonzalo; DeFelipe, Javier; Muñoz, Alberto

2015-01-01

Hibernating animals have been used as models to study several aspects of the plastic changes that occur in the metabolism and physiology of neurons. These models are also of interest in the study of Alzheimer's disease because the microtubule-associated protein tau is hyperphosphorylated during the hibernation state known as torpor, similar to the pretangle stage of Alzheimer's disease. Hibernating animals undergo torpor periods with drops in body temperature and metabolic rate, and a virtual cessation of neural activity. These processes are accompanied by morphological and neurochemical changes in neurons, which reverse a few hours after coming out of the torpor state. Since tau has been implicated in the structural regulation of the neuronal Golgi apparatus (GA) we have used Western Blot and immunocytochemistry to analyze whether the GA is modified in cortical neurons of the Syrian hamster at different hibernation stages. The results show that, during the hibernation cycle, the GA undergo important structural changes along with differential modifications in expression levels and distribution patterns of Golgi structural proteins. These changes were accompanied by significant transitory reductions in the volume and surface area of the GA elements during torpor and arousal stages as compared with euthermic animals. PMID:26696838

A polygalacturonase localized in the Golgi apparatus in Pisum sativum.

PubMed

Ohashi, Takao; Jinno, Jun; Inoue, Yoshiyuki; Ito, Shoko; Fujiyama, Kazuhito; Ishimizu, Takeshi

2017-09-01

Pectin is a plant cell wall constituent that is mainly composed of polygalacturonic acid (PGA), a linear α1,4-d-galacturonic acid (GalUA) backbone. Polygalacturonase (PG) hydrolyzes the α1,4-linkages in PGA. Nearly all plant PGs identified thus far are secreted as soluble proteins. Here we describe the microsomal PG activity in pea (Pisum sativum) epicotyls and present biochemical evidence that it was localized to the Golgi apparatus, where pectins are biosynthesized. The microsomal PG was purified, and it was enzymatically characterized. The purified enzyme showed maximum activity towards pyridylaminated oligogalacturonic acids with six degrees of polymerization (PA-GalUA6), with a Km value of 11 μM for PA-GalUA6. The substrate preference of the enzyme was complementary to that of PGA synthase. The main PG activity in microsomes was detected in the Golgi fraction by sucrose density gradient ultracentrifugation. The activity of the microsomal PG was lower in rapidly growing epicotyls, in contrast to the high expression of PGA synthase. The role of this PG in the regulation of pectin biosynthesis or plant growth is discussed. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Crystal Structure of the Golgi-Associated Human Nα-Acetyltransferase 60 Reveals the Molecular Determinants for Substrate-Specific Acetylation.

PubMed

Støve, Svein Isungset; Magin, Robert S; Foyn, Håvard; Haug, Bengt Erik; Marmorstein, Ronen; Arnesen, Thomas

2016-07-06

N-Terminal acetylation is a common and important protein modification catalyzed by N-terminal acetyltransferases (NATs). Six human NATs (NatA-NatF) contain one catalytic subunit each, Naa10 to Naa60, respectively. In contrast to the ribosome-associated NatA to NatE, NatF/Naa60 specifically associates with Golgi membranes and acetylates transmembrane proteins. To gain insight into the molecular basis for the function of Naa60, we developed an Naa60 bisubstrate CoA-peptide conjugate inhibitor, determined its X-ray structure when bound to CoA and inhibitor, and carried out biochemical experiments. We show that Naa60 adapts an overall fold similar to that of the catalytic subunits of ribosome-associated NATs, but with the addition of two novel elongated loops that play important roles in substrate-specific binding. One of these loops mediates a dimer to monomer transition upon substrate-specific binding. Naa60 employs a catalytic mechanism most similar to Naa50. Collectively, these data reveal the molecular basis for Naa60-specific acetyltransferase activity with implications for its Golgi-specific functions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fukutin-related protein localizes to the Golgi apparatus and mutations lead to mislocalization in muscle in vivo.

PubMed

Keramaris-Vrantsis, Elizabeth; Lu, Pei J; Doran, Timothy; Zillmer, Allen; Ashar, Jignya; Esapa, Christopher T; Benson, Matthew A; Blake, Derek J; Rosenfeld, Jeffrey; Lu, Qi L

2007-10-01

Mutations in the fukutin-related protein gene (FKRP) are associated with a spectrum of diseases from mild limb-girdle muscular dystrophy type 2I to severe congenital muscular dystrophy type 1C, muscle-eye-brain disease (MEB), and Walker-Warburg syndrome (WWS). The effect of mutations on the transportation of the mutant proteins may constitute the underlying mechanisms for the pathogenesis of these diseases. Here we examined the subcellular localization of mouse and human normal and mutant FKRP proteins in cells and in muscle in vivo. Both normal human and mouse FKRPs localize in part of the Golgi apparatus in muscle fibers. Mutations in the FKRP gene invariably altered the localization of the protein, leading to endoplasmic reticulum retention within cells and diminished Golgi localization in muscle fibers. Our results therefore suggest that an individual missense point mutation can confer at least two independent effects on the protein, causing (1) reduction or loss of the presumed glycosyltransferase activity directly and (2) mislocalization that could further alter the function of the protein. The complexity of the effect of individual missense point mutations may partly explain the wide variation of the FKRP-related myopathies.

Golgi- and Trans-Golgi Network-Mediated Vesicle Trafficking Is Required for Wax Secretion from Epidermal Cells1[W][OPEN

PubMed Central

McFarlane, Heather E.; Watanabe, Yoichiro; Yang, Weili; Huang, Yan; Ohlrogge, John; Samuels, A. Lacey

2014-01-01

Lipid secretion from epidermal cells to the plant surface is essential to create the protective plant cuticle. Cuticular waxes are unusual secretory products, consisting of a variety of highly hydrophobic compounds including saturated very-long-chain alkanes, ketones, and alcohols. These compounds are synthesized in the endoplasmic reticulum (ER) but must be trafficked to the plasma membrane for export by ATP-binding cassette transporters. To test the hypothesis that wax components are trafficked via the endomembrane system and packaged in Golgi-derived secretory vesicles, Arabidopsis (Arabidopsis thaliana) stem wax secretion was assayed in a series of vesicle-trafficking mutants, including gnom like1-1 (gnl1-1), transport particle protein subunit120-4, and echidna (ech). Wax secretion was dependent upon GNL1 and ECH. Independent of secretion phenotypes, mutants with altered ER morphology also had decreased wax biosynthesis phenotypes, implying that the biosynthetic capacity of the ER is closely related to its structure. These results provide genetic evidence that wax export requires GNL1- and ECH-dependent endomembrane vesicle trafficking to deliver cargo to plasma membrane-localized ATP-binding cassette transporters. PMID:24468625

The Amyloid Precursor Protein is rapidly transported from the Golgi apparatus to the lysosome and where it is processed into beta-amyloid

PubMed Central

2014-01-01

Background Alzheimer’s disease (AD) is characterized by cerebral deposition of β-amyloid peptide (Aβ). Aβ is produced by sequential cleavage of the Amyloid Precursor Protein (APP) by β- and γ-secretases. Many studies have demonstrated that the internalization of APP from the cell surface can regulate Aβ production, although the exact organelle in which Aβ is produced remains contentious. A number of recent studies suggest that intracellular trafficking also plays a role in regulating Aβ production, but these pathways are relatively under-studied. The goal of this study was to elucidate the intracellular trafficking of APP, and to examine the site of intracellular APP processing. Results We have tagged APP on its C-terminal cytoplasmic tail with photoactivatable Green Fluorescent Protein (paGFP). By photoactivating APP-paGFP in the Golgi, using the Golgi marker Galactosyltranferase fused to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to follow a population of nascent APP molecules from the Golgi to downstream compartments identified with compartment markers tagged with red fluorescent protein (mRFP or mCherry); including rab5 (early endosomes) rab9 (late endosomes) and LAMP1 (lysosomes). Because γ-cleavage of APP releases the cytoplasmic tail of APP including the photoactivated GFP, resulting in loss of fluorescence, we are able to visualize the cleavage of APP in these compartments. Using APP-paGFP, we show that APP is rapidly trafficked from the Golgi apparatus to the lysosome; where it is rapidly cleared. Chloroquine and the highly selective γ-secretase inhibitor, L685, 458, cause the accumulation of APP in lysosomes implying that APP is being cleaved by secretases in the lysosome. The Swedish mutation dramatically increases the rate of lysosomal APP processing, which is also inhibited by chloroquine and L685, 458. By knocking down adaptor protein 3 (AP-3; a heterotetrameric protein complex required for trafficking many proteins to the lysosome) using siRNA, we are able to reduce this lysosomal transport. Blocking lysosomal transport of APP reduces Aβ production by more than a third. Conclusion These data suggests that AP-3 mediates rapid delivery of APP to lysosomes, and that the lysosome is a likely site of Aβ production. PMID:25085554

Identification of a new B4GalNAcT1 (GM2/GD2/GA2 synthase) isoform, and regulation of enzyme stability and intracellular transport by arginine-based motif.

PubMed

Shishido, Fumi; Uemura, Satoshi; Kashimura, Madoka; Inokuchi, Jin-Ichi

2017-10-01

Glycosphingolipids (GSLs) are abundant in plasma membranes of mammalian cells, and their synthesis is strictly regulated in the Golgi apparatus. Disruption of GSL homeostasis is the cause of numerous diseases. Hundreds of molecular species of GSLs exist, and the detailed mechanisms underlying their homeostasis remain unclear. We investigated the physiological significance of isoform production for β1,4-N-acetyl-galactosaminyl transferase 1/B4GALNT1 (B4GN1), an enzyme involved in synthesis of ganglio-series GSLs GM2/GD2/GA2. We discovered a new mRNA variant (termed variant 2) of B4GN1 through EST clone search. A new isoform, M1-B4GN1, which has an NH 2 -terminal cytoplasmic tail longer than that of previously-known isoform M2-B4GN1, is translated from variant 2. M1-B4GN1 has R-based motif (a retrograde transport signal) in the cytoplasmic tail. M1-B4GN1 is partially localized in the endoplasmic reticulum (ER) depending on the R-based motif, whereas M2-B4GN1 is localized in the Golgi. Stability of M1-B4GN1 is higher than that of M2-B4GN1 because of the R-based motif. M2-B4GN1 forms a homodimer via disulfide bonding. When M1-B4GN1 and M2-B4GN1 were co-expressed in CHO-K1 cells, the two isoforms formed a heterodimer. The M1/M2-B4GN1 heterodimer was more stable than the M2-B4GN1 homodimer, but the heterodimer was not transported from the Golgi to the ER. Our findings indicate that stabilization of M1-B4GN1 homodimer and M1/M2-B4GN1 heterodimer by R-based motif is related to prolongation of Golgi retention, but not to retrograde transport from the Golgi to the ER. Coexistence of several B4GN1 isoforms having distinctive characteristics presumably helps maintain overall enzyme stability and GSL homeostasis. Copyright © 2017 Elsevier B.V. All rights reserved.

VPS35 in Dopamine Neurons Is Required for Endosome-to-Golgi Retrieval of Lamp2a, a Receptor of Chaperone-Mediated Autophagy That Is Critical for α-Synuclein Degradation and Prevention of Pathogenesis of Parkinson's Disease

PubMed Central

Tang, Fu-Lei; Erion, Joanna R.; Tian, Yun; Liu, Wei; Yin, Dong-Min; Ye, Jian; Tang, Baisha; Mei, Lin

2015-01-01

Vacuolar protein sorting-35 (VPS35) is essential for endosome-to-Golgi retrieval of membrane proteins. Mutations in the VPS35 gene have been identified in patients with autosomal dominant PD. However, it remains poorly understood if and how VPS35 deficiency or mutation contributes to PD pathogenesis. Here we provide evidence that links VPS35 deficiency to PD-like neuropathology. VPS35 was expressed in mouse dopamine (DA) neurons in substantia nigra pars compacta (SNpc) and STR (striatum)—regions that are PD vulnerable. VPS35-deficient mice exhibited PD-relevant deficits including accumulation of α-synuclein in SNpc-DA neurons, loss of DA transmitter and DA neurons in SNpc and STR, and impairment of locomotor behavior. Further mechanical studies showed that VPS35-deficient DA neurons or DA neurons expressing PD-linked VPS35 mutant (D620N) had impaired endosome-to-Golgi retrieval of lysosome-associated membrane glycoprotein 2a (Lamp2a) and accelerated Lamp2a degradation. Expression of Lamp2a in VPS35-deficient DA neurons reduced α-synuclein, supporting the view for Lamp2a as a receptor of chaperone-mediated autophagy to be critical for α-synuclein degradation. These results suggest that VPS35 deficiency or mutation promotes PD pathogenesis and reveals a crucial pathway, VPS35-Lamp2a-α-synuclein, to prevent PD pathogenesis. SIGNIFICANCE STATEMENT VPS35 is a key component of the retromer complex that is essential for endosome-to-Golgi retrieval of membrane proteins. Mutations in the VPS35 gene have been identified in patients with PD. However, if and how VPS35 deficiency or mutation contributes to PD pathogenesis remains unclear. We demonstrated that VPS35 deficiency or mutation (D620N) in mice leads to α-synuclein accumulation and aggregation in the substantia nigra, accompanied with DA neurodegeneration. VPS35-deficient DA neurons exhibit impaired endosome-to-Golgi retrieval of Lamp2a, which may contribute to the reduced α-synuclein degradation through chaperone-mediated autophagy. These results suggest that VPS35 deficiency or mutation promotes PD pathogenesis, and reveals a crucial pathway, VPS35-Lamp2a-α-synuclein, to prevent PD pathogenesis. PMID:26203154

Expression and localization of GLUT1 and GLUT12 in prostate carcinoma.

PubMed

Chandler, Jenalle D; Williams, Elizabeth D; Slavin, John L; Best, James D; Rogers, Suzanne

2003-04-15

Increased glucose consumption is a characteristic of malignant cells and in prostate carcinoma is associated with the proliferation of both androgen-dependent and independent cells. Transport of polar glucose across the nonpolar membrane relies on glucose transporter proteins, known as GLUTs. Increased expression of GLUT1 is a characteristic of many malignant cells. The authors characterized and cloned the cDNA for a novel glucose transporter, GLUT12, which was identified initially in malignant breast epithelial cells. To the authors' knowledge, there have been no reports on the expression of glucose transporters in the human prostate or human prostate carcinoma cells. The authors evaluated GLUT1 and GLUT12 expression in human prostate carcinoma cells. Reverse transcription-polymerase chain reaction was performed on total RNA extracted from cultured prostate carcinoma cells LNCaP, C4, C4-2, and C4-2B using primers to amplify GLUT1, GLUT12, or the housekeeping gene, 36B4. Total protein extracted from prostate carcinoma cell lines was assessed for GLUT12 protein by Western blot analysis. Cultured cell monolayers were incubated with antibodies to GLUT1 or GLUT12 and a peripheral Golgi protein, Golgi 58K, for detection by immunofluorescent confocal microscopy. Sections of benign prostatic hyperplasia and human prostate carcinoma were stained for immunohistochemical detection of GLUT1 and GLUT12. GLUT1 and GLUT12 mRNA and protein were detected in all cell lines evaluated. Immunofluorescence staining demonstrated both GLUT1 and GLUT12 on the plasma membrane and in the cytoplasm in all cultured prostate carcinoma cell lines, with GLUT1 but not GLUT12 appearing to colocalize with the Golgi. Immunohistochemical staining of benign prostatic hyperplasia indicated expression of GLUT1 but not GLUT12. Malignant tissue stained for GLUT12 but was negative for GLUT1. GLUT1 and GLUT12 are expressed in human prostate carcinoma cells. One possible rationale for the GLUT1 Golgi association is that it may supply glucose to the Golgi for byproduct incorporation into the prostatic secretory fluid. Further work will investigate the importance of glucose transport and GLUT1 and GLUT12 in prostate carcinoma cell growth. Copyright 2003 American Cancer Society.

Monkeypox Virus Host Factor Screen Using Haploid Cells Identifies Essential Role of GARP Complex in Extracellular Virus Formation.

PubMed

Realegeno, Susan; Puschnik, Andreas S; Kumar, Amrita; Goldsmith, Cynthia; Burgado, Jillybeth; Sambhara, Suryaprakash; Olson, Victoria A; Carroll, Darin; Damon, Inger; Hirata, Tetsuya; Kinoshita, Taroh; Carette, Jan E; Satheshkumar, Panayampalli Subbian

2017-06-01

Monkeypox virus (MPXV) is a human pathogen that is a member of the Orthopoxvirus genus, which includes Vaccinia virus and Variola virus (the causative agent of smallpox). Human monkeypox is considered an emerging zoonotic infectious disease. To identify host factors required for MPXV infection, we performed a genome-wide insertional mutagenesis screen in human haploid cells. The screen revealed several candidate genes, including those involved in Golgi trafficking, glycosaminoglycan biosynthesis, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. We validated the role of a set of vacuolar protein sorting (VPS) genes during infection, VPS51 to VPS54 (VPS51-54), which comprise the Golgi-associated retrograde protein (GARP) complex. The GARP complex is a tethering complex involved in retrograde transport of endosomes to the trans -Golgi apparatus. Our data demonstrate that VPS52 and VPS54 were dispensable for mature virion (MV) production but were required for extracellular virus (EV) formation. For comparison, a known antiviral compound, ST-246, was used in our experiments, demonstrating that EV titers in VPS52 and VPS54 knockout (KO) cells were comparable to levels exhibited by ST-246-treated wild-type cells. Confocal microscopy was used to examine actin tail formation, one of the viral egress mechanisms for cell-to-cell dissemination, and revealed an absence of actin tails in VPS52KO- or VPS54KO-infected cells. Further evaluation of these cells by electron microscopy demonstrated a decrease in levels of wrapped viruses (WVs) compared to those seen with the wild-type control. Collectively, our data demonstrate the role of GARP complex genes in double-membrane wrapping of MVs necessary for EV formation, implicating the host endosomal trafficking pathway in orthopoxvirus infection. IMPORTANCE Human monkeypox is an emerging zoonotic infectious disease caused by Monkeypox virus (MPXV). Of the two MPXV clades, the Congo Basin strain is associated with severe disease, increased mortality, and increased human-to-human transmission relative to the West African strain. Monkeypox is endemic in regions of western and central Africa but was introduced into the United States in 2003 from the importation of infected animals. The threat of MPXV and other orthopoxviruses is increasing due to the absence of routine smallpox vaccination leading to a higher proportion of naive populations. In this study, we have identified and validated candidate genes that are required for MPXV infection, specifically, those associated with the Golgi-associated retrograde protein (GARP) complex. Identifying host targets required for infection that prevents extracellular virus formation such as the GARP complex or the retrograde pathway can provide a potential target for antiviral therapy. Copyright © 2017 American Society for Microbiology.

Ultrastructure of meristem and root cap of pea seedlings under spaceflight conditions

NASA Technical Reports Server (NTRS)

Sytnyk, K. M.; Kordyum, E. L.; Bilyavska, N. O.; Tarasenko, V. O.

1983-01-01

Data of electron microscopic analysis of meristem and root cap of pea seedlings grown aboard the Salyut-6 orbital research station in the Oazis apparatus and in the laboratory are presented. The main morphological and anatomical characteristics of the test and control plants are shown to be similar. At the same time, some differences are found in the structural and functional organization of the experimental cells as compared to the controls. They concern first of all the plastic apparatus, mitochondria and Golgi apparatus. It is assumed that cell function for certain periods of weightlessness on the whole ensures execution of the cytodifferentiation programs genetically determined on the Earth. Biochemical and physiological processes vary rather markedly due to lack of initially rigorous determination.

Fission Yeast SCYL1/2 Homologue Ppk32: A Novel Regulator of TOR Signalling That Governs Survival during Brefeldin A Induced Stress to Protein Trafficking

PubMed Central

Petersen, Janni

2016-01-01

Target of Rapamycin (TOR) signalling allows eukaryotic cells to adjust cell growth in response to changes in their nutritional and environmental context. The two distinct TOR complexes (TORC1/2) localise to the cell’s internal membrane compartments; the endoplasmic reticulum (ER), Golgi apparatus and lysosomes/vacuoles. Here, we show that Ppk32, a SCYL family pseudo-kinase, is a novel regulator of TOR signalling. The absence of ppk32 expression confers resistance to TOR inhibition. Ppk32 inhibition of TORC1 is critical for cell survival following Brefeldin A (BFA) induced stress. Treatment of wild type cells with either the TORC1 specific inhibitor rapamycin or the general TOR inhibitor Torin1 confirmed that a reduction in TORC1 activity promoted recovery from BFA induced stress. Phosphorylation of Ppk32 on two residues that are conserved within the SCYL pseudo-kinase family are required for this TOR inhibition. Phosphorylation on these sites controls Ppk32 protein levels and sensitivity to BFA. BFA induced ER stress does not account for the response to BFA that we report here, however BFA is also known to induce Golgi stress and impair traffic to lysosomes. In summary, Ppk32 reduce TOR signalling in response to BFA induced stress to support cell survival. PMID:27191590

The statolith compartment in Chara rhizoids contains carbohydrate and protein.

PubMed

Wang-Cahill, F; Kiss, J Z

1995-02-01

In contrast to higher plants, the alga Chara has rhizoids with single membrane-bound compartments that function as statoliths in gravity perception. Previous work has demonstrated that these statoliths contain barium sulfate crystals. In this study, we show that statoliths in Chara rhizoids react with a Coomassie Brilliant Blue cytochemical stain for proteins. While statoliths did not react with silver methenamine carbohydrate cytochemistry, the monoclonal antibody CCRC-M2, which is against a carbohydrate (sycamore-maple rhamnogalacturonan I), labeled the statolith compartment. These results demonstrate that in addition to barium sulfate, statoliths in Chara rhizoids have an organic matrix that consists of protein and carbohydrate moieties. Since the statoliths were silver methenamine negative, the carbohydrate in this compartment could be a 3-linked polysaccharide. CCRC-M2 also labeled Golgi cisternae, Golgi-associated vesicles, apical vesicles, and cell walls in the rhizoids. The specificity of CCRC-M2 immunolabeling was verified by several control experiments, including the demonstration that labeling was abolished when the antibody was preabsorbed with its antigen. Since in this and a previous study (John Z. Kiss and L. Andrew Staehelin, American Journal of Botany 80: 273-282, 1993) antibodies against higher plant carbohydrates crossreacted with cell walls of Chara in a specific manner, Characean algae may be a useful model system in biochemical and molecular studies of cell walls.

The statolith compartment in Chara rhizoids contains carbohydrate and protein

NASA Technical Reports Server (NTRS)

Wang-Cahill, F.; Kiss, J. Z.

1995-01-01

In contrast to higher plants, the alga Chara has rhizoids with single membrane-bound compartments that function as statoliths in gravity perception. Previous work has demonstrated that these statoliths contain barium sulfate crystals. In this study, we show that statoliths in Chara rhizoids react with a Coomassie Brilliant Blue cytochemical stain for proteins. While statoliths did not react with silver methenamine carbohydrate cytochemistry, the monoclonal antibody CCRC-M2, which is against a carbohydrate (sycamore-maple rhamnogalacturonan I), labeled the statolith compartment. These results demonstrate that in addition to barium sulfate, statoliths in Chara rhizoids have an organic matrix that consists of protein and carbohydrate moieties. Since the statoliths were silver methenamine negative, the carbohydrate in this compartment could be a 3-linked polysaccharide. CCRC-M2 also labeled Golgi cisternae, Golgi-associated vesicles, apical vesicles, and cell walls in the rhizoids. The specificity of CCRC-M2 immunolabeling was verified by several control experiments, including the demonstration that labeling was abolished when the antibody was preabsorbed with its antigen. Since in this and a previous study (John Z. Kiss and L. Andrew Staehelin, American Journal of Botany 80: 273-282, 1993) antibodies against higher plant carbohydrates crossreacted with cell walls of Chara in a specific manner, Characean algae may be a useful model system in biochemical and molecular studies of cell walls.

Effect of monensin on the levels of tachykinins and their processing enzyme activity in rat dorsal root ganglia.

PubMed

Chikuma, Toshiyuki; Inomata, Yuji; Tsuchida, Ken; Hojo, Hiroshi; Kato, Takeshi

2002-06-28

Th effect of monensin, which inhibits trans-Golgi function, on the levels of tachykinins and their processing enzyme activity was examined in organ-cultured rat dorsal root ganglia (DRG). Using an enzyme immunoassay method, we measured neurokinin A and substance P immunoreactivity in the DRG cultured for 72 h with and without 0.1 microM monensin. Both tachykinins were reduced in the DRG treated with monensin. Treatment with monensin also reduced the activity of carboxypeptidase E, which is one of the proteolytic processing enzymes of neuropeptides. These data suggest that proteolytic processing enzymes may in part modulate the biological activity of neuropeptides within a trans-Golgi apparatus.

Active Dendrites and Differential Distribution of Calcium Channels Enable Functional Compartmentalization of Golgi Cells.

PubMed

Rudolph, Stephanie; Hull, Court; Regehr, Wade G

2015-11-25

Interneurons are essential to controlling excitability, timing, and synaptic integration in neuronal networks. Golgi cells (GoCs) serve these roles at the input layer of the cerebellar cortex by releasing GABA to inhibit granule cells (grcs). GoCs are excited by mossy fibers (MFs) and grcs and provide feedforward and feedback inhibition to grcs. Here we investigate two important aspects of GoC physiology: the properties of GoC dendrites and the role of calcium signaling in regulating GoC spontaneous activity. Although GoC dendrites are extensive, previous studies concluded they are devoid of voltage-gated ion channels. Hence, the current view holds that somatic voltage signals decay passively within GoC dendrites, and grc synapses onto distal dendrites are not amplified and are therefore ineffective at firing GoCs because of strong passive attenuation. Using whole-cell recording and calcium imaging in rat slices, we find that dendritic voltage-gated sodium channels allow somatic action potentials to activate voltage-gated calcium channels (VGCCs) along the entire dendritic length, with R-type and T-type VGCCs preferentially located distally. We show that R- and T-type VGCCs located in the dendrites can boost distal synaptic inputs and promote burst firing. Active dendrites are thus critical to the regulation of GoC activity, and consequently, to the processing of input to the cerebellar cortex. In contrast, we find that N-type channels are preferentially located near the soma, and control the frequency and pattern of spontaneous firing through their close association with calcium-activated potassium (KCa) channels. Thus, VGCC types are differentially distributed and serve specialized functions within GoCs. Interneurons are essential to neural processing because they modulate excitability, timing, and synaptic integration within circuits. At the input layer of the cerebellar cortex, a single type of interneuron, the Golgi cell (GoC), carries these functions. The extent of inhibition depends on both spontaneous activity of GoCs and the excitatory synaptic input they receive. In this study, we find that different types of calcium channels are differentially distributed, with dendritic calcium channels being activated by somatic activity, boosting synaptic inputs and enabling bursting, and somatic calcium cannels promoting regular firing. We therefore challenge the current view that GoC dendrites are passive and identify the mechanisms that contribute to GoCs regulating the flow of sensory information in the cerebellar cortex. Copyright © 2015 the authors 0270-6474/15/3515492-13$15.00/0.

An ABA-regulated and Golgi-localized protein phosphatase controls water loss during leaf senescence in Arabidopsis.

PubMed

Zhang, Kewei; Xia, Xiuying; Zhang, Yanyan; Gan, Su-Sheng

2012-02-01

It is known that a senescing leaf loses water faster than a non-senescing leaf and that ABA has an important role in promoting leaf senescence. However, questions such as why water loss is faster, how water loss is regulated, and how ABA functions in leaf senescence are not well understood. Here we report on the identification and functional analysis of a leaf senescence associated gene called SAG113. The RNA blot and GUS reporter analyses all show that SAG113 is expressed in senescing leaves and is induced by ABA in Arabidopsis. The SAG113 expression levels are significantly reduced in aba2 and abi4 mutants. A GFP fusion protein analysis revealed that SAG113 protein is localized in the Golgi apparatus. SAG113 encodes a protein phosphatase that belongs to the PP2C family and is able to functionally complement a yeast PP2C-deficient mutant TM126 (ptc1Δ). Leaf senescence is delayed in the SAG113 knockout mutant compared with that in the wild type, stomatal movement in the senescing leaves of SAG113 knockouts is more sensitive to ABA than that of the wild type, and the rate of water loss in senescing leaves of SAG113 knockouts is significantly reduced. In contrast, inducible over-expression of SAG113 results in a lower sensitivity of stomatal movement to ABA treatment, more rapid water loss, and precocious leaf senescence. No other aspects of growth and development, including seed germination, were observed. These findings suggest that SAG113, a negative regulator of ABA signal transduction, is specifically involved in the control of water loss during leaf senescence. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Herpes Simplex Virus Type 1 Neuronal Infection Perturbs Golgi Apparatus Integrity through Activation of Src Tyrosine Kinase and Dyn-2 GTPase

PubMed Central

Martin, Carolina; Leyton, Luis; Hott, Melissa; Arancibia, Yennyfer; Spichiger, Carlos; McNiven, Mark A.; Court, Felipe A.; Concha, Margarita I.; Burgos, Patricia V.; Otth, Carola

2017-01-01

Herpes simplex virus type 1 (HSV-1) is a ubiquitous pathogen that establishes a latent persistent neuronal infection in humans. The pathogenic effects of repeated viral reactivation in infected neurons are still unknown. Several studies have reported that during HSV-1 epithelial infection, the virus could modulate diverse cell signaling pathways remodeling the Golgi apparatus (GA) membranes, but the molecular mechanisms implicated, and the functional consequences to neurons is currently unknown. Here we report that infection of primary neuronal cultures with HSV-1 triggers Src tyrosine kinase activation and subsequent phosphorylation of Dynamin 2 GTPase, two players with a role in GA integrity maintenance. Immunofluorescence analyses showed that HSV-1 productive neuronal infection caused a scattered and fragmented distribution of the GA through the cytoplasm, contrasting with the uniform perinuclear distribution pattern observed in control cells. In addition, transmission electron microscopy revealed swollen cisternae and disorganized stacks in HSV-1 infected neurons compared to control cells. Interestingly, PP2, a selective inhibitor for Src-family kinases markedly reduced these morphological alterations of the GA induced by HSV-1 infection strongly supporting the possible involvement of Src tyrosine kinase. Finally, we showed that HSV-1 tegument protein VP11/12 is necessary but not sufficient to induce Dyn2 phosphorylation. Altogether, these results show that HSV-1 neuronal infection triggers activation of Src tyrosine kinase, phosphorylation of Dynamin 2 GTPase, and perturbation of GA integrity. These findings suggest a possible neuropathogenic mechanism triggered by HSV-1 infection, which could involve dysfunction of the secretory system in neurons and central nervous system. PMID:28879169

Herpes Simplex Virus Type 1 Neuronal Infection Perturbs Golgi Apparatus Integrity through Activation of Src Tyrosine Kinase and Dyn-2 GTPase.

PubMed

Martin, Carolina; Leyton, Luis; Hott, Melissa; Arancibia, Yennyfer; Spichiger, Carlos; McNiven, Mark A; Court, Felipe A; Concha, Margarita I; Burgos, Patricia V; Otth, Carola

2017-01-01

Herpes simplex virus type 1 (HSV-1) is a ubiquitous pathogen that establishes a latent persistent neuronal infection in humans. The pathogenic effects of repeated viral reactivation in infected neurons are still unknown. Several studies have reported that during HSV-1 epithelial infection, the virus could modulate diverse cell signaling pathways remodeling the Golgi apparatus (GA) membranes, but the molecular mechanisms implicated, and the functional consequences to neurons is currently unknown. Here we report that infection of primary neuronal cultures with HSV-1 triggers Src tyrosine kinase activation and subsequent phosphorylation of Dynamin 2 GTPase, two players with a role in GA integrity maintenance. Immunofluorescence analyses showed that HSV-1 productive neuronal infection caused a scattered and fragmented distribution of the GA through the cytoplasm, contrasting with the uniform perinuclear distribution pattern observed in control cells. In addition, transmission electron microscopy revealed swollen cisternae and disorganized stacks in HSV-1 infected neurons compared to control cells. Interestingly, PP2, a selective inhibitor for Src-family kinases markedly reduced these morphological alterations of the GA induced by HSV-1 infection strongly supporting the possible involvement of Src tyrosine kinase. Finally, we showed that HSV-1 tegument protein VP11/12 is necessary but not sufficient to induce Dyn2 phosphorylation. Altogether, these results show that HSV-1 neuronal infection triggers activation of Src tyrosine kinase, phosphorylation of Dynamin 2 GTPase, and perturbation of GA integrity. These findings suggest a possible neuropathogenic mechanism triggered by HSV-1 infection, which could involve dysfunction of the secretory system in neurons and central nervous system.

Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells.

PubMed

Morré, D M; Morre, D J

2000-06-23

Partitioning in dextran-poly(ethylene)glycol (PEG) aqueous-aqueous phase systems represents a mature technology with many applications to separations of cells and to the preparation of membranes from mammalian cells. Most applications to membrane isolation and purification have focused on plasma membranes, plasma membrane domains and separation of right side-out and inside-out plasma membrane vesicles. The method exploits a combination of membrane properties, including charge and hydrophobicity. Purification is based upon differential distributions of the constituents in a sample between the two principal compartments of the two phases (upper and lower) and at the interface. The order of affinity of animal cell membranes for the upper phase is: endoplasmic reticulum

Posttranslational processing of the prohormone-cleaving Kex2 protease in the Saccharomyces cerevisiae secretory pathway.

PubMed

Wilcox, C A; Fuller, R S

1991-10-01

The Kex2 protease of the yeast Saccharomyces cerevisiae is a prototypical eukaryotic prohormone-processing enzyme that cleaves precursors of secreted peptides at pairs of basic residues. Here we have established the pathway of posttranslational modification of Kex2 protein using immunoprecipitation of the biosynthetically pulse-labeled protein from a variety of wild-type and mutant yeast strains as the principal methodology. Kex2 protein is initially synthesized as a prepro-enzyme that undergoes cotranslational signal peptide cleavage and addition of Asn-linked core oligosaccharide and Ser/Thr-linked mannose in the ER. The earliest detectable species, I1 (approximately 129 kD), undergoes rapid amino-terminal proteolytic removal of a approximately 9-kD pro-segment yielding species I2 (approximately 120 kD) before arrival at the Golgi complex. Transport to the Golgi complex is marked by extensive elaboration of Ser/Thr-linked chains and minor modification of Asn-linked oligosaccharide. During the latter phase of its lifetime, Kex2 protein undergoes a gradual increase in apparent molecular weight. This final modification serves as a marker for association of Kex2 protease with a late compartment of the yeast Golgi complex in which it is concentrated about 27-fold relative to other secretory proteins.

Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted root tips of Nicotiana and Arabidopsis

NASA Technical Reports Server (NTRS)

Kiss, J. Z.; Giddings, T. H. Jr; Staehelin, L. A.; Sack, F. D.

1990-01-01

To circumvent the limitations of chemical fixation (CF) and to gain more reliable structural information about higher plant tissues, we have cryofixed root tips of Nicotiana and Arabidopsis by high pressure freezing (HPF). Whereas other freezing techniques preserve tissue to a relatively shallow depth, HPF in conjunction with freeze substitution (FS) resulted in excellent preservation of entire root tips. Compared to CF, in tissue prepared by HPF/FS: (1) the plasmalemma and all internal membranes were much smoother and often coated on the cytoplasmic side by a thin layer of stained material, (2) the plasmalemma was appressed to the cell wall, (3) organelle profiles were rounder, (4) the cytoplasmic, mitochondrial, and amyloplast matrices were denser, (5) vacuoles contained electron dense material, (6) microtubules appeared to be more numerous and straighter, with crossbridges observed between them, (7) cisternae of endoplasmic reticulum (ER) were wider and filled with material, (8) Golgi intercisternal elements were more clearly resolved and were observed between both Golgi vesicles and cisternae, and (9) larger vesicles were associated with Golgi stacks. This study demonstrates that HPF/FS can be used to successfully preserve the ultrastructure of relatively large plant tissues without the use of intracellular cryoprotectants.

Determining the Subcellular Location of Synthesis and Assembly of the Cell Wall Polysaccharide (1,3; 1,4)-β-d-Glucan in Grasses[OPEN

PubMed Central

Wilson, Sarah M.; Ho, Yin Ying; Lampugnani, Edwin R.; Van de Meene, Allison M.L.; Bain, Melissa P.; Bacic, Antony; Doblin, Monika S.

2015-01-01

The current dogma for cell wall polysaccharide biosynthesis is that cellulose (and callose) is synthesized at the plasma membrane (PM), whereas matrix phase polysaccharides are assembled in the Golgi apparatus. We provide evidence that (1,3;1,4)-β-d-glucan (mixed-linkage glucan [MLG]) does not conform to this paradigm. We show in various grass (Poaceae) species that MLG-specific antibody labeling is present in the wall but absent over Golgi, suggesting it is assembled at the PM. Antibodies to the MLG synthases, cellulose synthase-like F6 (CSLF6) and CSLH1, located CSLF6 to the endoplasmic reticulum, Golgi, secretory vesicles, and the PM and CSLH1 to the same locations apart from the PM. This pattern was recreated upon expression of VENUS-tagged barley (Hordeum vulgare) CSLF6 and CSLH1 in Nicotiana benthamiana leaves and, consistent with our biochemical analyses of native grass tissues, shown to be catalytically active with CSLF6 and CSLH1 in PM-enriched and PM-depleted membrane fractions, respectively. These data support a PM location for the synthesis of MLG by CSLF6, the predominant enzymatically active isoform. A model is proposed to guide future experimental approaches to dissect the molecular mechanism(s) of MLG assembly. PMID:25770111

The oculocerebrorenal syndrome gene product is a 105-kD protein localized to the Golgi complex.

PubMed Central

Olivos-Glander, I M; Jänne, P A; Nussbaum, R L

1995-01-01

The oculocerebrorenal syndrome of Lowe (OCRL) is a multisystem disorder affecting the lens, kidney, and CNS. The predicted amino acid sequence of the OCRL gene, OCRL-1, was used to develop antibodies against the OCRL-1 protein. Western blot analysis using affinity-purified serum against the amino terminus of the OCRL-1 gene product (ocrl-1) demonstrates a single protein of 105 kD in fibroblasts of a normal individual that is absent in fibroblasts of an OCRL patient who lacks OCRL-1 transcript. A single protein with the same electrophoretic mobility is found by western analysis in various human cultured cell lines, and approximately the same size protein is also found in all mouse tissues tested. Northern analysis of various human and mouse tissues demonstrate that OCRL-1 transcript is expressed in nearly all tissues examined. By immunofluorescence, the ocrl-1 antibody stains a juxtanuclear region in normal fibroblast cells, while no specific staining is evident in the OCRL patient who produces no transcript. Colocalization of the ocrl-1 protein to the Golgi complex was demonstrated using a known monoclonal antibody against a Golgi-specific coat protein, beta-COP (beta coatomer protein). Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7573041

Human CLEC16A regulates autophagy through modulating mTOR activity.

PubMed

Tam, Rachel Chun Yee; Li, Michelle Wing Man; Gao, Yan Pan; Pang, Yuen Ting; Yan, Sheng; Ge, Wei; Lau, Chak Sing; Chan, Vera Sau Fong

2017-03-15

CLEC16A is genetically linked with multiple autoimmune disorders but its functional relevance in autoimmunity remains obscure. Recent evidence has signposted the emerging role of autophagy in autoimmune disease development. Here, by ectopic expression and siRNA silencing, we show that CLEC16A has an inhibitory role in starvation-induced autophagy in human cells. Combining quantitative proteomics and immunoblotting analyses, we found that CLEC16A likely regulates autophagy by activating mTOR pathway. Overexpression of CLEC16A was found to sensitize cells towards the availability of nutrients, resulting in a heightened mTOR activity, which in turn diminished LC3 autophagic activity following nutrient deprivation. CLEC16A deficiency, on the other hand, delayed mTOR activity in response to nutrient sensing, thereby resulted in an augmented autophagic response. CLEC16A was found residing in cytosolic vesicles and the Golgi, and nutrient removal promoted a stronger clustering within the Golgi, where it was possibly in a vantage position to activate mTOR upon nutrient replenishment. These findings suggest that Golgi-associated CLEC16A negatively regulates autophagy via modulation of mTOR activity, and may provide support for a functional link between CLEC16A and autoimmunity. Copyright © 2017 Elsevier Inc. All rights reserved.

Reprint of: Chromogranin A: a new proposal for trafficking, processing and induction of granule biogenesis.

PubMed

Koshimizu, Hisatsugu; Kim, Taeyoon; Cawley, Niamh X; Loh, Y Peng

2010-11-30

Chromogranin A (CgA), a member of the granin family serves several important cell biological roles in (neuro)endocrine cells which are summarized in this review. CgA is a "prohormone" that is synthesized at the rough endoplasmic reticulum and transported into the cisternae of this organelle via its signal peptide. It is then trafficked to the Golgi complex and then to the trans-Golgi network (TGN) where CgA aggregates at low pH in the presence of calcium. The CgA aggregates provide the physical driving force to induce budding of the TGN membrane resulting in dense core granule (DCG) formation. Within the granule, a small amount of the CgA is processed to bioactive peptides, including a predicted C-terminal peptide, serpinin. Upon stimulation, DCGs undergo exocytosis and CgA and its derived peptides are released. Serpinin, acting extracellularly is able to signal the increase in transcription of a serine protease inhibitor, protease nexin-1 (PN-1) that protects DCG proteins against degradation in the Golgi complex, which then enhances DCG biogenesis to replenish those that were released. Thus CgA and its derived peptide, serpinin, plays a significant role in granule formation and regulation of granule biogenesis, respectively, in (neuro) endocrine cells. Copyright © 2010. Published by Elsevier B.V.

Reprint of: Chromogranin A: A new proposal for trafficking, processing and induction of granule biogenesis☆

PubMed Central

Koshimizu, Hisatsugu; Kim, Taeyoon; Cawley, Niamh X.; Loh, Y. Peng

2014-01-01

Chromogranin A (CgA), a member of the granin family serves several important cell biological roles in (neuro) endocrine cells which are summarized in this review. CgA is a “prohormone” that is synthesized at the rough endoplasmic reticulum and transported into the cisternae of this organelle via its signal peptide. It is then trafficked to the Golgi complex and then to the trans-Golgi network (TGN) where CgA aggregates at low pH in the presence of calcium. The CgA aggregates provide the physical driving force to induce budding of the TGN membrane resulting in dense core granule (DCG) formation. Within the granule, a small amount of the CgA is processed to bioactive peptides, including a predicted C-terminal peptide, serpinin. Upon stimulation, DCGs undergo exocytosis and CgA and its derived peptides are released. Serpinin, acting extracellularly is able to signal the increase in transcription of a serine protease inhibitor, protease nexin-1 (PN-1) that protects DCG proteins against degradation in the Golgi complex, which then enhances DCG biogenesis to replenish those that were released. Thus CgA and its derived peptide, serpinin, plays a significant role in granule formation and regulation of granule biogenesis, respectively, in (neuro) endocrine cells. PMID:20920534

Chromogranin A: A New Proposal for Trafficking, Processing and Induction of Granule Biogenesis

PubMed Central

Koshimizu, Hisatsugu; Kim, Taeyoon; Cawley, Niamh X.; Loh, Y. Peng

2009-01-01

Chromogranin A (CgA), a member of the granin family serves several important cell biological roles in (neuro)endocrine cells which are summarized in this review. CgA is a “prohormone” that is synthesized at the rough endoplasmic reticulum and transported into the cisternae of this organelle via its signal peptide. It is then trafficked to the Golgi complex and then to the trans-Golgi network (TGN) where CgA aggregates at low pH in the presence of calcium. The CgA aggregates provide the physical driving force to induce budding of the TGN membrane resulting in dense core granule (DCG) formation. Within the granule, a small amount of the CgA is processed to bioactive peptides, including a predicted C-terminal peptide, serpinin. Upon stimulation, DCGs undergo exocytosis and CgA and its derived peptides are released. Serpinin, acting extracellularly is able to signal the increase in transcription of a serine protease inhibitor, protease nexin-1 (PN-1) that protects DCG proteins against degradation in the Golgi complex, which then enhances DCG biogenesis to replenish those that were released. Thus CgA and its derived peptide, serpinin, plays a significant role in the formation and regulation, respectively, of granule biogenesis in (neuro)endocrine cells. PMID:20006653

Chromogranin A: a new proposal for trafficking, processing and induction of granule biogenesis.

PubMed

Koshimizu, Hisatsugu; Kim, Taeyoon; Cawley, Niamh X; Loh, Y Peng

2010-02-25

Chromogranin A (CgA), a member of the granin family serves several important cell biological roles in (neuro)endocrine cells which are summarized in this review. CgA is a "prohormone" that is synthesized at the rough endoplasmic reticulum and transported into the cisternae of this organelle via its signal peptide. It is then trafficked to the Golgi complex and then to the trans-Golgi network (TGN) where CgA aggregates at low pH in the presence of calcium. The CgA aggregates provide the physical driving force to induce budding of the TGN membrane resulting in dense core granule (DCG) formation. Within the granule, a small amount of the CgA is processed to bioactive peptides, including a predicted C-terminal peptide, serpinin. Upon stimulation, DCGs undergo exocytosis and CgA and its derived peptides are released. Serpinin, acting extracellularly is able to signal the increase in transcription of a serine protease inhibitor, protease nexin-1 (PN-1) that protects DCG proteins against degradation in the Golgi complex, which then enhances DCG biogenesis to replenish those that were released. Thus CgA and its derived peptide, serpinin, plays a significant role in granule formation and regulation of granule biogenesis, respectively, in (neuro) endocrine cells. Published by Elsevier B.V.

Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells

NASA Technical Reports Server (NTRS)

Morre, D. M.; Morre, D. J.

2000-01-01

Partitioning in dextran-poly(ethylene)glycol (PEG) aqueous-aqueous phase systems represents a mature technology with many applications to separations of cells and to the preparation of membranes from mammalian cells. Most applications to membrane isolation and purification have focused on plasma membranes, plasma membrane domains and separation of right side-out and inside-out plasma membrane vesicles. The method exploits a combination of membrane properties, including charge and hydrophobicity. Purification is based upon differential distributions of the constituents in a sample between the two principal compartments of the two phases (upper and lower) and at the interface. The order of affinity of animal cell membranes for the upper phase is: endoplasmic reticulum

[Effect of Golgi α-mannosidase 2 (GM2) gene knockdown on adhesion abilities of human gastric carcinoma cell line BGC-823 and its mechanism].

PubMed

Zeng, Bo; Zeng, Zhen; Liu, Chang; Yang, Yaying

2017-06-01

Objective To investigate the effect of Golgi α-mannosidase II (GM2) gene knockdown on adhesion abilities of BGC-823 human gastric carcinoma cells. Methods Three plasmid vectors expressing GM2 shRNAs and a negative control plasmid vector were designed, constructed and then transfected into BGC-823 cells by Lipofectamine TM 2000. After transfection, the mRNA and protein levels of GM2 in BGC-823 cells were detected by real-time quantitative PCR (qRT-PCR) and Western blotting to evaluate the transfection efficacy. The best plasmid for GM2 gene knockdown was selected and stably transfected into BGC-823 cells. Adhesion abilities of BGC-823 cells after GM2 gene silencing were observed by cell-cell, cell-matrix and cell-endothelial cell adhesion assays. At the same time, the expressions of E-cadherin, P-selectin, CD44v6 and intercellular adhesion molecule-1 (ICAM-1) proteins were detected by Western blotting after GM2 gene knockdown. Results The expression of GM2 was effectively knockdown in GM2-shRNA-2-transfected BGC-823 cells. Compared with the blank control group and the negative control group, the intercellular adhesion ability of the GM2-shRNA-2-transfected cells increased significantly, while their cell-matrix and cell-endothelium adhesion abilities markedly decreased. In GM2-shRNA-2 transfection group, E-cadherin expression was significantly elevated and the P-selectin expression was significantly reduced, while the expression levels of CD44v6 and ICAM-1 were not obviously changed. Conclusion After GM2 gene knockdown, the intercellular adhesion ability of gastric carcinoma BGC-823 cells is enhanced, while the adhesion abilities with the extracellular matrix and endothelial cells are weakened. The changes might be related to the up-regulated expression of E-cadherin and the down-regulation of P-selectin.

The calcium-binding protein ALG-2 regulates protein secretion and trafficking via interactions with MISSL and MAP1B proteins.

PubMed

Takahara, Terunao; Inoue, Kuniko; Arai, Yumika; Kuwata, Keiko; Shibata, Hideki; Maki, Masatoshi

2017-10-13

Mobilization of intracellular calcium is essential for a wide range of cellular processes, including signal transduction, apoptosis, and vesicular trafficking. Several lines of evidence have suggested that apoptosis-linked gene 2 (ALG-2, also known as PDCD6 ), a calcium-binding protein, acts as a calcium sensor linking calcium levels with efficient vesicular trafficking, especially at the endoplasmic reticulum (ER)-to-Golgi transport step. However, how ALG-2 regulates these processes remains largely unclear. Here, we report that M APK1- i nteracting and s pindle- s tabilizing (MISS)- l ike (MISSL), a previously uncharacterized protein, interacts with ALG-2 in a calcium-dependent manner. Live-cell imaging revealed that upon a rise in intracellular calcium levels, GFP-tagged MISSL (GFP-MISSL) dynamically relocalizes in a punctate pattern and colocalizes with ALG-2. MISSL knockdown caused disorganization of the components of the ER exit site, the ER-Golgi intermediate compartment, and Golgi. Importantly, knockdown of either MISSL or ALG-2 attenuated the secretion of se creted a lkaline p hosphatase (SEAP), a model secreted cargo protein, with similar reductions in secretion by single- and double-protein knockdowns, suggesting that MISSL and ALG-2 act in the same pathway to regulate the secretion process. Furthermore, ALG-2 or MISSL knockdown delayed ER-to-Golgi transport of procollagen type I. We also found that ALG-2 and MISSL interact with microtubule-associated protein 1B (MAP1B) and that MAP1B knockdown reverts the reduced secretion of SEAP caused by MISSL or ALG-2 depletion. These results suggest that a change in the intracellular calcium level plays a role in regulation of the secretory pathway via interaction of ALG-2 with MISSL and MAP1B. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanisms Regulating the Secretion of the Promalignancy Chemokine CCL5 by Breast Tumor Cells: CCL5's 40s Loop and Intracellular Glycosaminoglycans12

PubMed Central

Soria, Gali; Lebel-Haziv, Yaeli; Ehrlich, Marcelo; Meshel, Tsipi; Suez, Adva; Avezov, Edward; Rozenberg, Perri; Ben-Baruch, Adit

2012-01-01

The chemokine CCL5 (RANTES) plays active promalignancy roles in breast malignancy. The secretion of CCL5 by breast tumor cells is an important step in its tumor-promoting activities; therefore, inhibition of CCL5 secretion may have antitumorigenic effects. We demonstrate that, in breast tumor cells, CCL5 secretion necessitated the trafficking of CCL5-containing vesicles on microtubules from the endoplasmic reticulum (ER) to the post-Golgi stage, and CCL5 release was regulated by the rigidity of the actin cytoskeleton. Focusing on the 40s loop of CCL5, we found that the 43TRKN46 sequence of CCL5 was indispensable for its inclusion in motile vesicles, and for its secretion. The TRKN-mutated chemokine reached the Golgi, but trafficked along the ER-to-post-Golgi route differently than the wild-type (WT) chemokine. Based on the studies showing that the 40s loop of CCL5 mediates its binding to glycosaminoglycans (GAG), we analyzed the roles of GAG in regulating CCL5 secretion. TRKN-mutated CCL5 had lower propensity for colocalization with GAG in the Golgi compared to the WT chemokine. Secretion of WT CCL5 was significantly reduced in CHO mutant cells deficient in GAG synthesis, and the WT chemokine acquired an ER-like distribution in these cells, similar to that of TRKN-mutated CCL5 in GAG-expressing cells. The release of WT CCL5 was also reduced after inhibition of GAG presence/synthesis by intracellular expression of heparanase, inhibition of GAG sulfation, and sulfate deprivation. The need for a 43TRKN46 motif and for a GAG-mediated process in CCL5 secretion may enable the future design of modalities that prevent CCL5 release by breast tumor cells. PMID:22355269

A mucin-like glycoprotein identified by MAG (mouse ascites Golgi) antibodies. Menstrual cycle-dependent localization in human endometrium.

PubMed Central

Kliman, H. J.; Feinberg, R. F.; Schwartz, L. B.; Feinman, M. A.; Lavi, E.; Meaddough, E. L.

1995-01-01

Human endometrial glands synthesize and secrete a high molecular weight mucin-like glycoprotein in a menstrual cycle-dependent fashion. A novel moiety within this Golgi-associated glycoprotein is strongly reactive with IgG antibodies in numerous murine ascites, and has been termed MAG (mouse ascites Golgi). Immunohistochemical staining of 201 endometrial biopsies revealed the following patterns: MAG first appeared in the Golgi on cycle day 5, peaked on day 15, was present on the surface of the luminal epithelium between days 17 and 19, and was no longer detectable after day 19. MAG was also present in cervical, prostate, seminal vesicle, and lacrimal glands, pancreatic acinar cells, gall bladder and bile duct epithelium, and certain cells of the salivary and sweat glands. Interestingly, only tissues from blood group A individuals exhibited this staining. As a common link among all these cell types is the expression of mucins, we speculated that the MAG epitope could be a mucin-associated blood group A-related epitope. This hypothesis was tested by absorption experiments with a variety of glycoconjugates and erythrocytes and by immunoblots of MAG-rich material. The absorption studies demonstrated that only type III porcine mucin (< 1% sialic acid) and blood type A or AB erythrocytes were able to absorb the anti-MAG antibody. Inasmuch as N-acetyl-galactosamine alone, the terminal blood group A carbohydrate, did not block MAG antibody binding, the MAG epitope appears to involve N-acetylgalactosamine plus other determinants. Immunoblots of endometrial extracts and saliva from blood type A individuals revealed MAG-reactive material with a molecular weight > 200 kd under reducing conditions. Because the MAG epitope appears on the endometrial surface during the purported implantation window, we speculate that mucin-like epitopes could play a role in the earliest apposition phases of conceptus-endometrial interaction. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 PMID:7531946

Sec34p, a Protein Required for Vesicle Tethering to the Yeast Golgi Apparatus, Is in a Complex with Sec35p

PubMed Central

VanRheenen, Susan M.; Cao, Xiaochun; Sapperstein, Stephanie K.; Chiang, Elbert C.; Lupashin, Vladimir V.; Barlowe, Charles; Waters, M. Gerard

1999-01-01

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393–406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)–associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an ∼750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well. PMID:10562277

Internalization and sorting of a fluorescent analogue of glucosylceramide to the Golgi apparatus of human skin fibroblasts: utilization of endocytic and nonendocytic transport mechanisms

PubMed Central

1994-01-01

We examined the uptake and intracellular transport of the fluorescent glucosylceramide analogue N-[5-(5,7-dimethyl BODIPYTM)-1-pentanoyl]- glucosyl sphingosine (C5-DMB-GlcCer) in human skin fibroblasts, and we compared its behavior to that of the corresponding fluorescent analogues of sphingomyelin, galactosylceramide, and lactosylceramide. All four fluorescent analogues were readily transferred from defatted BSA to the plasma membrane during incubation at 4 degrees C. When cells treated with C5-DMB-GlcCer were washed, warmed to 37 degrees C, and subsequently incubated with defatted BSA to remove fluorescent lipid at the cell surface, strong fluorescence was observed at the Golgi apparatus, as well as weaker labeling at the nuclear envelope and other intracellular membranes. Similar results were obtained with C5-DMB- galactosylceramide, except that labeling of the Golgi apparatus was weaker than with C5-DMB-GlcCer. Internalization of C5-DMB-GlcCer was not inhibited by various treatments, including ATP depletion or warming to 19 degrees C, and biochemical analysis demonstrated that the lipid was not metabolized during its internalization. However, accumulation of C5-DMB-GlcCer at the Golgi apparatus was reduced when cells were treated with a nonfluorescent analogue of glucosylceramide, suggesting that accumulation of C5-DMB-GlcCer at the Golgi apparatus was a saturable process. In contrast, cells treated with C5-DMB-analogues of sphingomyelin or lactosylceramide internalized the fluorescent lipid into a punctate pattern of fluorescence during warming at 37 degrees C, and this process was temperature and energy dependent. These results with C5-DMB-sphingomyelin and C5-DMB-lactosylceramide were analogous to those obtained with another fluorescent analogue of sphingomyelin in which labeling of endocytic vesicles and plasma membrane lipid recycling were documented (Koval, M., and R. E. Pagano. 1990. J. Cell Biol. 111:429-442). Incubation of perforated cells with C5-DMB- sphingomyelin resulted in prominent labeling of the nuclear envelope and other intracellular membranes, similar to the pattern observed with C5-DMB-GlcCer in intact cells. These observations are consistent with the transbilayer movement of fluorescent analogues of glucosylceramide and galactosylceramide at the plasma membrane and early endosomes of human skin fibroblasts, and suggest that both endocytic and nonendocytic pathways are used in the internalization of these lipids from the plasma membrane. PMID:8188745

Characterization, expression profiles, intracellular distribution and association analysis of porcine PNAS-4 gene with production traits.

PubMed

Mo, Delin; Zhu, Zhengmao; te Pas, Marinus F W; Li, Xinyun; Yang, Shulin; Wang, Heng; Wang, Huanling; Li, Kui

2008-06-30

In a previous screen to identify differentially expressed genes associated with embryonic development, the porcine PNAS-4 gene had been found. Considering differentially expressed genes in early stages of muscle development are potential candidate genes to improve meat quality and production efficiency, we determined how porcine PNAS-4 gene regulates meat production. Therefore, this gene has been sequenced, expression analyzed and associated with meat production traits. We cloned the full-length cDNA of porcine PNAS-4 gene encoding a protein of 194 amino acids which was expressed in the Golgi complex. This gene was mapped to chromosome 10, q11-16, in a region of conserved synteny with human chromosome 1 where the human homologous gene was localized. Real-time PCR revealed that PNAS-4 mRNA was widely expressed with highest expression levels in skeletal muscle followed by lymph, liver and other tissues, and showed a down-regulated expression pattern during prenatal development while a up-regulated expression pattern after weaning. Association analysis revealed that allele C of SNP A1813C was prevalent in Chinese indigenous breeds whereas A was dominant allele in Landrace and Large White, and the pigs with homozygous CC had a higher fat content than those of the pigs with other genotypes (P < 0.05). Porcine PNAS-4 protein tagged with green fluorescent protein accumulated in the Golgi complex, and its mRNA showed a widespread expression across many tissues and organs in pigs. It may be an important factor affecting the meat production efficiency, because its down-regulated expression pattern during early embryogenesis suggests involvement in increase of muscle fiber number. In addition, the SNP A1813C associated with fat traits might be a genetic marker for molecular-assisted selection in animal breeding.

Maintenance of asymmetric cellular localization of an auxin transport protein through interaction with the actin cytoskeleton

NASA Technical Reports Server (NTRS)

Muday, G. K.

2000-01-01

In shoots, polar auxin transport is basipetal (that is, from the shoot apex toward the base) and is driven by the basal localization of the auxin efflux carrier complex. The focus of this article is to summarize the experiments that have examined how the asymmetric distribution of this protein complex is controlled and the significance of this polar distribution. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through localized secretion of Golgi vesicles, whereas an attachment of a subunit of the efflux carrier to the actin cytoskeleton may maintain this localization. In addition, the idea that this localization of the efflux carrier may control both the polarity of auxin movement and more globally regulate developmental polarity is explored. Finally, evidence indicating that the gravity vector controls auxin transport polarity is summarized and possible mechanisms for the environmentally induced changes in auxin transport polarity are discussed.

The Road not Taken: Less Traveled Roads from the TGN to the Plasma Membrane

PubMed Central

Spang, Anne

2015-01-01

The trans-Golgi network functions in the distribution of cargo into different transport vesicles that are destined to endosomes, lysosomes and the plasma membrane. Over the years, it has become clear that more than one transport pathway promotes plasma membrane localization of proteins. In spite of the importance of temporal and spatial control of protein localization at the plasma membrane, the regulation of sorting into and the formation of different transport containers are still poorly understood. In this review different transport pathways, with a special emphasis on exomer-dependent transport, and concepts of regulation and sorting at the TGN are discussed. PMID:25764365

The Road not Taken: Less Traveled Roads from the TGN to the Plasma Membrane.

PubMed

Spang, Anne

2015-03-10

The trans-Golgi network functions in the distribution of cargo into different transport vesicles that are destined to endosomes, lysosomes and the plasma membrane. Over the years, it has become clear that more than one transport pathway promotes plasma membrane localization of proteins. In spite of the importance of temporal and spatial control of protein localization at the plasma membrane, the regulation of sorting into and the formation of different transport containers are still poorly understood. In this review different transport pathways, with a special emphasis on exomer-dependent transport, and concepts of regulation and sorting at the TGN are discussed.

Glycosyltransferases in the Golgi membranes of onion stem

PubMed Central

Powell, Janet T.; Brew, Keith

1974-01-01

Cell fractions consisting largely of Golgi membranes were prepared from the meristematic region of the onion. Several enzyme activities were found to be localized in these fractions: inosine diphosphatase, galactosyltransferases and glucosyltransferases. The fractions catalysed the transfer of [14C]galactose from UDP-galactose to endogenous and cell-sap acceptors, to N-acetylglucosamine and to ovalbumin. In the presence of bovine α-lactalbumin, transfer to glucose (lactose synthesis) was catalysed. [14C]Glucose was transferred from UDP-glucose to endogenous and cell-sap acceptors, to cellobiose and to fructose (sucrose synthesis). All these activities were latent, being potentiated by detergents (Triton X-100 or sodium deoxycholate). The characteristics of some of these enzyme activities are described and their biological significance is discussed. ImagesPLATE 1 PMID:4374190

Cytochemical localization of phosphatases in the germ- and Sertoli cells of Odontophrynus cultripes (Amphibia, Anura, Leptodactylidae).

PubMed

Fernandes, A P; Báo, S N

1998-08-01

Ultrastructural cytochemical techniques were used for the localization of phosphatases in spermatid and spermatozoon, as well as in Sertoli cells of Odontophrynus cultripes (Amphibia, Anura, Leptodactylidae). Acid phosphatase was found in the acrosome. Thiamine pyrophosphatase was observed in the Golgi cisternae and in the tail spermatozoon surface. Glucose-6-phosphatase was located in the membrane complex of the acrosomal region. Already, in the Sertoli cells acid phosphatase was located in the lysosomes and glucose-6-phosphatase was observed in association with the endoplasmic reticulum and Golgi complex. These observations support the idea that various phosphatases may play some role in spermatid differentiation and in the interactions germ cells--Sertoli cells during spermiogenesis process.

Nonequilibrium description of de novo biogenesis and transport through Golgi-like cisternae

NASA Astrophysics Data System (ADS)

Sachdeva, Himani; Barma, Mustansir; Rao, Madan

2016-12-01

A central issue in cell biology is the physico-chemical basis of organelle biogenesis in intracellular trafficking pathways, its most impressive manifestation being the biogenesis of Golgi cisternae. At a basic level, such morphologically and chemically distinct compartments should arise from an interplay between the molecular transport and chemical maturation. Here, we formulate analytically tractable, minimalist models, that incorporate this interplay between transport and chemical progression in physical space, and explore the conditions for de novo biogenesis of distinct cisternae. We propose new quantitative measures that can discriminate between the various models of transport in a qualitative manner-this includes measures of the dynamics in steady state and the dynamical response to perturbations of the kind amenable to live-cell imaging.

Rab11 in Recycling Endosomes Regulates the Sorting and Basolateral Transport of E-CadherinV⃞

PubMed Central

Lock, John G.; Stow, Jennifer L.

2005-01-01

E-cadherin plays an essential role in cell polarity and cell-cell adhesion; however, the pathway for delivery of E-cadherin to the basolateral membrane of epithelial cells has not been fully characterized. We first traced the post-Golgi, exocytic transport of GFP-tagged E-cadherin (Ecad-GFP) in unpolarized cells. In live cells, Ecad-GFP was found to exit the Golgi complex in pleiomorphic tubulovesicular carriers, which, instead of moving directly to the cell surface, most frequently fused with an intermediate compartment, subsequently identified as a Rab11-positive recycling endosome. In MDCK cells, basolateral targeting of E-cadherin relies on a dileucine motif. Both E-cadherin and a targeting mutant, ΔS1-E-cadherin, colocalized with Rab11 and fused with the recycling endosome before diverging to basolateral or apical membranes, respectively. In polarized and unpolarized cells, coexpression of Rab11 mutants disrupted the cell surface delivery of E-cadherin and caused its mistargeting to the apical membrane, whereas apical ΔS1-E-cadherin was unaffected. We thus demonstrate a novel pathway for Rab11 dependent, dileucine-mediated, μ1B-independent sorting and basolateral trafficking, exemplified by E-cadherin. The recycling endosome is identified as an intermediate compartment for the post-Golgi trafficking and exocytosis of E-cadherin, with a potentially important role in establishing and maintaining cadherin-based adhesion. PMID:15689490

Distinct functions for IFT140 and IFT20 in opsin transport.

PubMed Central

Crouse, Jacquelin A.; Lopes, Vanda S.; SanAgustin, Jovenal T.; Keady, Brian T.; Williams, David S.; Pazour, Gregory J.

2014-01-01

In the vertebrate retina, light is detected by the outer segments of photoreceptor rods and cones, which are highly modified cilia. Like other cilia, outer segments have no protein synthetic capacity and depend on proteins made in the cell body for their formation and maintenance. The mechanism of transport into the outer segment is not fully understood but intraflagellar transport (IFT) is thought to be a major mechanism for moving protein from the cell body into the cilium. In the case of photoreceptor cells, the high density of receptors and the disk turnover that occurs daily necessitates much higher rates of transport than would be required in other cilia. In this work, we show that the IFT complex A protein IFT140 is required for development and maintenance of outer segments. In earlier work we found that acute deletion of Ift20 caused opsin to accumulate at the Golgi complex. In this work we find that acute deletion of Ift140 does not cause opsin to accumulate at the Golgi complex but rather it accumulates in the plasma membrane of the inner segments. This work is strong support of a model of opsin transport where IFT20 is involved in the movement from the Golgi complex to the base of the cilium. Then, once at the base, the opsin is carried through the connecting cilium by an IFT complex that includes IFT140. PMID:24619649

Microtubule-dependent association of AKAP350A and CCAR1 with RNA stress granules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kolobova, Elena; Efimov, Andrey; Kaverina, Irina

Recent investigations have highlighted the importance of subcellular localization of mRNAs to cell function. While AKAP350A, a multifunctional scaffolding protein, localizes to the Golgi apparatus and centrosomes, we have now identified a cytosolic pool of AKAP350A. Analysis of AKAP350A scaffolded complexes revealed two novel interacting proteins, CCAR1 and caprin-1. CCAR1, caprin-1 and AKAP350A along with G3BP, a stress granule marker, relocate to RNA stress granules after arsenite treatment. Stress also caused loss of AKAP350 from the Golgi and fragmentation of the Golgi apparatus. Disruption of microtubules with nocodazole altered stress granule formation and changed their morphology by preventing fusion ofmore » stress granules. In the presence of nocodazole, arsenite induced smaller granules with the vast majority of AKAP350A and CCAR1 separated from G3BP-containing granules. Similar to nocodazole treatment, reduction of AKAP350A or CCAR1 expression also altered the size and number of G3BP-containing stress granules induced by arsenite treatment. A limited set of 69 mRNA transcripts was immunoisolated with AKAP350A even in the absence of stress, suggesting the association of AKAP350A with mRNA transcripts. These results provide the first evidence for the microtubule dependent association of AKAP350A and CCAR1 with RNA stress granules.« less