scholarly journals Morphological and Functional Association of Sec22b/ERS-24 with the pre-Golgi Intermediate Compartment

1999 ◽  
Vol 10 (2) ◽  
pp. 435-453 ◽  
Author(s):  
Tao Zhang ◽  
Siew Heng Wong ◽  
Bor Luen Tang ◽  
Yue Xu ◽  
Wanjin Hong
Keyword(s):  
Golgi Apparatus ◽  
Mammalian Cells ◽  
Intact Cell ◽  
Brefeldin A ◽  
Cell System ◽  
Fusion Event ◽  
Homologous Proteins ◽  
Golgi Transport ◽  
Protein Receptor ◽  
Intermediate Compartment

Yeast Sec22p participates in both anterograde and retrograde vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus by functioning as a v-SNARE (solubleN-ethylmaleimide-sensitive factor [NSF] attachment protein receptor) of transport vesicles. Three mammalian proteins homologous to Sec22p have been identified and are referred to as Sec22a, Sec22b/ERS-24, and Sec22c, respectively. The existence of three homologous proteins in mammalian cells calls for detailed cell biological and functional examinations of each individual protein. The epitope-tagged forms of all three proteins have been shown to be primarily associated with the ER, although functional examination has not been carefully performed for any one of them. In this study, using antibodies specific for Sec22b/ERS-24, it is revealed that endogenous Sec22b/ERS-24 is associated with vesicular structures in both the perinuclear Golgi and peripheral regions. Colabeling experiments for Sec22b/ERS-24 with Golgi mannosidase II, the KDEL receptor, and the envelope glycoprotein G (VSVG) of vesicular stomatitis virus (VSV) en route from the ER to the Golgi under normal, brefeldin A, or nocodazole-treated cells suggest that Sec22b/ERS-24 is enriched in the pre-Golgi intermediate compartment (IC). In a well-established semi-intact cell system that reconstitutes transport from the ER to the Golgi, transport of VSVG is inhibited by antibodies against Sec22b/ERS-24. EGTA is known to inhibit ER–Golgi transport at a stage after vesicle/transport intermediate docking but before the actual fusion event. Antibodies against Sec22b/ERS-24 inhibit ER–Golgi transport only when they are added before the EGTA-sensitive stage. Transport of VSVG accumulated in pre-Golgi IC by incubation at 15°C is also inhibited by Sec22b/ERS-24 antibodies. Morphologically, VSVG is transported from the ER to the Golgi apparatus via vesicular intermediates that scatter in the peripheral as well as the Golgi regions. In the presence of antibodies against Sec22b/ERS-24, VSVG is seen to accumulate in these intermediates, suggesting that Sec22b/ERS-24 functions at the level of the IC in ER–Golgi transport.

scholarly journals The mammalian homolog of yeast Sec13p is enriched in the intermediate compartment and is essential for protein transport from the endoplasmic reticulum to the Golgi apparatus.

1997 ◽  
Vol 17 (1) ◽  
pp. 256-266 ◽  
Author(s):  
B L Tang ◽  
F Peter ◽  
J Krijnse-Locker ◽  
S H Low ◽  
G Griffiths ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Intact Cell ◽  
Predicted Amino Acid Sequence ◽  
Restrictive Temperature ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Golgi Transport ◽  
Intermediate Compartment

The role of COPII components in endoplasmic reticulum (ER)-Golgi transport, first identified in the yeast Saccharomyces cerevisiae, has yet to be fully characterized in higher eukaryotes. A human cDNA whose predicted amino acid sequence showed 70% similarity to the yeast Sec13p has previously been cloned. Antibodies raised against the human SEC13 protein (mSEC13) recognized a cellular protein of 35 kDa in both the soluble and membrane fractions. Like the yeast Sec13p, mSEC13 exist in the cytosol in both monomeric and higher-molecular-weight forms. Immunofluorescence microscopy localized mSEC13 to the characteristic spotty ER-Golgi intermediate compartment (ERGIC) in cells of all species examined, where it colocalized well with the KDEL receptor, an ERGIC marker, at 15 degrees C. Immunoelectron microscopy also localized mSEC13 to membrane structures close to the Golgi apparatus. mSEC13 is essential for ER-to-Golgi transport, since both the His6-tagged mSEC13 recombinant protein and the affinity-purified mSEC13 antibody inhibited the transport of restrictive temperature-arrested vesicular stomatitis virus G protein from the ER to the Golgi apparatus in a semi-intact cell assay. Moreover, cytosol immunodepleted of mSEC13 could no longer support ER-Golgi transport. Transport could be restored in a dose-dependent manner by a cytosol fraction enriched in the high-molecular-weight mSEC13 complex but not by a fraction enriched in either monomeric mSEC13 or recombinant mSEC13. As a putative component of the mammalian COPII complex, mSEC13 showed partially overlapping but mostly different properties in terms of localization, membrane recruitment, and dynamics compared to that of beta-COP, a component of the COPI complex.

scholarly journals Molecular cloning, characterization, subcellular localization and dynamics of p23, the mammalian KDEL receptor.

1993 ◽  
Vol 120 (2) ◽  
pp. 325-338 ◽  
Author(s):  
B L Tang ◽  
S H Wong ◽  
X L Qi ◽  
S H Low ◽  
W Hong
Keyword(s):  
Golgi Apparatus ◽  
Brefeldin A ◽  
Cell Types ◽  
In Vitro Translation ◽  
Tubular Structures ◽  
Human Sequence ◽  
Intermediate Compartment ◽  
Membrane Spanning ◽  
Membrane Spanning Domains

We have isolated a cDNA clone (mERD2) for the mammalian (bovine) homologue of the yeast ERD2 gene, which codes for the yeast HDEL receptor. The deduced amino acid sequence bears extensive homology to its yeast counterpart and is almost identical to a previously described human sequence. The sequence predicts a very hydrophobic protein with multiple membrane spanning domains, as confirmed by analysis of the in vitro translation product. The protein encoded by mERD2 (p23) has widespread occurrence, being present in all the cell types examined. p23 was localized to the cis-side of the Golgi apparatus and to a spotty intermediate compartment which mediates ER to Golgi transport. A majority of the intracellular staining could be accumulated in the intermediate compartment by a low temperature (15 degrees C) or brefeldin A. During recovery from these treatments, the spotty intermediate compartment staining of p23 was shifted to the perinuclear staining of the Golgi apparatus and tubular structures marked by p23 were observed. These tubular structures may serve to mediate transport between the intermediate compartment and the Golgi apparatus.

scholarly journals ER to Golgi Transport

1999 ◽  
Vol 147 (6) ◽  
pp. 1205-1222 ◽  
Author(s):  
Cecilia Alvarez ◽  
Hideaki Fujita ◽  
Ann Hubbard ◽  
Elizabeth Sztul
Keyword(s):  
G Protein ◽  
Golgi Complex ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Intact Cell ◽  
Cell Transport ◽  
Golgi Stack ◽  
Golgi Transport ◽  
Transport Assay ◽  
Golgi Structure

The membrane transport factor p115 functions in the secretory pathway of mammalian cells. Using biochemical and morphological approaches, we show that p115 participates in the assembly and maintenance of normal Golgi structure and is required for ER to Golgi traffic at a pre-Golgi stage. Injection of antibodies against p115 into intact WIF-B cells caused Golgi disruption and inhibited Golgi complex reassembly after BFA treatment and wash-out. Addition of anti–p115 antibodies or depletion of p115 from a VSVtsO45 based semi-intact cell transport assay inhibited transport. The inhibition occurred after VSV glycoprotein (VSV-G) exit from the ER but before its delivery to the Golgi complex, and resulted in VSV-G protein accumulating in peripheral vesicular tubular clusters (VTCs). The p115-requiring step of transport followed the rab1-requiring step and preceded the Ca2+-requiring step. Unexpectedly, mannosidase I redistributed from the Golgi complex to colocalize with VSV-G protein arrested in pre-Golgi VTCs by p115 depletion. Redistribution of mannosidase I was also observed in cells incubated at 15°C. Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack. This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.

scholarly journals The translocation, folding, assembly and redox-dependent degradation of secretory and membrane proteins in semi-permeabilized mammalian cells

1995 ◽  
Vol 307 (3) ◽  
pp. 679-687 ◽  
Author(s):  
R Wilson ◽  
A J Allen ◽  
J Oliver ◽  
J L Brookman ◽  
S High ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Intact Cell ◽  
Cell System ◽  
In Vitro Translation ◽  
Tissue Type ◽  
Translation System ◽  
Secretory Proteins ◽  
Permeabilized Cell ◽  
Permeabilized Cells

We describe here a semi-permeabilized cell-system which reconstitutes the efficient synthesis, translocation, folding, assembly and degradation of membrane and secretory proteins. Cells grown in culture were treated with the detergent digitonin which selectively permeabilized the plasma membrane leaving the cellular organelles, such as the endoplasmic reticulum (ER) and trans-Golgi network intact. These permeabilized cells were added to an in vitro translation system, either wheatgerm or reticulocyte lysate, supplemented with RNA coding for either membrane or secretory proteins. Efficient translocation and modification of proteins by these cells was demonstrated by protease protection, photocross-linking of nascent chains to components of the translocation apparatus and by post-translational modifications such as glycosylation or hydroxylation. A comparison was made between the ability of semi-permeabilized cells and microsomal vesicles to fold and assemble proteins. The results show that the intact ER within these cells can assemble proteins much more efficiently than vesicularized ER. Furthermore, the semi-permeabilized cells carried out the redox-dependent degradation of tissue-type plasminogen activator. This system has all the advantages of conventional cell-free systems, including speed and, importantly, the ability to manipulate the components of the assay, while retaining intracellular organelles and, therefore, allowing cellular processes to occur as they would in the intact cell.

scholarly journals Phospholipase D2 Is Localized to the Rims of the Golgi Apparatus in Mammalian Cells

2002 ◽  
Vol 13 (11) ◽  
pp. 3930-3942 ◽  
Author(s):  
Zachary Freyberg ◽  
Sylvain Bourgoin ◽  
Dennis Shields
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Mammalian Cells ◽  
Intracellular Localization ◽  
Brefeldin A ◽  
Caveolin 1 ◽  
Nuclear Signaling ◽  
Golgi Region ◽  
Phospholipase D2 ◽  
Golgi Network

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid, a molecule known to have multiple physiological roles, including release of nascent secretory vesicles from thetrans-Golgi network. In mammalian cells two forms of the enzyme, PLD1 and PLD2, have been described. We recently demonstrated that PLD1 is localized to the Golgi apparatus, nuclei, and to a lesser extent, plasma membrane. Due to its low abundance, the intracellular localization of PLD2 has been characterized only indirectly through overexpression of chimeric proteins. Using antibodies specific to PLD2, together with immunofluorescence microscopy, herein we demonstrate that a significant fraction of endogenous PLD2 localized to the perinuclear Golgi region and was also distributed throughout cells in dense cytoplasmic puncta; a fraction of which colocalized with caveolin-1 and the plasma membrane. On treatment with brefeldin A, PLD2 translocated into the nucleus in a manner similar to PLD1, suggesting a potential role in nuclear signaling. Most significantly, cryoimmunogold electron microscopy demonstrated that in pituitary GH3 cells >90% of PLD2 present in the Golgi apparatus was localized to cisternal rims and peri-Golgi vesicles exclusively. The data are consistent with a model whereby PLD2 plays a role in Golgi vesicular transport.

scholarly journals The Mammalian Protein (rbet1) Homologous to Yeast Bet1p Is Primarily Associated with the Pre-Golgi Intermediate Compartment and Is Involved in Vesicular Transport from the Endoplasmic Reticulum to the Golgi Apparatus

1997 ◽  
Vol 139 (5) ◽  
pp. 1157-1168 ◽  
Author(s):  
Tao Zhang ◽  
Siew Heng Wong ◽  
Bor Luen Tang ◽  
Yue Xu ◽  
Frank Peter ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
G Protein ◽  
Vesicular Transport ◽  
Dependent Manner ◽  
Golgi Transport ◽  
Golgi Region ◽  
Intermediate Compartment ◽  
Kdel Receptor ◽  
Mammalian Protein

Yeast Bet1p participates in vesicular transport from the endoplasmic reticulum to the Golgi apparatus and functions as a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells. Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL receptor and ERGIC-53, which are known to be enriched in the intermediate compartment. When the Golgi apparatus is fragmented by nocodazole treatment, a significant portion of rbet1 is not colocalized with structures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered by preincubation of cells at 15°C. However, upon warming up from 15 to 37°C, rbet1 concentrates into the peri-Golgi region. Furthermore, rbet1 colocalizes with vesicular stomatitis virus G-protein en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincubation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the actual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These results suggest that rbet1 may be involved in the docking process of ER- derived vesicles with the cis-Golgi membrane.

scholarly journals 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

2012 ◽  
Vol 23 (16) ◽  
pp. 3203-3214 ◽  
Author(s):  
Yoko Ito ◽  
Tomohiro Uemura ◽  
Keiko Shoda ◽  
Masaru Fujimoto ◽  
Takashi Ueda ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Fluorescent Proteins ◽  
Plant Cells ◽  
Brefeldin A ◽  
Eukaryotic Cells ◽  
Er Exit Sites ◽  
Intermediate Compartment ◽  
Golgi Protein ◽  
And Function

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.

scholarly journals Regulation of vascular endothelial growth factor receptor 2 trafficking and angiogenesis by Golgi localized t-SNARE syntaxin 6

Blood ◽  
2011 ◽  
Vol 117 (4) ◽  
pp. 1425-1435 ◽  
Author(s):  
Venkatraman Manickam ◽  
Ajit Tiwari ◽  
Jae-Joon Jung ◽  
Resham Bhattacharya ◽  
Apollina Goel ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Golgi Apparatus ◽  
Growth Factor Receptor ◽  
Brefeldin A ◽  
Tube Formation ◽  
Vascular Endothelial ◽  
Protein Receptor ◽  
Mouse Ear ◽  
Endothelial Growth Factor

Abstract Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in physiologic and pathologic angiogenesis. Plasma membrane (PM) levels of VEGFR2 are regulated by endocytosis and secretory transport through the Golgi apparatus. To date, the mechanism whereby the VEGFR2 traffics through the Golgi apparatus remains incompletely characterized. We show in human endothelial cells that binding of VEGF to the cell surface localized VEGFR2 stimulates exit of intracellular VEGFR2 from the Golgi apparatus. Brefeldin A treatment reduced the level of surface VEGFR2, confirming that VEGFR2 traffics through the Golgi apparatus en route to the PM. Mechanistically, we show that inhibition of syntaxin 6, a Golgi-localized target membrane-soluble N-ethylmaleimide attachment protein receptor (t-SNARE) protein, interferes with VEGFR2 trafficking to the PM and facilitates lysosomal degradation of the VEGFR2. In cell culture, inhibition of syntaxin 6 also reduced VEGF-induced cell proliferation, cell migration, and vascular tube formation. Furthermore, in a mouse ear model of angiogenesis, an inhibitory form of syntaxin 6 reduced VEGF-induced neovascularization and permeability. Our data demonstrate the importance of syntaxin 6 in the maintenance of cellular VEGFR2 levels, and suggest that the inhibitory form of syntaxin 6 has good potential as an antiangiogenic agent.

scholarly journals Branched Actin Maintains Acetylated Microtubule Network in the Early Secretory Pathway

Cells ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 15
Author(s):  
Azumi Yoshimura ◽  
Stéphanie Miserey-Lenkei ◽  
Evelyne Coudrier ◽  
Bruno Goud
Keyword(s):  
Golgi Apparatus ◽  
Transport Process ◽  
Actin Polymerization ◽  
Secretory Pathway ◽  
Microtubule Network ◽  
Early Secretory Pathway ◽  
Golgi Transport ◽  
Er Exit Sites ◽  
Intermediate Compartment ◽  
Stable Network

In the early secretory pathway, the delivery of anterograde cargoes from the endoplasmic reticulum (ER) exit sites (ERES) to the Golgi apparatus is a multi-step transport process occurring via the ER-Golgi intermediate compartment (IC, also called ERGIC). While the role microtubules in ER-to-Golgi transport has been well established, how the actin cytoskeleton contributes to this process remains poorly understood. Here, we report that Arp2/3 inhibition affects the network of acetylated microtubules around the Golgi and induces the accumulation of unusually long RAB1/GM130-positive carriers around the centrosome. These long carriers are less prone to reach the Golgi apparatus, and arrival of anterograde cargoes to the Golgi is decreased upon Arp2/3 inhibition. Our data suggest that Arp2/3-dependent actin polymerization maintains a stable network of acetylated microtubules, which ensures efficient cargo trafficking at the late stage of ER to Golgi transport.

scholarly journals Phosphorylation and membrane dissociation of the ARF exchange factor GBF1 in mitosis

2010 ◽  
Vol 427 (3) ◽  
pp. 401-412 ◽  
Author(s):  
Yuichi Morohashi ◽  
Zita Balklava ◽  
Matthew Ball ◽  
Helen Hughes ◽  
Martin Lowe
Keyword(s):  
Golgi Apparatus ◽  
Protein Trafficking ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Brefeldin A ◽  
Secretory Protein ◽  
Golgi Membrane ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Mitotic Cells

Secretory protein trafficking is arrested and the Golgi apparatus fragmented when mammalian cells enter mitosis. These changes are thought to facilitate cell-cycle progression and Golgi inheritance, and are brought about through the actions of mitotically active protein kinases. To better understand how the Golgi apparatus undergoes mitotic fragmentation we have sought to identify novel Golgi targets for mitotic kinases. We report in the present paper the identification of the ARF (ADP-ribosylation factor) exchange factor GBF1 (Golgi-specific brefeldin A-resistant guanine nucleotide-exchange factor 1) as a Golgi phosphoprotein. GBF1 is phosphorylated by CDK1 (cyclin-dependent kinase 1)–cyclin B in mitosis, which results in its dissociation from Golgi membranes. Consistent with a reduced level of GBF1 activity at the Golgi membrane there is a reduction in levels of membrane-associated GTP-bound ARF in mitotic cells. Despite the reduced levels of membrane-bound GBF1 and ARF, COPI (coat protein I) binding to the Golgi membrane appears unaffected in mitotic cells. Surprisingly, this pool of COPI is dependent upon GBF1 for its recruitment to the membrane, suggesting that a low level of GBF1 activity persists in mitosis. We propose that the phosphorylation and membrane dissociation of GBF1 and the consequent reduction in ARF-GTP levels in mitosis are important for changes in Golgi dynamics and possibly other mitotic events mediated through effectors other than the COPI vesicle coat.

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