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 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 TFG facilitates outer coat disassembly on COPII transport carriers to promote tethering and fusion with ER–Golgi intermediate compartments

2017 ◽  
Vol 114 (37) ◽  
pp. E7707-E7716 ◽  
Author(s):  
Michael G. Hanna ◽  
Samuel Block ◽  
E. B. Frankel ◽  
Feng Hou ◽  
Adam Johnson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Protein Trafficking ◽  
Secretory Protein ◽  
Dependent Manner ◽  
Outer Coat ◽  
Concentration Dependent Manner ◽  
C Terminus ◽  
Intermediate Compartment ◽  
Fused Gene

The conserved coat protein complex II (COPII) mediates the initial steps of secretory protein trafficking by assembling onto subdomains of the endoplasmic reticulum (ER) in two layers to generate cargo-laden transport carriers that ultimately fuse with an adjacent ER–Golgi intermediate compartment (ERGIC). Here, we demonstrate that Trk-fused gene (TFG) binds directly to the inner layer of the COPII coat. Specifically, the TFG C terminus interacts with Sec23 through a shared interface with the outer COPII coat and the cargo receptor Tango1/cTAGE5. Our findings indicate that TFG binding to Sec23 outcompetes these other associations in a concentration-dependent manner and ultimately promotes outer coat dissociation. Additionally, we demonstrate that TFG tethers vesicles harboring the inner COPII coat, which contributes to their clustering between the ER and ERGIC in cells. Together, our studies define a mechanism by which COPII transport carriers are retained locally at the ER/ERGIC interface after outer coat disassembly, which is a prerequisite for fusion with ERGIC membranes.

scholarly journals Peri-Golgi vesicles contain retrograde but not anterograde proteins consistent with the cisternal progression model of intra-Golgi transport

2001 ◽  
Vol 155 (7) ◽  
pp. 1213-1224 ◽  
Author(s):  
José A. Martínez-Menárguez ◽  
Rytis Prekeris ◽  
Viola M.J. Oorschot ◽  
Richard Scheller ◽  
Jan W. Slot ◽  
...  
Keyword(s):  
Virus Protein ◽  
Anterograde Transport ◽  
Golgi Vesicles ◽  
Progression Model ◽  
Golgi Transport ◽  
Golgi Region ◽  
Cargo Proteins ◽  
Vesicular Stomatitis ◽  
Kdel Receptor

A cisternal progression mode of intra-Golgi transport requires that Golgi resident proteins recycle by peri-Golgi vesicles, whereas the alternative model of vesicular transport predicts anterograde cargo proteins to be present in such vesicles. We have used quantitative immuno-EM on NRK cells to distinguish peri-Golgi vesicles from other vesicles in the Golgi region. We found significant levels of the Golgi resident enzyme mannosidase II and the transport machinery proteins giantin, KDEL-receptor, and rBet1 in coatomer protein I–coated cisternal rims and peri-Golgi vesicles. By contrast, when cells expressed vesicular stomatitis virus protein G this anterograde marker was largely absent from the peri-Golgi vesicles. These data suggest a role of peri-Golgi vesicles in recycling of Golgi residents, rather than an important role in anterograde transport.

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 Golgi Apparatus Maintains Its Organization Independent of the Endoplasmic Reticulum

2006 ◽  
Vol 17 (12) ◽  
pp. 5372-5380 ◽  
Author(s):  
Matthew Y. Pecot ◽  
Vivek Malhotra
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Binding Protein ◽  
Physiological Conditions ◽  
Fk506 Binding Protein ◽  
Golgi Membranes ◽  
Intermediate Compartment ◽  
Tight Association

Under artificial conditions Golgi enzymes have the capacity to rapidly accumulate in the endoplasmic reticulum (ER). These observations prompted the idea that Golgi enzymes constitutively recycle through the ER. We have tested this hypothesis under physiological conditions through use of a procedure that captures Golgi enzymes in the ER. In the presence of rapamycin, which induces a tight association between FKBP (FK506-binding protein) and FRAP (FKBP-rapamycin–associated protein), an FKBP-tagged Golgi enzyme can be trapped when it visits the ER by an ER-retained protein fused to FRAP. We find that although FKBP-ERGIC-53 of the ER-Golgi intermediate compartment (ERGIC) rapidly cycles through the ER (30 min), FKBP-Golgi enzyme chimeras remain stably associated with Golgi membranes. We also demonstrate that Golgi dispersion upon nocodazole treatment mainly occurs through a mechanism that does not involve the recycling of Golgi membranes through the ER. Our findings suggest that the Golgi apparatus, as defined by its collection of resident enzymes, exists independent of the ER.

scholarly journals Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane.

1984 ◽  
Vol 99 (3) ◽  
pp. 1101-1109 ◽  
Author(s):  
A A Rogalski ◽  
J E Bergmann ◽  
S J Singer
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
G Protein ◽  
Rough Endoplasmic Reticulum ◽  
Intracellular Transport ◽  
Temperature Shift ◽  
Surface Expression ◽  
Microtubule Assembly ◽  
In Cells

We studied the effects of changes in microtubule assembly status upon the intracellular transport of an integral membrane protein from the rough endoplasmic reticulum to the plasma membrane. The protein was the G glycoprotein of vesicular stomatitis virus in cells infected with the Orsay-45 temperature-sensitive mutant of the virus; the synchronous intracellular transport of the G protein could be initiated by a temperature shift-down protocol. The intracellular and surface-expressed G protein were separately detected and localized in the same cells at different times after the temperature shift, by double-immunofluorescence microscopic measurements, and the extent of sialylation of the G protein at different times was quantitated by immunoprecipitation and SDS PAGE of [35S]methionine-labeled cell extracts. Neither complete disassembly of the cytoplasmic microtubules by nocodazole treatment, nor the radical reorganization of microtubules upon taxol treatment, led to any perceptible changes in the rate or extent of G protein sialylation, nor to any marked changes in the rate or extent of surface appearance of the G protein. However, whereas in control cells the surface expression of G was polarized, at membrane regions in juxtaposition to the perinuclear compact Golgi apparatus, in cells with disassembled microtubules the surface expression of the G protein was uniform, corresponding to the intracellular dispersal of the elements of the Golgi apparatus. The mechanisms of transfer of integral proteins from the rough endoplasmic reticulum to the Golgi apparatus, and from the Golgi apparatus to the plasma membrane, are discussed in the light of these observations, and compared with earlier studies of the intracellular transport of secretory proteins.

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 The G protein of vesicular stomatitis virus has free access into and egress from the smooth endoplasmic reticulum of UT-1 cells.

1990 ◽  
Vol 110 (3) ◽  
pp. 625-635 ◽  
Author(s):  
J E Bergmann ◽  
P J Fusco
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Smooth Endoplasmic Reticulum ◽  
Free Access ◽  
First Order ◽  
First Order Kinetics ◽  
Vesicular Stomatitis ◽  
Endoglycosidase H

We have investigated the role of the smooth endoplasmic reticulum (SER) of UT-1 cells in the biogenesis of the glycoprotein (G) of vesicular stomatitis virus (VSV). Using immunofluorescence microscopy, we observed the wild type G protein in the SER of infected cells. When these cells were infected with the mutant VSV strain ts045, the G protein was unable to reach the Golgi apparatus at 40 degrees C, but was able to exit the rough endoplasmic reticulum (RER) and accumulate in the SER. Ribophorin II, a RER marker, remained excluded from the SER during the viral infection, ruling out the possibility that the infection had destroyed the separate identities of these two organelles. Thus, the mechanism that results in the retention of this mutant glycoprotein in the ER at 39.9 degrees C does not limit its lateral mobility within the ER system. We have also localized GRP78/BiP to the SER of UT-1 cells indicating that other mutant proteins may also have access to this organelle. Upon incubation at 32 degrees C, the mutant G protein was able to leave the SER and move to the Golgi apparatus. To measure how rapidly this transfer occurs, we assayed the conversion of the G protein's N-linked oligosaccharides from endoglycosidase H-sensitive to endoglycosidase H-resistant forms. After a 5-min lag, transport of the G protein followed first order kinetics (t1/2 = 15 min). In contrast, no lag was seen in the transport of G protein that had accumulated in the RER of control UT-1 cells lacking extensive SER. In these cells, the transport of G protein also exhibited first order kinetics (t1/2 = 17 min). Possible implications of this lag are discussed.

scholarly journals ER exit sites in Drosophila display abundant ER-Golgi vesicles and pearled tubes but no megacarriers

2021 ◽  
Author(s):  
Ke Yang ◽  
Min Liu ◽  
Zhi Feng ◽  
Marta Rojas ◽  
Lingjian Zhou ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Fat Body ◽  
Retrograde Transport ◽  
Imaginal Discs ◽  
Membrane Structures ◽  
Molecular Events ◽  
Golgi Region ◽  
Er Exit Sites ◽  
Essential Questions ◽  
Intermediate Compartment

AbstractSecretory cargos are collected at ER exit sites (ERES) before transport to the Golgi apparatus. Decades of research have provided many details of the molecular events underlying ER-Golgi exchanges. Essential questions, however, remain about the organization of the ER-Golgi interface in cells and the type of membrane structures mediating traffic from ERES. To investigate these, we used transgenic tagging in Drosophila flies, 3D-SIM and FIB-SEM to characterize ERES-Golgi units in collagen-producing fat body, imaginal discs and imaginal discs overexpressing ERES determinant Tango1. We found in front of ERES a pre-cis-Golgi region involved in both anterograde and retrograde transport. This pre-cis-Golgi is continuous with the rest of the Golgi, not a separate intermediate compartment or collection of large carriers, for which we found no evidence. We found, however, many vesicles, as well as pearled tubules connecting ERES and Golgi.

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.

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