Biogenesis of endoplasmic reticulum transport vesicles transferring gastric apomucin from ER to Golgi.

Rough endoplasmic reticulum (RER) transport vesicles were generated from gastric mucous cell RER microsomes in the presence of labeled precursors of phospholipids. The vesicles contained 7-10% of their proteins in the form of apomucin (cargo), and 80% of de novo synthesized phosphatidylcholine (PC) was incorporated into the vesicular membrane. In the absence of choline and ethanolamine precursors or in the presence of 3 mM N-ethylmaleimide (NEM), an inhibitor of CTP:phosphocholine cytidylyltransferase, formation of the transport vesicles, their enrichment in the newly synthesized PC, and the total synthesis of PC decreased by 86%, whereas in the presence of 3 mM Zn2+, complete blockage of vesicle formation and PC synthesis was observed. Analysis of the mucin-transporting vesicles indicated that the CTP:phosphocholine cytidylyltransferase and 1,2-diacyl-sn-glycerol:CDP-choline phosphotransferase remained associated with transport vesicles released from ER. The enzymes and other proteins separated from the vesicle surface prior to vesicle fusion with Golgi and the process was induced by phosphorylation. Based on the results of this study, it is proposed that the formation of the ER transport vesicles of gastric mucosal cells is in concert with synthesis of phospholipids and thus in part is regulated by phospholipid-synthesizing enzymes that reside on the membrane during its biogenesis and dissociate from its surface once the task is completed.     

Role of glyceraldehyde-3-phosphate dehydrogenase in vesicular transport from Golgi apparatus to endoplasmic reticulum

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a well-studied glycolytic protein with energy production as its implied occupation. It has established itself lately as a multifunctional protein. Recent studies have found GAPDH to be involved in a variety of nuclear and cytosolic pathways ranging from its role in apoptosis and regulation of gene expression to its involvement in regulation of Ca2+ influx from endoplasmic reticulum. Numerous studies also indicate that GAPDH interacts with microtubules and participates in cell membrane fusion. This review is focused on the cytosolic functions of the protein related to vesicular transport. Suggestions for future directions as well as the model of protein polymer structure and possible post-translational modifications as a basis for its multifunctional activities in the early secretory pathway are given.     

Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus

We have studied the role of a previously described tubulovesicular compartment near the cis-Golgi apparatus in endoplasmic reticulum (ER)-to-Golgi protein transport by light and immunoelectron microscopy in Vero cells. The compartment is defined by a 53-kDa transmembrane protein designated p53. When transport of the vesicular stomatitis virus strain ts045 G protein was arrested at 39.5 degrees C, the G protein accumulated in the ER but had access to the p53 compartment. At 15 degrees C, the G protein was exported from the ER into the p53 compartment which formed a compact structure composed of vesicular and tubular profiles in close proximity to the Golgi. Upon raising the temperature to 32 degrees C, the G protein migrated through the Golgi apparatus while the p53 compartment resumed its normal structure again. These results establish the p53 compartment as the 15 degrees C intermediate of the ER-to-Golgi protein transport pathway.     

Deubiquitination, a new player in Golgi to endoplasmic reticulum retrograde transport

Modification by ubiquitin plays a major role in a broad array of cellular functions. Although reversal of this process, deubiquitination, likely represents an important regulatory step contributing to cellular homeostasis, functions of deubiquitination enzymes still remain poorly characterized. We have previously shown that the ubiquitin protease Ubp3p requires a co-factor, Bre5p, to specifically deubiquitinate the coat protein complex II (COPII) subunit Sec23p, which is involved in anterograde transport between endoplasmic reticulum and Golgi compartiments. In the present report, we show that disruption of BRE5 gene also led to a defect in the retrograde transport from the Golgi to the endoplasmic reticulum. Further analysis indicate that the COPI subunit beta'-COP represents another substrate of the Ubp3p.Bre5p complex. All together, our results indicate that the Ubp3p.Bre5p deubiquitination complex co-regulates anterograde and retrograde transports between endoplasmic reticulum and Golgi compartments.     

Molecular machinery required for protein transport from the endoplasmic reticulum to the Golgi complex

The cellular machinery responsible for conveying proteins between the endoplasmic reticulum and the Golgi is being investigated using genetics and biochemistry. A role for vesicles in mediating protein traffic between the ER and the Golgi has been established by characterizing yeast mutants defective in this process, and by using recently developed cell-free assays that measure ER to Golgi transport. These tools have also allowed the identification of several proteins crucial to intracellular protein trafficking. The characterization and possible functions of several GTP-binding proteins, peripheral membrane proteins, and an integral membrane protein during ER to Golgi transport are discussed here.     

Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum

The cytosolic coat-protein complex COP-I interacts with cytoplasmic 'retrieval' signals present in membrane proteins that cycle between the endoplasmic reticulum (ER) and the Golgi complex, and is required for both anterograde and retrograde transport in the secretory pathway. Here we study the role of COP-I in Golgi-to-ER transport of several distinct marker molecules. Microinjection of anti-COP-I antibodies inhibits retrieval of the lectin-like molecule ERGIC-53 and of the KDEL receptor from the Golgi to the ER. Transport to the ER of protein toxins, which contain a sequence that is recognized by the KDEL receptor, is also inhibited. In contrast, microinjection of anti-COP-I antibodies or expression of a GTP-restricted Arf-1 mutant does not interfere with Golgi-to-ER transport of Shiga toxin/Shiga-like toxin-1 or with the apparent recycling to the ER of Golgi-resident glycosylation enzymes. Overexpression of a GDP-restricted mutant of Rab6 blocks transport to the ER of Shiga toxin/Shiga-like toxin-1 and glycosylation enzymes, but not of ERGIC-53, the KDEL receptor or KDEL-containing toxins. These data indicate the existence of at least two distinct pathways for Golgi-to-ER transport, one COP-I dependent and the other COP-I independent. The COP-I-independent pathway is specifically regulated by Rab6 and is used by Golgi glycosylation enzymes and Shiga toxin/Shiga-like toxin-1.     

Beta-COP is essential for transport of protein from the endoplasmic reticulum to the Golgi in vitro

Using a novel in vitro assay which allows us to distinguish vesicle budding from subsequent targeting and fusion steps, we provide the first biological evidence that beta-COP, a component of non-clathrin- coated vesicles believed to mediate intraGolgi transport, is essential for transport of protein from the ER to the cis-Golgi compartment. Incubation in the presence of beta-COP specific antibodies and F(ab) fragments prevents the exit of vesicular stomatitis virus glycoprotein (VSV-G) from the ER. These results demonstrate that beta-COP is required for the assembly of coat complexes mediating vesicle budding. Fractionation of rat liver cytosol revealed that a major biologically active form of beta-COP was found in a high molecular pool (> 1,000 kD) distinct from coatomer and which promoted efficient vesicle budding from the ER. Surprisingly, rab1B could be quantitatively coprecipitated with this beta-COP containing complex and was also essential for function. We suggest that beta-COP functions in an early step during vesicle formation and that rab1B may be recruited as a component of a precoat complex which participates in the export of protein from the ER via vesicular carriers.     

Retrograde transport from the Golgi region to the endoplasmic reticulum is sensitive to GTP gamma S

The involvement of GTP-binding proteins in the intracellular transport of the secretory glycoprotein alpha 1-antitrypsin was investigated in streptolysin O-permeabilized HepG2 cells. This permeabilization procedure allows ready access to the intracellular milieu of the membrane-impermeant, nonhydrolyzable GTP analog GTP gamma S. In streptolysin O-permeabilized HepG2 cells, the constitutive secretory pathway remains functional and is sensitive to GTP gamma S. Exposure of HepG2 cells to brefeldin A resulted in redistribution of Golgi-resident glycosyltransferases (including both alpha 2----3 and alpha 2----6 sialyltransferases) to the ER. This redistribution was sensitive to GTP gamma S. Our results suggest that GTP-binding proteins are involved in the regulation not only of the anterograde, but also of the retrograde, pathway.     

Requirement for neo1p in retrograde transport from the Golgi complex to the endoplasmic reticulum

Neo1p from Saccharomyces cerevisiae is an essential P-type ATPase and potential aminophospholipid translocase (flippase) in the Drs2p family. We have previously implicated Drs2p in protein transport steps in the late secretory pathway requiring ADP-ribosylation factor (ARF) and clathrin. Here, we present evidence that epitope-tagged Neo1p localizes to the endoplasmic reticulum (ER) and Golgi complex and is required for a retrograde transport pathway between these organelles. Using conditional alleles of NEO1, we find that loss of Neo1p function causes cargo-specific defects in anterograde protein transport early in the secretory pathway and perturbs glycosylation in the Golgi complex. Rer1-GFP, a protein that cycles between the ER and Golgi complex in COPI and COPII vesicles, is mislocalized to the vacuole in neo1-ts at the nonpermissive temperature. These phenotypes suggest that the anterograde protein transport defect is a secondary consequence of a defect in a COPI-dependent retrograde pathway. We propose that loss of lipid asymmetry in the cis Golgi perturbs retrograde protein transport to the ER.     

Membrane topology of the endoplasmic reticulum to Golgi transport factor Erv29p

Secretory proteins are transported from the endoplasmic reticulum to the Golgi apparatus via COPII-coated intermediates. Yeast Erv29p is a transmembrane protein cycling between these compartments. It is conserved across species, with one ortholog found in each genome studied, including the surf-4 protein in mammals. Yeast Erv29p acts as a receptor, loading a specific subset of soluble cargo, including glycosylated alpha factor pheromone precursor and carboxypeptidase Y, into vesicles. As the eukaryotic secretory pathway is highly conserved, mammalian surf-4 may perform a similar role in the transport of unknown substrates. Here we report the membrane topology of yeast Erv29p, which we solved by minimally invasive cysteine accessibility scanning using thiol-specific biotinylation and fluorescent labeling methods. Erv29p contains four transmembrane domains with both termini exposed to the cytosol. Two luminal loops may contain a recognition site for hydrophobic export signals on soluble cargo.     

Membrane topology of the endoplasmic reticulum to Golgi transport factor Erv29p

Secretory proteins are transported from the endoplasmic reticulum to the Golgi apparatus via COPII-coated intermediates. Yeast Erv29p is a transmembrane protein cycling between these compartments. It is conserved across species, with one ortholog found in each genome studied, including the surf-4 protein in mammals. Yeast Erv29p acts as a receptor, loading a specific subset of soluble cargo, including glycosylated alpha factor pheromone precursor and carboxypeptidase Y, into vesicles. As the eukaryotic secretory pathway is highly conserved, mammalian surf-4 may perform a similar role in the transport of unknown substrates. Here we report the membrane topology of yeast Erv29p, which we solved by minimally invasive cysteine accessibility scanning using thiol-specific biotinylation and fluorescent labeling methods. Erv29p contains four transmembrane domains with both termini exposed to the cytosol. Two luminal loops may contain a recognition site for hydrophobic export signals on soluble cargo.     

Src regulates Golgi structure and KDEL receptor-dependent retrograde transport to the endoplasmic reticulum

The tyrosine kinase Src is present on the Golgi membranes. Its role, however, in the overall function and organization of the Golgi apparatus is unclear. We have found that in a cell line called SYF, which lacks the three ubiquitous Src-like kinases (Src, Yes, and Fyn), the organization of the Golgi apparatus is perturbed. The Golgi apparatus is composed of collapsed stacks and bloated cisternae in these cells. Expression of an activated form of Src relocated the KDEL receptor (KDEL-R) from the Golgi apparatus to the endoplasmic reticulum. Other Golgi-specific marker proteins were not affected under these conditions. Because of the specific effect of Src on the location of KDEL-R, we tested whether protein transport between ER and the Golgi apparatus involves Src. Transport of Pseudomonas exotoxin, which is transported to the ER by binding to the KDEL-R is accelerated by inhibition or genetic ablation of Src. Protein transport from ER to the Golgi apparatus however, is unaffected by Src deletion or inhibition. We propose that Src has an appreciable role in the organization of the Golgi apparatus, which may be linked to its involvement in protein transport from the Golgi apparatus to the endoplasmic reticulum.     

Sequential coupling between COPII and COPI vesicle coats in endoplasmic reticulum to Golgi transport

COPI and COPII are vesicle coat complexes whose assembly is regulated by the ARF1 and Sar1 GTPases, respectively. We show that COPI and COPII coat complexes are recruited separately and independently to ER (COPII), pre-Golgi (COPI, COPII), and Golgi (COPI) membranes of mammalian cells. To address their individual roles in ER to Golgi transport, we used stage specific in vitro transport assays to synchronize movement of cargo to and from pre-Golgi intermediates, and GDP- and GTP-restricted forms of Sar1 and ARF1 proteins to control coat recruitment. We find that COPII is solely responsible for export from the ER, is lost rapidly following vesicle budding and mediates a vesicular step required for the build-up of pre-Golgi intermediates composed of clusters of vesicles and small tubular elements. COPI is recruited onto pre-Golgi intermediates where it initiates segregation of the anterograde transported protein vesicular stomatitis virus glycoprotein (VSV-G) from the retrograde transported protein p58, a protein which actively recycles between the ER and pre-Golgi intermediates. We propose that sequential coupling between COPII and COPI coats is essential to coordinate and direct bi-directional vesicular traffic between the ER and pre-Golgi intermediates involved in transport of protein to the Golgi complex.     

Crossing the divide--transport between the endoplasmic reticulum and Golgi apparatus in plants

The transport of proteins between the endoplasmic reticulum (ER) and the Golgi apparatus in plants is an exciting and constantly expanding topic, which has attracted much attention in recent years. The study of protein transport within the secretory pathway is a relatively new field, dating back to the 1970s for mammalian cells and considerably later for plants. This may explain why COPI- and COPII-mediated transport between the ER and the Golgi in plants is only now becoming clear, while the existence of these pathways in other organisms is relatively well documented. We summarize current knowledge of these protein transport routes, as well as highlighting key differences between those of plant systems and those of mammals and yeast. These differences have necessitated the study of plant-specific aspects of protein transport in the early secretory pathway, and this review discusses recent developments in this area. Advances in live-cell-imaging technology have allowed the observation of protein movement in vivo, giving a new insight into many of the processes involved in vesicle formation and protein trafficking. The use of these new technologies has been combined with more traditional methods, such as protein biochemistry and electron microscopy, to increase our understanding of the transport routes in the cell.     

Protein transport from endoplasmic reticulum to the Golgi complex can occur during meiotic metaphase in Xenopus oocytes

We have previously shown that Xenopus oocytes arrested at second meiotic metaphase lost their characteristic multicisternal Golgi apparati and cannot secrete proteins into the surrounding medium. In this paper, we extend these studies to ask whether intracellular transport events affecting the movement of secretory proteins from the endoplasmic reticulum to the Golgi apparatus are also similarly inhibited in such oocytes. Using the acquisition of resistance to endoglycosidase H (endo H) as an assay for movement to the Golgi, we find that within 6 h, up to 66% of the influenza virus membrane protein, hemagglutinin (HA), synthesized from injected synthetic RNA, can move to the Golgi apparati in nonmatured oocytes; indeed after longer periods some correctly folded HA can be detected at the cell surface where it distributes in a nonpolarized fashion. In matured oocytes, up to 49% of the HA becomes endo H resistant in the same 6-h period. We conclude that movement from the endoplasmic reticulum to the Golgi can occur in matured oocytes despite the dramatic fragmentation of the Golgi apparati that we observe to occur on maturation. This observation of residual protein movement during meiotic metaphase contrasts with the situation at mitotic metabphase in cultured mammalian cells where all movement ceases, but resembles that in the budding yeast Saccharomyces cerevisiae where transport is unaffected.     

Actin microfilaments facilitate the retrograde transport from the Golgi complex to the endoplasmic reticulum in mammalian cells

The morphology and subcellular positioning of the Golgi complex depend on both microtubule and actin cytoskeletons. In contrast to microtubules, the role of actin cytoskeleton in the secretory pathway in mammalian cells has not been clearly established. Using cytochalasin D, we have previously shown that microfilaments are not involved in the endoplasmic reticulum–Golgi membrane dynamics. However, it has been reported that, unlike botulinum C2 toxin and latrunculins, cytochalasin D does not produce net depolymerization of actin filaments. Therefore, we have reassessed the functional role of actin microfilaments in the early steps of the biosynthetic pathway using C2 toxin and latrunculin B. The anterograde endoplasmic reticulum-to-Golgi transport monitored with the vesicular stomatitis virus-G protein remained unaltered in cells treated with cytochalasin D, latrunculin B or C2 toxin. Conversely, the brefeldin A-induced Golgi membrane fusion into the endoplasmic reticulum, the Golgi-to-endoplasmic reticulum transport of a Shiga toxin mutant form, and the subcellular distribution of the KDEL receptor were all impaired when actin microfilaments were depolymerized by latrunculin B or C2 toxin. These findings, together with the fact that COPI-coated and uncoated vesicles contain β/γ-actin isoforms, indicate that actin microfilaments are involved in the endoplasmic reticulum/Golgi interface, facilitating the retrograde Golgi-to-endoplasmic reticulum membrane transport, which could be mediated by the orchestrated movement of transport intermediates along microtubule and microfilament tracks.     

A mammalian homologue of SLY1 a yeast gene required for transport from endoplasmic reticulum to Golgi

We characterized rat cDNAs that predict a protein, r-Slyl, which is similar to SLY1, a yeast protein that plays a critical role in endoplasmic reticulum to Golgi apparatus vesicle trafficking. The r-Slyl gene is expressed in all tissues examined     

Protein recycling from the Golgi apparatus to the endoplasmic reticulum in plants and its minor contribution to calreticulin retention

Using pulse-chase experiments combined with immunoprecipitation and N-glycan structural analysis, we showed that the retrieval mechanism of proteins from post-endoplasmic reticulum (post-ER) compartments is active in plant cells at levels similar to those described previously for animal cells. For instance, recycling from the Golgi apparatus back to the ER is sufficient to block the secretion of as much as 90% of an extracellular protein such as the cell wall invertase fused with an HDEL C-terminal tetrapeptide. Likewise, recycling can sustain fast retrograde transport of Golgi enzymes into the ER in the presence of brefeldin A. However, on the basis of our data, we propose that this retrieval mechanism in plants has little impact on the ER retention of a soluble ER protein such as calreticulin. Indeed, the latter is retained in the ER without any N-glycan-related evidence for a recycling through the Golgi apparatus. Taken together, these results indicate that calreticulin and perhaps other plant reticuloplasmins are possibly largely excluded from vesicles exported from the ER. Instead, they are probably retained in the ER by mechanisms that rely primarily on signals other than H/KDEL motifs.     

Regulation of protein transport from the Golgi complex to the endoplasmic reticulum by CDC42 and N-WASP

Actin is involved in the organization of the Golgi complex and Golgi-to-ER protein transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER protein transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and non-coated Golgi-associated transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgi-to-ER transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(Delta WA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-N-WASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER protein transport in an N-WASP-dependent manner.