Syntaxin 17 cycles between the ER and ERGIC and is required to maintain the architecture of ERGIC and Golgi

Background information. Syntaxin 17 is a SNARE (soluble N‐ethylmaleimide‐sensitive‐factor‐attachment protein receptor) protein that predominantly localizes to the ER (endoplasmic reticulum) and to some extent in the ERGIC (ER—Golgi intermediate compartment). Syntaxin 17 has been suggested to function as a receptor at the ER membrane that mediates trafficking between the ER and post‐ER compartments. It has a unique 33 amino acid luminal tail whose function is not known. Here we have investigated the structural requirements for localization of syntaxin 17 to the ERGIC and its role in trafficking. Results. Deletion analysis showed that syntaxin 17 required its cytoplasmic domain to exit the ER and localize to the ERGIC. Mutation of a conserved tyrosine residue in the cytoplasmic domain resulted in reduced localization of syntaxin 17 in the ERGIC and ER‐exit sites, suggesting the presence of a tyrosine‐based ER export motif. Syntaxin 17 also required its C‐terminal tail to localize to the ERES (ER exit sites) and ERGIC. Knockdown of syntaxin 17 destabilized the ERGIC organization and also caused fragmentation of the Golgi complex. Syntaxin 17 showed direct interaction with transmembrane proteins p23 and p25 (cargo receptors that cycle between the ER and Golgi) with the help of its C‐terminal tail. Overexpression of syntaxin 17 redistributed β‐COP (β‐coatomer protein) which required its C‐terminal tail. Overexpression of syntaxin 17 also blocked the anterograde transport of VSVG (vesicular stomatitis virus G‐protein) in the ERGIC. Conclusions. We show that syntaxin 17 has a tyrosine‐based motif which is required for its incorporation into COPII (coatomer protein II) vesicles, exit from the ER and localization to the ERGIC. Our results suggest that syntaxin 17 cycles between the ER and ERGIC through classical trafficking pathways involving COPII and COPI (coatomer protein I) vesicles, which requires its unique C‐terminal tail. We also show that syntaxin 17 is essential for maintaining the architecture of ERGIC and Golgi.     

COPII collar defines the boundary between ER and ER exit site and does not coat cargo containers

COPII and COPI mediate the formation of membrane vesicles translocating in opposite directions within the secretory pathway. Live-cell and electron microscopy revealed a novel mode of function for COPII during cargo export from the ER. COPII is recruited to membranes defining the boundary between the ER and ER exit sites, facilitating selective cargo concentration. Using direct observation of living cells, we monitored cargo selection processes, accumulation, and fission of COPII-free ERES membranes. CRISPR/Cas12a tagging, the RUSH system, and pharmaceutical and genetic perturbations of ER-Golgi transport demonstrated that the COPII coat remains bound to the ER–ERES boundary during protein export. Manipulation of the cargo-binding domain in COPII Sec24B prohibits cargo accumulation in ERES. These findings suggest a role for COPII in selecting and concentrating exported cargo rather than coating Golgi-bound carriers. These findings transform our understanding of coat proteins’ role in ER-to-Golgi transport.     

COPII‐mediated trafficking at the ER/ERGIC interface

Coat proteins play multiple roles in the life cycle of a membrane‐bound transport intermediate, functioning in lipid bilayer remodeling, cargo selection and targeting to an acceptor compartment. The Coat Protein complex II (COPII) coat is known to act in each of these capacities, but recent work highlights the necessity for numerous accessory factors at all stages of transport carrier existence. Here, we review recent findings that highlight the roles of COPII and its regulators in the biogenesis of tubular COPII‐coated carriers in mammalian cells that enable cargo transport between the endoplasmic reticulum and ER‐Golgi intermediate compartments, the first step in a series of trafficking events that ultimately allows for the distribution of biosynthetic secretory cargoes throughout the entire endomembrane system.     

Regulation of ER–Golgi Intermediate Compartment Tubulation and Mobility by COPI Coats,Motor Proteins and Microtubules

Little is known about the formation and regulation of endoplasmic reticulum (ER)–Golgi transport intermediates, although previous studies suggest that cargo is the main regulator of their morphology. In this study, we analyze the role of coat protein I (COPI) and cytoskeleton in the formation of tubular ER–Golgi intermediate compartment (ERGIC) and also show that partial COPI detachment by means of low temperature (15°C) or brefeldin A induces the formation of transient tubular ERGIC elements. Most of them moved from the cell periphery to the perinuclear area and were 2.5× slower than vesicles. Time‐lapse analysis of living cells demonstrates that the ERGIC elements are able to shift very fast from tubular to vesicular forms and vice versa, suggesting that the amount of cargo is not the determining factor for ERGIC morphology. Both the partial microtubule depolymerization and the inhibition of uncoating of the membranes result in the formation of long tubules that grow from round ERGICs and form at complex network. Interestingly, both COPI detachment and microtubule depolymerization induce a redistribution of kinesin from peripheral ERGIC elements to the Golgi area, while dynein distribution is not affected. However, both kinesin and dynein downregulation by RNA interference induced ERGIC tubulation. The tubules induced by kinesin depletion were static, whereas those resulting from dynein depletion were highly mobile. Our results strongly suggest that the interaction of motor proteins with COPI‐coated membranes and microtubules is a key regulator of ERGIC morphology and mobility.     

Evidence against an essential role of COPII-mediated cargo transport to the endoplasmic reticulum-Golgi intermediate compartment in the formation of the primary membrane of vaccinia virus

Vaccinia virus assembles two distinct lipoprotein membranes. The primary membrane contains nonglycosylated proteins, appears as crescents in the cytoplasm, and delimits immature and mature intracellular virions. The secondary or wrapping membrane contains glycoproteins, is derived from virus-modified trans-Golgi or endosomal cisternae, forms a loose coat around some intracellular mature virions, and becomes the envelope of extracellular virions. Although the mode of formation of the wrapping membrane is partially understood, we know less about the primary membrane. Recent reports posit that the primary membrane originates from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). According to this model, viral primary membrane proteins are cotranslationally inserted into the ER and accumulate in the ERGIC. To test the ERGIC model, we employed Sar1(H79G), a dominant negative form of the Sar1 protein, which is an essential component of coatomer protein II (COPII)-mediated cargo transport from the ER to the ERGIC and other post-ER compartments. Overexpression of Sar1(H79G) by transfection or by a novel recombinant vaccinia virus with an inducible Sar1(H79G) gene resulted in retention of ERGIC 53 in the ER but did not interfere with localization of viral primary membrane proteins in factory regions or with formation of viral crescent membranes and infectious intracellular mature virions. Wrapping of intracellular mature virions and formation of extracellular virions did not occur, however, because some proteins that are essential for the secondary membrane were retained in the ER as a consequence of Sar1(H79G) overexpression. Our data argue against an essential role of COPII-mediated cargo transport and the ERGIC in the formation of the viral primary membrane. Instead, viral membranes may be derived directly from the ER or by a novel mechanism.     

Silencing of Mammalian Sar1 Isoforms Reveals COPII‐Independent Protein Sorting and Transport

The Sar1 GTPase coordinates the assembly of coat protein complex‐II (COPII) at specific sites of the endoplasmic reticulum (ER). COPII is required for ER‐to‐Golgi transport, as it provides a structural and functional framework to ship out protein cargoes produced in the ER. To investigate the requirement of COPII‐mediated transport in mammalian cells, we used small interfering RNA (siRNA)‐mediated depletion of Sar1A and Sar1B. We report that depletion of these two mammalian forms of Sar1 disrupts COPII assembly and the cells fail to organize transitional elements that coordinate classical ER‐to‐Golgi protein transfer. Under these conditions, minimal Golgi stacks are seen in proximity to juxtanuclear ER membranes that contain elements of the intermediate compartment, and from which these stacks coordinate biosynthetic transport of protein cargo, such as the vesicular stomatitis virus G protein and albumin. Here, transport of procollagen‐I is inhibited. These data provide proof‐of‐principle for the contribution of alternative mechanisms that support biosynthetic trafficking in mammalian cells, providing evidence of a functional boundary associated with a bypass of COPII .     

Kinase signaling initiates coat complex II (COPII) recruitment and export from the mammalian endoplasmic reticulum

The events regulating coat complex II (COPII) vesicle formation involved in the export of cargo from the endoplasmic reticulum (ER) are unknown. COPII recruitment to membranes is initiated by the activation of the small GTPase Sar1. We have utilized purified COPII components in both membrane recruitment and cargo export assays to analyze the possible role of kinase regulation in ER export. We now demonstrate that Sar1 recruitment to membranes requires ATP. We find that the serine/threonine kinase inhibitor H89 abolishes membrane recruitment of Sar1, thereby preventing COPII polymerization by interfering with the recruitment of the cytosolic Sec23/24 COPII coat complex. Inhibition of COPII recruitment prevents export of cargo from the ER. These results demonstrate that ER export and initiation of COPII vesicle formation in mammalian cells is under kinase regulation.     

ER-to-Golgi carriers arise through direct en bloc protrusion and multistage maturation of specialized ER exit domains

Protein transport between the ER and the Golgi in mammalian cells occurs via large pleiomorphic carriers, and most current models suggest that these are formed by the fusion of small ER-derived COPII vesicles. We have examined the dynamics and structural features of these carriers during and after their formation from the ER by correlative video/light electron microscopy and tomography. We found that saccular carriers containing either the large supramolecular cargo procollagen or the small diffusible cargo protein VSVG arise through cargo concentration and direct en bloc protrusion of specialized ER domains in the vicinity of COPII-coated exit sites. This formation process is COPII dependent but does not involve budding and fusion of COPII-dependent vesicles. Fully protruded saccules then move centripetally, evolving into one of two types of carriers (with distinct kinetic and structural features). These findings provide an alternative framework for analysis of ER-to-Golgi traffic.     

Want to leave the ER? We offer vesicles,tubules, and tunnels

Export from the ER is COPII-dependent. However, there is disagreement on the nature of the cargo-containing carriers that exit the ER. Two new studies from Shomron et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.201907224) and Weigel et al. (2021. Cell. https://doi.org/10.1016/j.cell.2021.03.035) present a new model, where COPII helps to select secretory cargo but does not coat the carriers leaving the ER.

An analogy for ER-to-Golgi transport is day-to-day logistics, whereby we have developed different types of carriers to accommodate cargo of varying shapes, quantities, and sizes. Following the same logic, we could assume that our cells are equipped with carriers of different shapes and sizes to shuttle small or bulky cargo or different cargo quantities out of the ER. Although this appears straightforward, the molecular details of ER-to-Golgi transport has been the subject of intensive debate.Early work on secretory trafficking from the 1960s noted that secretory proteins leave the ER at ribosome free regions, which were termed transitional elements (or transitional ER; 1). These transitional elements were postulated to give rise to “transport vesicles with fuzzy coats,” which mediate transport from the ER (1). Sec23 was found to be enriched at these transitional elements (2) and was later shown to be part of the COPII complex that mediates export from the ER in small vesicles (3). Since then, the standard model for export from the ER was that small and round COPII vesicles (60–80 nm in diameter) leave the ER from transitional elements, now referred to as ER exit sites (ERES; Fig. 1 A). This model was later expanded to include pre-Golgi intermediates such as the ER-Golgi-intermediate compartment (ERGIC) generated from the homotypic fusion of COPII vesicles (4).Open in a separate windowFigure 1.Depiction of different possible modes of ER export. (A) The classical (vesicular) transport model that includes budding of a coated vesicle followed by homotypic fusion of COPII vesicles. (B) ERES can give rise to tubular structures, which either depart as tubules followed by COPI recruitment or may form tunnels with distal compartments.Despite its wide acceptance, experimental evidence for the presence of COPII vesicles at ERES in intact cells was scarce. The main evidence for COPII vesicles came from in vitro assays from which such vesicles were isolated. Furthermore, the relatively small size of COPII vesicles (60–80 nm) could not explain transport of bulky cargo such as type I collagen (with a length of 300 nm). A major challenge to the vesicular transport model came from a paper by Mironov et al. (5), who presented evidence that ER-derived carriers are large uncoated saccules that mature toward the Golgi. Notably, round and small COPII vesicles as carriers mediating the ER export were absent during synchronized transport of type I collagen and VSVG-ts045 (a temperature-sensitive mutant of the vesicular stomatitis virus glycoprotein). Both types of cargo rely on retention in the ER at 40°C followed by export upon lowering the temperature (and addition of ascorbic acid in the case of type I collagen). Supporters of the conventional vesicular transport model argued that the size of type I collagen as well as the large quantities of molecules queuing to leave the ER simultaneously might have led the ER export machinery to adapt to this situation, thereby leading to formation of the observed carriers. Further support for the vesicle transport model came from work using 3D electron tomography showing that ERES are domains that are continuous with the ER, which are surrounded by COPII vesicles (6). The electron tomography was performed in both chemically fixed as well as high-pressure-frozen cells in the absence of secretory cargo overexpression or synchronized trafficking waves. Because ERES exhibited budding profiles coated with COPII, it was concluded that the COPII vesicles bud as coated carriers from ERES. Despite this support for the vesicular transport model, it was becoming increasingly clear that ER-to-Golgi transport cannot be explained solely by small COPII vesicles. Thus, the idea that different types of carriers operate in the ER-Golgi route began to ripen in the community.In yeast, cis-Golgi compartments were shown to touch ERES to “pick up” secretory cargo (7). Whether this “hug and kiss” model involves fusion of the cis-Golgi with budded COPII vesicles or whether it forms a “tunnel” between the ERES and the cis-Golgi remains unclear. In support of the existence of tunnels is work from the Malhotra group showing that collagen transport might occur via recruitment of ERGIC membranes to ERES enriched in pro-collagen (8). The observation that a tunnel is formed between the ERES and ERGIC necessitates the preexistence of an ERGIC membrane container. As discussed, the ERGIC might form by homotypic fusion of COPII vesicles, and thus the tunnel model proposed by Raote and Malhotra (9) can easily be reconciled with the vesicle transport model.In this issue, Shomron et al. (10) present a major challenge to the vesicle transport model. They suggest that COPII complexes only decorate the neck of an ERES, where they solely serve to concentrate cargo into transport containers. This confirms earlier papers showing that COPII mediates concentrative ER export (11, 12). Strikingly, Shomron et al. observe with live imaging that secretory cargo enters a tubule that segregates from COPII at the level of ERES, indicating that the departing transport carrier is not coated. Furthermore, COPII was confined to the neck of the tubular carrier. This finding agrees with previously observed (noncoated) saccules that leave the ER (5). A concurrent study from Weigel et al. (13) reached a similar conclusion. To overcome prior difficulties associated with fixation, low sampling, and thick sections, they aimed at imaging ERES in living cells by combining focused ion beam scanning electron microscopy with cryo-structured illumination microscopy. Furthermore, they used the retention using selective hook technology (14) to perform synchronized cargo release experiments, thus avoiding problems associated with temperature shifts. In agreement with Shomron et al., they show that ERES give rise to a network of tubules that contain secretory cargo devoid of COPII components. Again, COPII components were only found to localize to the neck of these tubules, implicating that the main role of COPII is to concentrate cargo into carriers. They also showed that ERES are structures continuous with the ER (confirming the earlier data from 3D electron-tomography; 6) that adapt in size to accommodate the load of secretory cargo (again confirming earlier work by others; 15, 16).Another interesting finding by both groups was that the tubule acquired COPI as it moved toward the Golgi (10, 13). Therefore, they independently conclude that this presents evidence for a role of COPI in anterograde ER-to-Golgi transport, which challenges the classical model whereby COPI is thought exclusively to mediate retrograde transport from the Golgi back to the ER. It remains unclear what role COPI would precisely play in anterograde transport. Simply because the tubular membrane container is positive for COPI does not necessarily mean that COPI regulates anterograde transport. Carriers need tethering factors such as p115/Uso1, which are recruited by Rab1 to deliver their content to the next compartment. No role for COPI is known in this process. An alternative explanation for the recruitment of COPI to the ER-derived carriers is that this marks the beginning of retrograde transport back to the ER. This is supported by the observation that a mutant of ERGIC-53 (LMAN1) that does not bind COPI is capable of leaving the ER and without exhibiting any defect in anterograde transport (17). Strikingly, this mutant ERGIC-53 mislocalizes to the plasma membrane because it cannot use COPI for retrograde transport (16). Thus, recruitment of COPI might contribute to the maturation of the forward moving membrane carrier by retrieving back ER proteins.Altogether, it appears that several types of carriers (tubules, saccules, tunnels, and coated vesicles) may coexist and operate along the ER-to-Golgi route. The papers by Shomron et al. and Weigel el al. do not cancel or revoke the other models of trafficking. Rather, they add a new model and show us how diverse and flexible this trafficking route is. It is possible that our cells are equipped with all types of carriers, which cells use depending on the size, quantity, or type of cargo, as well as on the cellular and the environmental context. This diversity might confer robustness of the ER-to-Golgi transport pathway. This might explain why different groups reached sometimes opposing conclusions. For instance, papers that relied on waves of synchronized trafficking or on bulky cargo might have shifted the balance toward a certain type of carrier. Most cells contain several hundred ERES, with some of them at several microns’ distance to the Golgi. It is therefore possible that different types of carriers might operate in a manner depending on the type of ERES. Future work will clarify and reconcile all these open questions.     

Sorting of plant vacuolar proteins is initiated in the ER

Transport of soluble cargo molecules to the lytic vacuole of plants requires vacuolar sorting receptors (VSRs) to divert transport of vacuolar cargo from the default secretory route to the cell surface. Just as important is the trafficking of the VSRs themselves, a process that encompasses anterograde transport of receptor–ligand complexes from a donor compartment, dissociation of these complexes upon arrival at the target compartment, and recycling of the receptor back to the donor compartment for a further round of ligand transport. We have previously shown that retromer‐mediated recycling of the plant VSR BP80 starts at the trans‐Golgi network (TGN). Here we demonstrate that inhibition of retromer function by either RNAi knockdown of sorting nexins (SNXs) or co‐expression of mutants of SNX1/2a specifically inhibits the ER export of VSRs as well as soluble vacuolar cargo molecules, but does not influence cargo molecules destined for the COPII‐mediated transport route. Retention of soluble cargo despite ongoing COPII‐mediated bulk flow can only be explained by an interaction with membrane‐bound proteins. Therefore, we examined whether VSRs are capable of binding their ligands in the lumen of the ER by expressing ER‐anchored VSR derivatives. These experiments resulted in drastic accumulation of soluble vacuolar cargo molecules in the ER. This demonstrates that the ER, rather than the TGN, is the location of the initial VSR–ligand interaction. It also implies that the retromer‐mediated recycling route for the VSRs leads from the TGN back to the ER.     

Remodeling of ER‐exit sites initiates a membrane supply pathway for autophagosome biogenesis

Autophagosomes are double‐membrane vesicles generated during autophagy. Biogenesis of the autophagosome requires membrane acquisition from intracellular compartments, the mechanisms of which are unclear. We previously found that a relocation of COPII machinery to the ER–Golgi intermediate compartment (ERGIC) generates ERGIC‐derived COPII vesicles which serve as a membrane precursor for the lipidation of LC3, a key membrane component of the autophagosome. Here we employed super‐resolution microscopy to show that starvation induces the enlargement of ER‐exit sites (ERES) positive for the COPII activator, SEC12, and the remodeled ERES patches along the ERGIC. A SEC12 binding protein, CTAGE5, is required for the enlargement of ERES, SEC12 relocation to the ERGIC, and modulates autophagosome biogenesis. Moreover, FIP200, a subunit of the ULK protein kinase complex, facilitates the starvation‐induced enlargement of ERES independent of the other subunits of this complex and associates via its C‐terminal domain with SEC12. Our data indicate a pathway wherein FIP200 and CTAGE5 facilitate starvation‐induced remodeling of the ERES, a prerequisite for the production of COPII vesicles budded from the ERGIC that contribute to autophagosome formation.     

Proteomics of endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membranes from brefeldin A-treated HepG2 cells identifies ERGIC-32, a new cycling protein that interacts with human Erv46

Cycling proteins play important roles in the organization and function of the early secretory pathway by participating in membrane traffic and selective transport of cargo between the endoplasmic reticulum (ER), the intermediate compartment (ERGIC), and the Golgi. To identify new cycling proteins, we have developed a novel procedure for the purification of ERGIC membranes from HepG2 cells treated with brefeldin A, a drug known to accumulate cycling proteins in the ERGIC. Membranes enriched 110-fold over the homogenate for ERGIC-53 were obtained and analyzed by mass spectrometry. Major proteins corresponded to established and putative cargo receptors and components mediating protein maturation and membrane traffic. Among the uncharacterized proteins, a 32-kDa protein termed ERGIC-32 is a novel cycling membrane protein with sequence homology to Erv41p and Erv46p, two proteins enriched in COPII vesicles of yeast. ERGIC-32 localizes to the ERGIC and partially colocalizes with the human homologs of Erv41p and Erv46p, which mainly localize to the cis-Golgi. ERGIC-32 interacts with human Erv46 (hErv46) as revealed by covalent cross-linking and mistargeting experiments, and silencing of ERGIC-32 by small interfering RNAs increases the turnover of hErv46. We propose that ERGIC-32 functions as a modulator of the hErv41-hErv46 complex by stabilizing hErv46. Our novel approach for the isolation of the ERGIC from BFA-treated cells may ultimately lead to the identification of all proteins rapidly cycling early in the secretory pathway.     

Reconstitution of coat protein complex II (COPII) vesicle formation from cargo-reconstituted proteoliposomes reveals the potential role of GTP hydrolysis by Sar1p in protein sorting

Secretory proteins are transported from the endoplasmic reticulum (ER) in vesicles coated with coat protein complex II (COPII). To investigate the molecular mechanism of protein sorting into COPII vesicles, we have developed an in vitro budding reaction comprising purified coat proteins and cargo reconstituted proteolipsomes. Emp47p, a type-I membrane protein, is specifically required for the transport of an integral membrane protein, Emp46p, from the ER. Recombinant Emp46/47p proteins and the ER resident protein Ufe1p were reconstituted into liposomes whose composition resembles yeast ER membranes. When the proteoliposomes were mixed with COPII proteins and GMP-PNP, Emp46/47p, but not Ufe1p, were concentrated into COPII vesicles. We also show here that reconstituted Emp47p accelerates the GTP hydrolysis by Sar1p as stimulated by its GTPase-activating protein, Sec23/24p, both of which are components of the COPII coat. Furthermore, this GTP hydrolysis decreases the error of cargo sorting. We suggest that GTP hydrolysis by Sar1p promotes exclusion of improper proteins from COPII vesicles.     

De novo formation of plant endoplasmic reticulum export sites is membrane cargo induced and signal mediated

The plant endoplasmic reticulum (ER) contains functionally distinct subdomains at which cargo molecules are packed into transport carriers. To study these ER export sites (ERES), we used tobacco (Nicotiana tabacum) leaf epidermis as a model system and tested whether increased cargo dosage leads to their de novo formation. We have followed the subcellular distribution of the known ERES marker based on a yellow fluorescent protein (YFP) fusion of the Sec24 COPII coat component (YFP-Sec24), which, differently from the previously described ERES marker, tobacco Sar1-YFP, is visibly recruited at ERES in both the presence and absence of overexpressed membrane cargo. This allowed us to quantify variation in the ERES number and in the recruitment of Sec24 to ERES upon expression of cargo. We show that increased synthesis of membrane cargo leads to an increase in the number of ERES and induces the recruitment of Sec24 to these ER subdomains. Soluble proteins that are passively secreted were found to leave the ER with no apparent up-regulation of either the ERES number or the COPII marker, showing that bulk flow transport has spare capacity in vivo. However, de novo ERES formation, as well as increased recruitment of Sec24 to ERES, was found to be dependent on the presence of the diacidic ER export motif in the cytosolic domain of the membrane cargo. Our data suggest that the plant ER can adapt to a sudden increase in membrane cargo-stimulated secretory activity by signal-mediated recruitment of COPII machinery onto existing ERES, accompanied by de novo generation of new ERES.     

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

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.     

Regulated Oligomerization Induces Uptake of a Membrane Protein into COPII Vesicles Independent of Its Cytosolic Tail

Export of transmembrane proteins from the endoplasmic reticulum (ER) is driven by directed incorporation into coat protein complex II (COPII)‐coated vesicles. The sorting of some cargo proteins into COPII vesicles was shown to be mediated by specific interactions between transmembrane and COPII‐coat‐forming proteins. But even though some signals for ER exit have been identified on the cytosolic domains of membrane proteins, the general signaling and sorting mechanisms of ER export are still poorly understood. To investigate the role of cargo protein oligomer formation in the export process, we have created a transmembrane fusion protein that – owing to its FK506‐binding protein domains – can be oligomerized in isolated membranes by addition of a small‐molecule dimerizer. Packaging of the fusion protein into COPII vesicles is strongly enhanced in the presence of the dimerizer, demonstrating that the oligomeric state is an ER export signal for this membrane protein. Surprisingly, the cytosolic tail is not required for this oligomerization‐dependent effect on protein sorting. Thus, an alternative mechanism, such as membrane bending, must account for ER export of the fusion protein.      

Immuno-electron tomography of ER exit sites reveals the existence of free COPII-coated transport carriers

Transport from the endoplasmic reticulum (ER) to the Golgi complex requires assembly of the COPII coat complex at ER exit sites. Recent studies have raised the question as to whether in mammalian cells COPII coats give rise to COPII-coated transport vesicles or instead form ER sub-domains that collect proteins for transport via non-coated carriers. To establish whether COPII-coated vesicles do exist in vivo, we developed approaches to combine quantitative immunogold labelling (to identify COPII) and three-dimensional electron tomography (to reconstruct entire membrane structures). In tomograms of both chemically fixed and high-pressure-frozen HepG2 cells, immuno-labelled COPII was found on ER-associated buds as well as on free approximately 50-nm diameter vesicles. In addition, we identified a novel type of COPII-coated structure that consists of partially COPII-coated, 150-200-nm long, dumb-bell-shaped tubules. Both COPII-coated carriers also contain the SNARE protein Sec22b, which is necessary for downstream fusion events. Our studies unambiguously establish the existence of free, bona fide COPII-coated transport carriers at the ER-Golgi interface, suggesting that assembly of COPII coats in vivo can result in vesicle formation.     

Low temperature (15°C) induces COPII dissociation from membranes and slow exit from the endoplasmic reticulum in HeLa cells

Low temperature induces a transport blockade at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in cultured cells. Our previous studies support that the primary effect of low temperature is the detachment of COPI complexes from membranes. In the present study, we have used immunofluorescence and cryoimmunoelectron microscopy to investigate the effects of low temperature on both COPII and clathrin coat complexes in HeLa cells. Strikingly, COPII proteins moved from membranes to the cytosol at 15°C, accumulating into electron-dense areas. In agreement with this observation, we also showed that ER exit is delayed in cells cultured at this temperature. In contrast, clathrin coat is not affected. Together, our results demonstrate that low temperature induces COPII dissociation from membranes and slow exit from the endoplasmic reticulum. Emma Martínez-Alonso and Mónica Tomás contributed equally to this study.     

Uncoupled packaging of amyloid precursor protein and presenilin 1 into coat protein complex II vesicles

Mutant forms of presenilin (PS) 1 and 2 and amyloid precursor protein (APP) lead to familial Alzheimer's disease. Several reports indicate that PS may modulate APP export from the endoplasmic reticulum (ER). To develop a test of this possibility, we reconstituted the capture of APP and PS1 in COPII (coat protein complex II) vesicles formed from ER membranes in permeabilized cultured cells. The recombinant forms of mammalian COPII proteins were active in a reaction that measures coat subunit assembly and coated vesicle budding on chemically defined synthetic liposomes. However, the recombinant COPII proteins were not active in cargo capture and vesicle budding from microsomal membranes. In contrast, rat liver cytosol was active in stimulating the sorting and packaging of APP, PS1, and p58 (an itinerant ER to Golgi marker protein) into transport vesicles from donor ER membranes. Budding was stimulated in dilute cytosol by the addition of recombinant COPII proteins. Fractionation of the cytosol suggested one or more additional proteins other than the COPII subunits may be essential for cargo selection or vesicle formation from the mammalian ER membrane. The recombinant Sec24C specifically recognized the APP C-terminal region for packaging. Titration of Sarla distinguished the packaging requirements of APP and PS1. Furthermore, APP packaging was not affected by deletion of PS1 or PS1 and 2, suggesting APP and PS1 trafficking from the ER are normally uncoupled.     

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.