Stage-specific assays to study biosynthetic cargo selection and role of SNAREs in export from the endoplasmic reticulum and delivery to the Golgi

To analyze the role of coat protein type II (COPII) coat components and targeting and fusion factors in selective export from the endoplasmic reticulum (ER) and transport to the Golgi, we have developed three novel, stage-specific assays. Cargo selection can be measured using a "stage 1 cargo capture assay," in which ER microsomes are incubated in the presence of glutathione S-transferase (GST)-tagged Sar1 GTPase and purified Sec23/24 components to follow recruitment of biosynthetic cargo to prebudding complexes. This cargo recruitment assay can be followed by two sequential assays that measure separately the budding of COPII-coated vesicles from ER microsomes (stage 2) and, finally, delivery of cargo-containing vesicles to the Golgi (stage 3). We show how these assays provide a means to identify the snap receptor (SNARE) protein rBet1 as an essential component that is not required for vesicle formation, but is required for vesicle targeting and fusion during ER-to-Golgi transport. In general, these assays provide an approach to characterize the biochemical basis for the recruitment of a wide variety of biosynthetic cargo proteins to COPII vesicles and the role of different transport components in the early secretory pathway of mammalian cells.     

COPII coat assembly and selective export from the endoplasmic reticulum

The coat protein complex II (COPII) generates transport vesicles that mediate protein transport from the endoplasmic reticulum (ER). Recent structural and biochemical studies have suggested that the COPII coat is responsible for direct capture of membrane cargo proteins and for the physical deformation of the ER membrane that drives the transport vesicle formation. The COPII-coated vesicle formation at the ER membrane is triggered by the activation of the Ras-like small GTPase Sar1 by GDP/GTP exchange, and activated Sar1 in turn promotes COPII coat assembly. Subsequent GTP hydrolysis by Sar1 leads to disassembly of the coat proteins, which are then recycled for additional rounds of vesicle formation. Thus, the Sar1 GTPase cycle is thought to regulate COPII coat assembly and disassembly. Emerging evidence suggests that the cargo proteins modulate the Sar1 GTP hydrolysis to coordinate coat assembly with cargo selection. Here, I discuss the possible roles of the GTP hydrolysis by Sar1 in COPII coat assembly and selective uptake of cargo proteins into transport vesicles.     

Sar1p N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle

Secretory proteins traffic from the ER to the Golgi via COPII-coated transport vesicles. The five core COPII proteins (Sar1p, Sec23/24p, and Sec13/31p) act in concert to capture cargo proteins and sculpt the ER membrane into vesicles of defined geometry. The molecular details of how the coat proteins deform the lipid bilayer into vesicles are not known. Here we show that the small GTPase Sar1p directly initiates membrane curvature during vesicle biogenesis. Upon GTP binding by Sar1p, membrane insertion of the N-terminal amphipathic alpha helix deforms synthetic liposomes into narrow tubules. Replacement of bulky hydrophobic residues in the alpha helix with alanine yields Sar1p mutants that are unable to generate highly curved membranes and are defective in vesicle formation from native ER membranes despite normal recruitment of coat and cargo proteins. Thus, the initiation of vesicle budding by Sar1p couples the generation of membrane curvature with coat-protein assembly and cargo capture.     

Endoplasmic reticulum export sites and Golgi bodies behave as single mobile secretory units in plant cells

In contrast with animals, plant cells contain multiple mobile Golgi stacks distributed over the entire cytoplasm. However, the distribution and dynamics of protein export sites on the plant endoplasmic reticulum (ER) surface have yet to be characterized. A widely accepted model for ER-to-Golgi transport is based on the sequential action of COPII and COPI coat complexes. The COPII complex assembles by the ordered recruitment of cytosolic components on the ER membrane. Here, we have visualized two early components of the COPII machinery, the small GTPase Sar1p and its GTP exchanging factor Sec12p in live tobacco (Nicotiana tabacum) leaf epidermal cells. By in vivo confocal laser scanning microscopy and fluorescence recovery after photobleaching experiments, we show that Sar1p cycles on mobile punctate structures that track with the Golgi bodies in close proximity but contain regions that are physically separated from the Golgi bodies. By contrast, Sec12p is uniformly distributed along the ER network and does not accumulate in these structures, consistent with the fact that Sec12p does not become part of a COPII vesicle. We propose that punctate accumulation of Sar1p represents ER export sites (ERES). The sites may represent a combination of Sar1p-coated ER membranes, nascent COPII membranes, and COPII vectors in transit, which have yet to lose their coats. ERES can be induced by overproducing Golgi membrane proteins but not soluble bulk-flow cargos. Few punctate Sar1p loci were observed that are independent of Golgi bodies, and these may be nascent ERES. The vast majority of ERES form secretory units that move along the surface of the ER together with the Golgi bodies, but movement does not influence the rate of cargo transport between these two organelles. Moreover, we could demonstrate using the drug brefeldin A that formation of ERES is strictly dependent on a functional retrograde transport route from the Golgi apparatus.     

A role for Yip1p in COPII vesicle biogenesis

Yeast Ypt1p-interacting protein (Yip1p) belongs to a conserved family of transmembrane proteins that interact with Rab GTPases. We encountered Yip1p as a constituent of ER-derived transport vesicles, leading us to hypothesize a direct role for this protein in transport through the early secretory pathway. Using a cell-free assay that recapitulates protein transport from the ER to the Golgi complex, we find that affinity-purified antibodies directed against the hydrophilic amino terminus of Yip1p potently inhibit transport. Surprisingly, inhibition is specific to the COPII-dependent budding stage. In support of this in vitro observation, strains bearing the temperature-sensitive yip1-4 allele accumulate ER membranes at a nonpermissive temperature, with no apparent accumulation of vesicle intermediates. Genetic interaction analyses of the yip1-4 mutation corroborate a function in ER budding. Finally, ordering experiments show that preincubation of ER membranes with COPII proteins decreases sensitivity to anti-Yip1p antibodies, indicating an early requirement for Yip1p in vesicle formation. We propose that Yip1p has a previously unappreciated role in COPII vesicle biogenesis.     

Molecular Dissection of Erv26p Identifies Separable Cargo Binding and Coat Protein Sorting Activities

Efficient export of secretory alkaline phosphatase (ALP) from the endoplasmic reticulum depends on the conserved transmembrane sorting adaptor Erv26p/Svp26p. In the present study we investigated the mechanism by which Erv26p couples pro-ALP to the coat protein complex II (COPII) export machinery. Site-specific mutations were introduced into Erv26p, and mutant proteins were assessed in cell-free assays that monitor interactions with pro-ALP cargo and packaging into COPII vesicles. Mutations in the second and third loop domains of Erv26p inhibited interaction with pro-ALP, whereas mutations in the C-terminal tail sequence influenced incorporation into COPII vesicles and subcellular distribution. Interestingly mutations in the second loop domain also influenced Erv26p homodimer associations. Finally we demonstrated that Ktr3p, a cis-Golgi-localized mannosyltransferase, also relies on Erv26p for efficient COPII-dependent export from the endoplasmic reticulum. These findings demonstrate that Erv26p acts as a protein sorting adaptor for a variety of Type II transmembrane cargo proteins and requires domain-specific interactions with both cargo and coat subunits to promote efficient secretory protein transport.Anterograde transport in the eukaryotic secretory pathway is initiated by the formation of COPII²-coated vesicles that emerge from transitional ER sites. The COPII coat, which consists of the small GTPase Sar1p, Sec23/24 complex, and Sec13/31 complex, selects vesicle cargo through recognition of export signals and forms ER-derived vesicles through assembly of an outer layer cage structure (1, 2). Cytoplasmically exposed ER export signals have been identified in secretory cargo including the C-terminal dihydrophic and diacidic motifs (3, 4). Structural studies indicate that the Sec24p subunit of the COPII coat contains distinct binding sites for some of the molecularly defined export signals (5, 6). Thus a cycle of cargo-coat interactions regulated by the Sar1p GTPase directs anterograde movement of secretory proteins into ER-derived transport vesicles (7).Although many secretory proteins contain known export signals that interact directly with COPII subunits, the diverse array of secretory cargo that depends on this export route requires additional machinery for efficient collection of all cargo into COPII vesicles (1). For instance certain soluble secretory proteins as well as transmembrane cargo require protein sorting adaptors for efficient ER export. These membrane-spanning adaptors, or sorting receptors, interact directly with secretory cargo and with coat subunits to efficiently couple cargo to the COPII budding machinery. For example, ERGIC-53 acts as a protein sorting adaptor for several glycoproteins and has a large N-terminal lumenal domain that interacts with secretory proteins including blood coagulation factors, cathepsins, and α₁-antitrypsin (8–10). The cytoplasmic C-terminal tail of ERGIC-53 contains a diphenylalanine export signal that is necessary for COPII export as well as a dilysine motif required for COPI-dependent retrieval to the ER (11). Additional ER vesicle proteins identified in yeast have been shown to interact with the COPII coat as well as specific secretory proteins (12). For example Erv29p acts as a protein sorting adaptor for the soluble secretory proteins glyco-pro-α-factor and carboxypeptidase Y (13). Erv29p also contains COPII and COPI sorting signals that shuttle the protein between ER and Golgi compartments. More recently Erv26p was identified as a cargo receptor that escorts the pro-form of secretory alkaline phosphatase (ALP) into COPII-coated vesicles (14).Although COPII sorting receptors have been identified, the molecular mechanisms by which these receptors link cargo to coat remain poorly understood. Moreover it is not clear how cargo binding is regulated to promote interaction in the ER and then trigger dissociation in the Golgi complex. We have shown previously that Erv26p binds to pro-ALP and is required for efficient export of this secretory protein from the ER (14). Therefore specific lumenal regions of Erv26p are proposed to interact with pro-ALP, whereas cytosolically exposed sorting signals are presumably recognized and bound by coat subunits. To gain insight on the molecular contacts required for Erv26p sorting function, we undertook a systematic mutational analysis of this multispanning membrane protein. After generating a series of Erv26p mutants, we observed that mutation of specific residues in the third loop domain affect pro-ALP interaction and that residues in the C-terminal cytosolic tail are required for COPII and COPI transport. Finally mutation of residues in the second loop domain influenced Erv26p homodimer formation and sorting activity.     

Sed4p stimulates Sar1p GTP hydrolysis and promotes limited coat disassembly

The coat protein complex II (COPII) generates transport vesicles that mediate protein export from the endoplasmic reticulum (ER). The first step of COPII vesicle formation involves conversion of Sar1p-GDP to Sar1p-GTP by guanine-nucleotide-exchange factor (GEF) Sec12p. In Saccharomyces cerevisiae, Sed4p is a structural homolog of Sec12p, but no GEF activity toward Sar1p has been found. Although the role of Sed4p in COPII vesicle formation is implied by the genetic interaction with SAR1, the molecular basis by which Sed4p contributes to this process is unclear. This study showed that the cytoplasmic domain of Sed4p preferentially binds the nucleotide-free form of Sar1p and that Sed4p binding stimulates both the intrinsic and Sec23p GTPase-activating protein (GAP)-accelerated GTPase activity of Sar1p. This stimulation of Sec23p GAP activity by Sed4p leads to accelerated dissociation of coat proteins from membranes. However, Sed4p binding to Sar1p occurs only when cargo is not associated with Sar1p. On the basis of these findings, Sed4p appears to accelerate the dissociation of the Sec23/24p coat from the membrane, but the effect is limited to Sar1p molecules that do not capture cargo protein. We speculate that this restricted coat disassembly may contribute to the concentration of specific cargo molecules into the COPII vesicles.     

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.     

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.     

Exiting the endoplasmic reticulum

Vesicular transport from the endoplasmic reticulum (ER) to the Golgi complex constitutes the initial step in protein secretion. COPII-coated vesicles mediate the export of newly synthesized proteins from the ER, and this transport step is coupled with COPI-mediated retrograde traffic to form a transport circuit that supports the compositional asymmetry of the ER-Golgi system. Biochemical and structural studies have advanced our understanding of the mechanisms that control vesicle formation and cargo-protein capture. Recent work has highlighted the function of transitional ER regions in specifying the location of COPII budding.     

Traffic of Kv4 K+ channels mediated by KChIP1 is via a novel post-ER vesicular pathway

The traffic of Kv4 K+ channels is regulated by the potassium channel interacting proteins (KChIPs). Kv4.2 expressed alone was not retained within the ER, but reached the Golgi complex. Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2-4)-EYFP. The EF-hand mutant had no effect on general exocytic traffic. Traffic of Kv4.2 was coat protein complex I (COPI)-dependent, but KChIP1-containing vesicles were not COPII-coated, and expression of a GTP-loaded Sar1 mutant to block COPII function more effectively inhibited traffic of vesicular stomatitis virus glycoprotein (VSVG) than did KChIP1/Kv4.2 through the secretory pathway. Therefore, KChIP1seems to be targeted to post-ER transport vesicles, different from COPII-coated vesicles and those involved in traffic of VSVG. When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.     

The genetic basis of a craniofacial disease provides insight into COPII coat assembly

Proteins trafficking through the secretory pathway must first exit the endoplasmic reticulum (ER) through membrane vesicles created and regulated by the COPII coat protein complex. Cranio-lenticulo-sutural dysplasia (CLSD) was recently shown to be caused by a missense mutation in SEC23A, a gene encoding one of two paralogous COPII coat proteins. We now elucidate the molecular mechanism underlying this disease. In vitro assays reveal that the mutant form of SEC23A poorly recruits the Sec13-Sec31 complex, inhibiting vesicle formation. Surprisingly, this effect is modulated by the Sar1 GTPase paralog used in the reaction, indicating distinct affinities of the two human Sar1 paralogs for the Sec13-Sec31 complex. Patient cells accumulate numerous tubular cargo-containing ER exit sites devoid of observable membrane coat, likely representing an intermediate step in COPII vesicle formation. Our results indicate that the Sar1-Sec23-Sec24 prebudding complex is sufficient to form cargo-containing tubules in vivo, whereas the Sec13-Sec31 complex is required for membrane fission.     

Concentration of Sec12 at ER exit sites via interaction with cTAGE5 is required for collagen export

Mechanisms for exporting variably sized cargo from the endoplasmic reticulum (ER) using the same machinery remain poorly understood. COPII-coated vesicles, which transport secretory proteins from the ER to the Golgi apparatus, are typically 60–90 nm in diameter. However, collagen, which forms a trimeric structure that is too large to be accommodated by conventional transport vesicles, is also known to be secreted via a COPII-dependent process. In this paper, we show that Sec12, a guanine-nucleotide exchange factor for Sar1 guanosine triphosphatase, is concentrated at ER exit sites and that this concentration of Sec12 is specifically required for the secretion of collagen VII but not other proteins. Furthermore, Sec12 recruitment to ER exit sites is organized by its direct interaction with cTAGE5, a previously characterized collagen cargo receptor component, which functions together with TANGO1 at ER exit sites. These findings suggest that the export of large cargo requires high levels of guanosine triphosphate–bound Sar1 generated by Sec12 localized at ER exit sites.     

Mechanisms of COPII vesicle formation and protein sorting

The evolutionarily conserved coat protein complex II (COPII) generates transport vesicles that mediate protein transport from the endoplasmic reticulum (ER). COPII coat is responsible for direct capture of cargo proteins and for the physical deformation of the ER membrane that drives the COPII vesicle formation. In addition to coat proteins, recent data have indicated that the Ras-like small GTPase Sar1 plays multiple roles, such as COPII coat recruitment, cargo sorting, and completion of the final fission. In the present review, we summarize current knowledge of COPII-mediated vesicle formation from the ER, as well as highlighting non-canonical roles of COPII components.     

Visualization of cargo concentration by COPII minimal machinery in a planar lipid membrane

Selective protein export from the endoplasmic reticulum is mediated by COPII vesicles. Here, we investigated the dynamics of fluorescently labelled cargo and non‐cargo proteins during COPII vesicle formation using single‐molecule microscopy combined with an artificial planar lipid bilayer. Single‐molecule analysis showed that the Sar1p–Sec23/24p‐cargo complex, but not the Sar1p–Sec23/24p complex, undergoes partial dimerization before Sec13/31p recruitment. On addition of a complete COPII mixture, cargo molecules start to assemble into fluorescent spots and clusters followed by vesicle release from the planar membrane. We show that continuous GTPase cycles of Sar1p facilitate cargo concentration into COPII vesicle buds, and at the same time, non‐cargo proteins are excluded from cargo clusters. We propose that the minimal set of COPII components is required not only to concentrate cargo molecules, but also to mediate exclusion of non‐cargo proteins from the COPII vesicles.     

A vacuolar sorting domain may also influence the way in which proteins leave the endoplasmic reticulum

Protein sorting to plant vacuoles is known to be dependent on a considerable variety of protein motifs recognized by a family of sorting receptors. This can involve either traffic from the endoplasmic reticulum (ER) through the Golgi apparatus or direct ER-to-vacuole transport. Barley aspartic protease (Phytepsin) was shown previously to reach the vacuole via trafficking through the Golgi apparatus. Here we show that Phytepsin normally exits the ER in a COPII-mediated manner, because the Phytepsin precursor accumulates in the ER upon specific inhibition of the formation of COPII vesicles in vivo. Phytepsin differs from its yeast and mammalian counterparts by the presence of a saposin-like plant-specific insert (PSI). Deletion of this domain comprising 104 amino acids causes efficient secretion of the truncated molecule (Phytepsin Delta PSI) without affecting the enzymatic activity of the enzyme. Interestingly, deletion of the PSI also changes the way in which Phytepsin exits the ER. Inhibition of COPII vesicle formation causes accumulation of the Phytepsin precursor in the ER but has no effect on the secretion of Phytepsin Delta PSI. This suggests either that vacuolar sorting commences at the ER export step and involves recruitment into COPII vesicles or that the PSI domain carries two signals, one for COPII-dependent export from the ER and one for vacuolar delivery from the Golgi. The relevance of these observations with respect to the bulk flow model of secretory protein synthesis is discussed.     

Insights into structural and regulatory roles of Sec16 in COPII vesicle formation at ER exit sites

COPII-coated buds are formed at endoplasmic reticulum exit sites (ERES) to mediate ER-to-Golgi transport. Sec16 is an essential factor in ERES formation, as well as in COPII-mediated traffic in vivo. Sec16 interacts with multiple COPII proteins, although the functional significance of these interactions remains unknown. Here we present evidence that full-length Sec16 plays an important role in regulating Sar1 GTPase activity at the late steps of COPII vesicle formation. We show that Sec16 interacts with Sec23 and Sar1 through its C-terminal conserved region and hinders the ability of Sec31 to stimulate Sec23 GAP activity toward Sar1. We also find that purified Sec16 alone can self-assemble into homo-oligomeric complexes on a planar lipid membrane. These features ensure prolonged COPII coat association within a preformed Sec16 cluster, which may lead to the formation of ERES. Our results indicate a mechanistic relationship between COPII coat assembly and ERES formation.     

Activation of phospholipase D by the small GTPase Sar1p is required to support COPII assembly and ER export

The small GTPase Sar1p controls the assembly of the cytosolic COPII coat that mediates export from the endoplasmic reticulum (ER). Here we demonstrate that phospholipase D (PLD) activation is required to support COPII-mediated ER export. PLD activity by itself does not lead to the recruitment of COPII to the membranes or ER export. However, PLD activity is required to support Sar1p-dependent membrane tubulation, the subsequent Sar1p-dependent recruitment of Sec23/24 and Sec13/31 COPII complexes to ER export sites and ER export. Sar1p recruitment to the membrane is PLD independent, yet activation of Sar1p is required to stimulate PLD activity on ER membranes, thus PLD is temporally regulated to support ER export. Regulated modification of membrane lipid composition is required to support the cooperative interactions that enable selective transport, as we demonstrate here for the mammalian COPII coat.