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.     

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.     

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.     

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.     

Vesicle-mediated ER export of proteins and lipids

In eukaryotic cells, the endoplasmic reticulum (ER) is a major site of synthesis of both lipids and proteins, many of which must be transported to other organelles. The COPII coat-comprising Sar1, Sec23/24, Sec13/31-generates transport vesicles that mediate the bulk of protein/lipid export from the ER. The coat exhibits remarkable flexibility in its ability to specifically select and accommodate a large number of cargoes with diverse properties. In this review, we discuss the fundamentals of COPII vesicle production and describe recent advances that further our understanding of just how flexible COPII cargo recruitment and vesicle formation may be. Large or bulky cargo molecules (e.g. collagen rods and lipoprotein particles) exceed the canonical size for COPII vesicles and seem to rely on the additional action of recently identified accessory molecules. Although the bulk of the research has focused on the fate of protein cargo, the mechanisms and regulation of lipid transport are equally critical to cellular survival. From their site of synthesis in the ER, phospholipids, sphingolipids and sterols exit the ER, either accompanying cargo in vesicles or directly across the cytoplasm shielded by lipid-transfer proteins. Finally, we highlight the current challenges to the field in addressing the physiological regulation of COPII vesicle production and the molecular details of how diverse cargoes, both proteins and lipids, are accommodated. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

New Insights into the Structural Mechanisms of the COPII Coat

In eukaryotes, coat protein complex II (COPII) proteins are involved in transporting cargo proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. The COPII proteins, Sar1, Sec23/24, and Sec13/31 polymerize into a coat that gathers cargo proteins into a coated vesicle. Structures have been recently solved of individual COPII proteins, COPII proteins in complex with cargo, and higher‐order COPII coat assemblies. In this review, we will summarize the latest developments in COPII structure and discuss how these structures shed light on the functional mechanisms of the COPII coat.     

Carbon tetrachloride suppresses ER-Golgi transport by inhibiting COPII vesicle formation on the ER membrane in the RLC-16 hepatocyte cell line

Carbon tetrachloride (CCl₄) causes hepatotoxicity in mammals, with its hepatocytic metabolism producing radicals that attack the intracellular membrane system and destabilize intracellular vesicle transport. Inhibition of intracellular transport causes lipid droplet retention and abnormal protein distribution. The intracellular transport of synthesized lipids and proteins from the endoplasmic reticulum (ER) to the Golgi apparatus is performed by coat complex II (COPII) vesicle transport, but how CCl₄ inhibits COPII vesicle transport has not been elucidated. COPII vesicle formation on the ER membrane is initiated by the recruitment of Sar1 protein from the cytoplasm to the ER membrane, followed by that of the COPII coat constituent proteins (Sec23, Sec24, Sec13, and Sec31). In this study, we evaluated the effect of CCl₄ on COPII vesicle formation using the RLC-16 rat hepatocyte cell line. Our results showed that CCl₄ suppressed ER-Golgi transport in RLC-16 cells. Using a reconstituted system of rat liver tissue-derived cytoplasm and RLC-16 cell-derived ER membranes, CCl₄ treatment inhibited the recruitment of Sar1 and Sec13 from the cytosolic fraction to ER membranes. CCl₄-induced changes in the ER membrane accordingly inhibited the accumulation of COPII vesicle-coated constituent proteins on the ER membrane, as well as the formation of COPII vesicles, which suppressed lipid and protein transport between the ER and Golgi apparatus. Our data suggest that CCl₄ inhibits ER-Golgi intracellular transport by inhibiting COPII vesicle formation on the ER membrane in hepatocytes.     

Distinct stages in the recognition,sorting, and packaging of proTGFα into COPII-coated transport vesicles

In addition to its role in forming vesicles from the endoplasmic reticulum (ER), the coat protein complex II (COPII) is also responsible for selecting specific cargo proteins to be packaged into COPII transport vesicles. Comparison of COPII vesicle formation in mammalian systems and in yeast suggested that the former uses more elaborate mechanisms for cargo recognition, presumably to cope with a significantly expanded repertoire of cargo that transits the secretory pathway. Using proTGFα, the transmembrane precursor of transforming growth factor α (TGFα), as a model cargo protein, we demonstrate in cell-free assays that at least one auxiliary cytosolic factor is specifically required for the efficient packaging of proTGFα into COPII vesicles. Using a knockout HeLa cell line generated by CRISPR/Cas9, we provide functional evidence showing that a transmembrane protein, Cornichon-1 (CNIH), acts as a cargo receptor of proTGFα. We show that both CNIH and the auxiliary cytosolic factor(s) are required for efficient recruitment of proTGFα to the COPII coat in vitro. Moreover, we provide evidence that the recruitment of cargo protein by the COPII coat precedes and may be distinct from subsequent cargo packaging into COPII vesicles.     

A rapid and quantitative coat protein complex II vesicle formation assay using luciferase reporters

The majority of protein export from the endoplasmic reticulum (ER) is facilitated by coat protein complex II (COPII). The COPII proteins deform the ER membrane into vesicles at the ER exit sites. During the vesicle formation step, the COPII proteins load cargo molecules into the vesicles. Formation of COPII vesicles has been reconstituted in vitro in yeast and in mammalian systems. These in vitro COPII vesicle formation assays involve incubation of microsomal membranes and purified COPII proteins with nucleotides. COPII vesicles are separated from the microsomes by differential centrifugation. Interestingly, the efficiency of the COPII vesicle formation with purified recombinant mammalian COPII proteins is lower than that with cytosol, suggesting that an additional cytosolic factor(s) is involved in this process. Indeed, other studies have also implicated additional factors. To facilitate biochemical identification of such regulators, a rapid and quantitative COPII vesicle formation assay is necessary because the current assay is lengthy. To expedite this assay, we generated luciferase reporter constructs. The reporter proteins were packaged into COPII vesicles and yielded quantifiable luminescent signals, resulting in a rapid and quantitative COPII vesicle formation assay.     

Emergent properties of proteostasis-COPII coupled systems in human health and disease

In eukaryotic membrane trafficking, emergent protein folding pathways dictated by the proteostasis network (the 'PN') in each cell type are linked to the coat protein complex II (COPII) system that initiates transport through the exocytic pathway. These coupled pathways direct the transit of protein cargo from the endoplasmic reticulum (ER) to diverse subcellular and extracellular destinations. Understanding how the COPII system selectively manages the trafficking of distinct folded states of nascent cargo (comprising one-third of the proteins synthesized by the eukaryotic genome) in close cooperation with the PN remains a formidable challenge to the field. Whereas the PN may contain a thousand component, the minimal COPII coat components that drive all vesicle budding from the ER include Sar1 (a GTPase), Sec12 (a guanine nucleotide exchange factor), Sec23-Sec24 complexes (protein cargo selectors) and the Sec13-Sec31 complex (that functions as a protein cargo collector and as a polymeric lattice generator to promote vesicle budding). A wealth of data suggests a hierarchical role of the PN and COPII components in coupling protein folding with recruitment and assembly of vesicle coats on the ER. In this minireview, we focus on insights recently gained from the study of inherited human disease states of the COPII machinery. We explore the relevance of the COPII system to human biology in the context of its inherent link with the remarkably flexible folding capacity of the PN in each cell type and in response to the environment. The pharmacological manipulation of this coupled system has important therapeutic implications for restoration of function in human disease.     

COPII vesicles derived from mammalian endoplasmic reticulum microsomes recruit COPI

ER to Golgi transport requires the function of two distinct vesicle coat complexes, termed COPI (coatomer) and COPII, whose assembly is regulated by the small GTPases ADP-ribosylation factor 1 (ARF1) and Sar1, respectively. To address their individual roles in transport, we have developed a new assay using mammalian microsomes that reconstitute the formation of ER-derived vesicular carriers. Vesicles released from the ER were found to contain the cargo molecule vesicular stomatitis virus glycoprotein (VSV-G) and p58, an endogenous protein that continuously recycles between the ER and pre-Golgi intermediates. Cargo was efficiently sorted from resident ER proteins during vesicle formation in vitro. Export of VSV-G and p58 were found to be exclusively mediated by COPII. Subsequent movement of ER-derived carriers to the Golgi stack was blocked by a trans-dominant ARF1 mutant restricted to the GDP-bound state, which is known to prevent COPI recruitment. To establish the initial site of coatomer assembly after export from the ER, we immunoisolated the vesicular intermediates and tested their ability to recruit COPI. Vesicles bound coatomer in a physiological fashion requiring an ARF1-guanine nucleotide exchange activity. These results suggest that coat exchange is an early event preceding the targeting of ER-derived vesicles to pre-Golgi intermediates.     

CideB Protein Is Required for the Biogenesis of Very Low Density Lipoprotein (VLDL) Transport Vesicle

Nascent very low density lipoprotein (VLDL) exits the endoplasmic reticulum (ER) in a specialized ER-derived vesicle, the VLDL transport vesicle (VTV). Similar to protein transport vesicles (PTVs), VTVs require coat complex II (COPII) proteins for their biogenesis from the ER membranes. Because the size of the VTV is large, we hypothesized that protein(s) in addition to COPII components might be required for VTV biogenesis. Our proteomic analysis, supported by Western blotting data, shows that a 26-kDa protein, CideB, is present in the VTV but not in other ER-derived vesicles such as PTV and pre-chylomicron transport vesicle. Western blotting and immunoelectron microscopy analyses suggest that CideB is concentrated in the VTV. Our co-immunoprecipitation data revealed that CideB specifically interacts with VLDL structural protein, apolipoprotein B100 (apoB100), but not with albumin, a PTV cargo protein. Confocal microscopic data indicate that CideB co-localizes with apoB100 in the ER. Additionally, CideB interacts with COPII components, Sar1 and Sec24. To investigate the role of CideB in VTV biogenesis, we performed an in vitro ER budding assay. We show that the blocking of CideB inhibits VTV budding, indicating a direct requirement of CideB in VTV formation. To confirm our findings, we knocked down CideB in primary hepatocytes and isolated ER and cytosol to examine whether they support VTV budding. Our data suggest that CideB knockdown significantly reduces VTV biogenesis. These findings suggest that CideB forms an intricate COPII coat and regulates the VTV biogenesis.     

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.     

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.     

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.     

Coordination of COPII vesicle trafficking by Sec23

Coat protein complex II (COPII) is a multi-subunit protein complex responsible for the formation of membrane vesicles at the endoplasmic reticulum. The assembly of this complex on the endoplasmic reticulum membrane needs to be tightly regulated to ensure efficient and specific incorporation of cargo proteins into nascent vesicles. Recent studies of a genetic disease affecting COPII function, and a structural analysis of COPII subunit interactions emphasize the central role of the Sec23 subunit in COPII coat assembly. Similarly, the demonstration that Sec23 interacts physically and functionally with proteins involved in both vesicle tethering and the transport along microtubules indicates that the Sec23 subunit is crucially important in linking COPII vesicle formation to anterograde transport events.     

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.     

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.     

Erv26p directs pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles

Secretory proteins are exported from the endoplasmic reticulum (ER) in transport vesicles formed by the coat protein complex II (COPII). We detected Erv26p as an integral membrane protein that was efficiently packaged into COPII vesicles and cycled between the ER and Golgi compartments. The erv26Delta mutant displayed a selective secretory defect in which the pro-form of vacuolar alkaline phosphatase (pro-ALP) accumulated in the ER, whereas other secretory proteins were transported at wild-type rates. In vitro budding experiments demonstrated that Erv26p was directly required for packaging of pro-ALP into COPII vesicles. Moreover, Erv26p was detected in a specific complex with pro-ALP when immunoprecipitated from detergent-solublized ER membranes. Based on these observations, we propose that Erv26p serves as a transmembrane adaptor to link specific secretory cargo to the COPII coat. Because ALP is a type II integral membrane protein in yeast, these findings imply that an additional class of secretory cargo relies on adaptor proteins for efficient export from the ER.     

Plasmodium falciparum Sec24 marks transitional ER that exports a model cargo via a diacidic motif

Exit from the endoplasmic reticulum (ER) often occurs at distinct sites of vesicle formation known as transitional ER (tER) that are enriched for COPII vesicle coat proteins. We have characterized the organization of ER export in the malaria parasite, Plasmodium falciparum , by examining the localization of two components of the COPII machinery, PfSec12 and PfSec24a. PfSec12 was found throughout the ER, whereas the COPII cargo adaptor, PfSec24a, was concentrated at distinct foci that likely correspond to tER sites. These foci were closely apposed to cis -Golgi sites marked by PfGRASP–GFP, and upon treatment with brefeldin A they accumulated a model cargo protein via a process dependent on the presence of an intact diacidic export motif. Our data suggest that the cargo-binding function of PfSec24a is conserved and that accumulation of cargo in discrete tER sites depends upon positive sorting signals. Furthermore, the number and position of tER sites with respect to the cis -Golgi suggests a co-ordinated biogenesis of these domains.