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.     

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.     

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.     

Secretory cargo regulates the turnover of COPII subunits at single ER exit sites

The COPII coat complex mediates the formation of transport carriers at specialized sites of the endoplasmic reticulum (ERES). It consists of the Sar1p GTPase and the Sec23/24p and the Sec13/31p subcomplexes . Both stimulate the GTPase activity of Sar1p , which itself triggers coat disassembly. This built-in GAP activity makes the COPII complex in principle unstable and raises the question of how sufficient stability required for cargo capture and carrier formation is achieved. To address this, we analyzed COPII turnover at single ERES in living cells. The half times for Sar1p, Sec23p, and Sec24p turnover are 1.1, 3.7, and 3.9 s, respectively. Decreasing the amount of transport-competent cargo in the endoplasmic reticulum accelerates turnover of the Sec23/24p and slows down that of Sar1p. A mathematical model of COPII membrane turnover that reproduces the experimental in vivo FRAP kinetics and is consistent with existing in vitro data predicts that Sec23/24p remains membrane associated even after GTP hydrolysis by Sar1p for a duration that is strongly increased by the presence of cargo. We conclude that secretory cargo retains the COPII complex on membranes, after Sar1p release has occurred, and prevents premature disassembly of COPII during cargo sorting and transport carrier formation.     

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.     

Cargo Selection by the COPII Budding Machinery during Export from the ER

Cargo is selectively exported from the ER in COPII vesicles. To analyze the role of COPII in selective transport from the ER, we have purified components of the mammalian COPII complex from rat liver cytosol and then analyzed their role in cargo selection and ER export. The purified mammalian Sec23–24 complex is composed of an 85-kD (Sec23) protein and a 120-kD (Sec24) protein. Although the Sec23–24 complex or the monomeric Sec23 subunit were found to be the minimal cytosolic components recruited to membranes after the activation of Sar1, the addition of the mammalian Sec13–31 complex is required to complete budding. To define possible protein interactions between cargo and coat components, we recruited either glutathione-S-transferase (GST)–tagged Sar1 or GST– Sec23 to ER microsomes. Subsequently, we solubilized and reisolated the tagged subunits using glutathione-Sepharose beads to probe for interactions with cargo. We find that activated Sar1 in combination with either Sec23 or the Sec23–24 complex is necessary and sufficient to recover with high efficiency the type 1 transmembrane cargo protein vesicular stomatitis virus glycoprotein in a detergent-soluble prebudding protein complex that excludes ER resident proteins. Supplementing these minimal cargo recruitment conditions with the mammalian Sec13–31 complex leads to export of the selected cargo into COPII vesicles. The ability of cargo to interact with a partial COPII coat demonstrates that these proteins initiate cargo sorting on the ER membrane before budding and establishes the role of GTPase-dependent coat recruitment in cargo selection.     

SNARE selectivity of the COPII coat

The COPII coat buds transport vesicles from the endoplasmic reticulum that incorporate cargo and SNARE molecules. Here, we show that recognition of the ER-Golgi SNAREs Bet1, Sed5, and Sec22 occurs through three binding sites on the Sec23/24 subcomplex of yeast COPII. The A site binds to the YNNSNPF motif of Sed5. The B site binds to Lxx-L/M-E sequences present in both the Bet1 and Sed5 molecules, as well as to the DxE cargo-sorting signal. A third, spatially distinct site binds to Sec22. COPII selects the free v-SNARE form of Bet1 because the LxxLE sequence is sequestered in the four-helix bundle of the v-/t-SNARE complex. COPII favors Sed5 within the Sed5/Bos1/Sec22 t-SNARE complex because t-SNARE assembly removes autoinhibitory contacts to expose the YNNSNPF motif. The COPII coat seems to be a specific conductor of the fusogenic forms of these SNAREs, suggesting how vesicle fusion specificity may be programmed during budding.     

Lst1p and Sec24p cooperate in sorting of the plasma membrane ATPase into COPII vesicles in Saccharomyces cerevisiae

Formation of ER-derived protein transport vesicles requires three cytosolic components, a small GTPase, Sar1p, and two heterodimeric complexes, Sec23/24p and Sec13/31p, which comprise the COPII coat. We investigated the role of Lst1p, a Sec24p homologue, in cargo recruitment into COPII vesicles in Saccharomyces cerevisiae. A tagged version of Lst1p was purified and eluted as a heterodimer complexed with Sec23p comparable to the Sec23/24p heterodimer. We found that cytosol from an lst1-null strain supported the packaging of alpha-factor precursor into COPII vesicles but was deficient in the packaging of Pma1p, the essential plasma membrane ATPase. Supplementation of mutant cytosol with purified Sec23/Lst1p restored Pma1p packaging into the vesicles. When purified COPII components were used in the vesicle budding reaction, Pma1p packaging was optimal with a mixture of Sec23/24p and Sec23/Lst1p; Sec23/Lst1p did not replace Sec23/24p. Furthermore, Pma1p coimmunoprecipitated with Lst1p and Sec24p from vesicles. Vesicles formed with a mixture of Sec23/Lst1p and Sec23/24p were similar morphologically and in their buoyant density, but larger than normal COPII vesicles (87-nm vs. 75-nm diameter). Immunoelectronmicroscopic and biochemical studies revealed both Sec23/Lst1p and Sec23/24p on the membranes of the same vesicles. These results suggest that Lst1p and Sec24p cooperate in the packaging of Pma1p and support the view that biosynthetic precursors of plasma membrane proteins must be sorted into ER-derived transport vesicles. Sec24p homologues may comprise a more complex coat whose combinatorial subunit composition serves to expand the range of cargo to be packaged into COPII vesicles. By changing the geometry of COPII coat polymerization, Lst1p may allow the transport of bulky cargo molecules, polymers, or particles.     

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.     

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.     

A structural view of the COPII vesicle coat

The COPII vesicle coat coordinates the budding of transport vesicles from the endoplasmic reticulum in the initial step of the secretory pathway. The coat orchestrates a sequence of events including self-assembly on the membrane, cargo and SNARE molecule selection, and deformation of the membrane into a bud to drive vesicle fission. Recent molecular-level studies have helped to explain how the three components of yeast COPII - Sar1 GTPase, the Sec23/24 subcomplex and the Sec13/31 subcomplex - combine to organize this complex process.     

Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat

Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly.     

Sfb2p, a yeast protein related to Sec24p, can function as a constituent of COPII coats required for vesicle budding from the endoplasmic reticulum

The COPII coat is required for vesicle budding from the endoplasmic reticulum (ER), and consists of two heterodimeric subcomplexes, Sec23p/Sec24p, Sec13p/Sec31p, and a small GTPase, Sar1p. We characterized a yeast mutant, anu1 (abnormal nuclear morphology) exhibiting proliferated ER as well as abnormal nuclear morphology at the restrictive temperature. Based on the finding that ANU1 is identical to SEC24, we confirmed a temperature-sensitive protein transport from the ER to the Golgi in anu1-1/sec24-20 cells. Overexpression of SFB2, a SEC24 homologue with 56% identity, partially suppressed not only the mutant phenotype of sec24-20 cells but also rescued the SEC24-disrupted cells. Moreover, the yeast two-hybrid assay revealed that Sfb2p, similarly to Sec24p, interacted with Sec23p. In SEC24-disrupted cells rescued by overexpression of SFB2, some cargo proteins were still retained in the ER, while most of the protein transport was restored. Together, these findings strongly suggest that Sfb2p functions as the component of COPII coats in place of Sec24p, and raise the possibility that each member of the SEC24 family of proteins participates directly and/or indirectly in cargo-recognition events with its own cargo specificity at forming ER-derived vesicles.     

Endoplasmic reticulum export of glycosyltransferases depends on interaction of a cytoplasmic dibasic motif with Sar1

Membrane proteins exit the endoplasmic reticulum (ER) in COPII-transport vesicles. ER export is a selective process in which transport signals present in the cytoplasmic tail (CT) of cargo membrane proteins must be recognized by coatomer proteins for incorporation in COPII vesicles. Two classes of ER export signals have been described for type I membrane proteins, the diacidic and the dihydrophobic motifs. Both motifs participate in the Sar1-dependent binding of Sec23p-Sec24p complex to the CTs during early steps of cargo selection. However, information concerning the amino acids in the CTs that interact with Sar1 is lacking. Herein, we describe a third class of ER export motif, [RK](X)[RK], at the CT of Golgi resident glycosyltransferases that is required for these type II membrane proteins to exit the ER. The dibasic motif is located proximal to the transmembrane border, and experiments of cross-linking in microsomal membranes and of binding to immobilized peptides showed that it directly interacts with the COPII component Sar1. Sar1GTP-bound to immobilized peptides binds Sec23p. Collectively, the present data suggest that interaction of the dibasic motif with Sar1 participates in early steps of selection of Golgi resident glycosyltransferases for transport in COPII vesicles.     

The Sar1 GTPase coordinates biosynthetic cargo selection with endoplasmic reticulum export site assembly

Cargo selection and export from the endoplasmic reticulum is mediated by the COPII coat machinery that includes the small GTPase Sar1 and the Sec23/24 and Sec13/31 complexes. We have analyzed the sequential events regulated by purified Sar1 and COPII coat complexes during synchronized export of cargo from the ER in vitro. We find that activation of Sar1 alone, in the absence of other cytosolic components, leads to the formation of ER-derived tubular domains that resemble ER transitional elements that initiate cargo selection. These Sar1-generated tubular domains were shown to be transient, functional intermediates in ER to Golgi transport in vitro. By following cargo export in live cells, we show that ER export in vivo is also characterized by the formation of dynamic tubular structures. Our results demonstrate an unanticipated and novel role for Sar1 in linking cargo selection with ER morphogenesis through the generation of transitional tubular ER export sites.     

The transport signal on Sec22 for packaging into COPII-coated vesicles is a conformational epitope

The mechanism of cargo concentration into ER-derived vesicles involves interactions between the COPII vesicular coat complex and cargo transport signals--peptide sequences of 10-15 residues. The SNARE protein Sec22 contains a signal that binds the COPII subcomplex Sec23/24 and specifies its endoplasmic reticulum (ER) exit as an unassembled SNARE. The 200 kDa crystal structure of Sec22 bound to Sec23/24 reveals that the transport signal is a folded epitope rather than a conventional short peptide sequence. The NIE segment of the SNARE motif folds against the N-terminal longin domain, and this closed form of Sec22 binds at the Sec23/24 interface. Thus, COPII recognizes unassembled Sec22 via a folded epitope, whereas Sec22 assembly into SNARE complexes would mask the NIE segment. The concept of a conformational epitope as a transport signal suggests packaging mechanisms in which a coat is sensitive to the folded state of a cargo protein or the assembled state of a multiprotein complex.     

GTP/GDP exchange by Sec12p enables COPII vesicle bud formation on synthetic liposomes

The generation of COPII vesicles from synthetic liposome membranes requires the minimum coat components Sar1p, Sec23/24p, Sec13/31p, and a nonhydrolyzable GTP analog such as GMP-PNP. However, in the presence of GTP and the full complement of coat subunits, nucleotide hydrolysis by Sar1p renders the coat insufficiently stable to sustain vesicle budding. In order to recapitulate a more authentic, GTP-dependent budding event, we introduced the Sar1p nucleotide exchange catalyst, Sec12p, and evaluated the dynamics of coat assembly and disassembly by light scattering and tryptophan fluorescence measurements. The catalytic, cytoplasmic domain of Sec12p (Sec12DeltaCp) activated Sar1p with a turnover 10-fold higher than the GAP activity of Sec23p stimulated by the full coat. COPII assembly was stabilized on liposomes incubated with Sec12DeltaCp and GTP. Numerous COPII budding profiles were visualized on membranes, whereas a parallel reaction conducted in the absence of Sec12DeltaCp produced no such profiles. We suggest that Sec12p participates actively in the growth of COPII vesicles by charging new Sar1p-GTP molecules that insert at the boundary between a bud and the surrounding endoplasmic reticulum membrane.     

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.     

Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro

In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.     

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.