Sit4p/PP6 regulates ER-to-Golgi traffic by controlling the dephosphorylation of COPII coat subunits

Traffic from the endoplasmic reticulum (ER) to the Golgi complex is initiated when the activated form of the GTPase Sar1p recruits the Sec23p-Sec24p complex to ER membranes. The Sec23p-Sec24p complex, which forms the inner shell of the COPII coat, sorts cargo into ER-derived vesicles. The coat inner shell recruits the Sec13p-Sec31p complex, leading to coat polymerization and vesicle budding. Recent studies revealed that the Sec23p subunit sequentially interacts with three different binding partners to direct a COPII vesicle to the Golgi. One of these binding partners is the serine/threonine kinase Hrr25p. Hrr25p phosphorylates the COPII coat, driving the membrane-bound pool into the cytosol. The phosphorylated coat cannot rebind to the ER to initiate a new round of vesicle budding unless it is dephosphorylated. Here we screen all known protein phosphatases in yeast to identify one whose loss of function alters the cellular distribution of COPII coat subunits. This screen identifies the PP2A-like phosphatase Sit4p as a regulator of COPII coat dephosphorylation. Hyperphosphorylated coat subunits accumulate in the sit4Δ mutant in vivo. In vitro, Sit4p dephosphorylates COPII coat subunits. Consistent with a role in coat recycling, Sit4p and its mammalian orthologue, PP6, regulate traffic from the ER to the Golgi complex.     

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.     

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.     

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.     

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.     

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.     

The lysophospholipid acyltransferase antagonist CI-976 inhibits a late step in COPII vesicle budding

The mechanism of coat protein (COP)II vesicle fission from the endoplasmic reticulum (ER) remains unclear. Lysophospholipid acyltransferases (LPATs) catalyze the conversion of various lysophospholipids to phospholipids, a process that can promote spontaneous changes in membrane curvature. Here, we show that 2,2-methyl- N -(2,4,6,-trimethoxyphenyl)dodecanamide (CI-976), a potent LPAT inhibitor, reversibly inhibited export from the ER in vivo and the formation of COPII vesicles in vitro . Moreover, CI-976 caused the rapid and reversible accumulation of cargo at ER exit sites (ERESs) containing the COPII coat components Sec23/24 and Sec13/31 and a marked enhancement of Sar1p-mediated tubule formation from ERESs, suggesting that CI-976 inhibits the fission of assembled COPII budding elements. These results identify a small molecule inhibitor of a very late step in COPII vesicle formation, consistent with fission inhibition, and demonstrate that this step is likely facilitated by an ER-associated LPAT.     

Crystal structure of Sar1-GDP at 1.7 A resolution and the role of the NH2 terminus in ER export.

The Sar1 GTPase is an essential component of COPII vesicle coats involved in export of cargo from the ER. We report the 1.7-A structure of Sar1 and find that consistent with the sequence divergence of Sar1 from Arf family GTPases, Sar1 is structurally distinct. In particular, we show that the Sar1 NH2 terminus contains two regions: an NH2-terminal extension containing an evolutionary conserved hydrophobic motif that facilitates membrane recruitment and activation by the mammalian Sec12 guanine nucleotide exchange factor, and an alpha1' amphipathic helix that contributes to interaction with the Sec23/24 complex that is responsible for cargo selection during ER export. We propose that the hydrophobic Sar1 NH2-terminal activation/recruitment motif, in conjunction with the alpha1' helix, mediates the initial steps in COPII coat assembly for export from the ER.     

p125A exists as part of the mammalian Sec13/Sec31 COPII subcomplex to facilitate ER-Golgi transport

Coat protein II (COPII)–mediated export from the endoplasmic reticulum (ER) involves sequential recruitment of COPII complex components, including the Sar1 GTPase, the Sec23/Sec24 subcomplex, and the Sec13/Sec31 subcomplex. p125A was originally identified as a Sec23A-interacting protein. Here we demonstrate that p125A also interacts with the C-terminal region of Sec31A. The Sec31A-interacting domain of p125A is between residues 260–600, and is therefore a distinct domain from that required for interaction with Sec23A. Gel filtration and immunodepletion studies suggest that the majority of cytosolic p125A exists as a ternary complex with the Sec13/Sec31A subcomplex, suggesting that Sec 13, Sec31A, and p125A exist in the cytosol primarily as preassembled Sec13/Sec31A/p125A heterohexamers. Golgi morphology and protein export from the ER were affected in p125A-silenced cells. Our results suggest that p125A is part of the Sec13/Sec31A subcomplex and facilitates ER export in mammalian cells.     

Dissection of COPII subunit-cargo assembly and disassembly kinetics during Sar1p-GTP hydrolysis

COPII coat proteins are required for direct capture of cargo and SNARE proteins into transport vesicles from the endoplasmic reticulum (ER). Cargo and SNARE capture occurs during the formation of a 'prebudding complex' comprising a cargo, Sar1p-GTP and the COPII subunits Sec23/24p. The assembly and disassembly cycle of the prebudding complex on ER membranes is coupled to the Sar1p GTPase cycle. Using FRET to monitor a single round of Sec23/24p binding and dissociation from SNAREs in reconstituted liposomes, we show that Sec23/24p dissociates from v-SNARE and complexed t-SNARE with kinetics slower than Sar1p-GTP hydrolysis. Once Sec23/24p becomes associated with v-SNARE or complexed t-SNARE, the complex remains assembled during multiple rounds of Sar1p-GTP hydrolysis mediated by the GDP-GTP exchange factor Sec12p. These data suggest a model for the maintenance of kinetically stable prebudding complexes during the Sar1p GTPase cycle that regulates cargo sorting into transport 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.     

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.     

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.     

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.     

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.     

Mammalian Sec16/p250 plays a role in membrane traffic from the endoplasmic reticulum

Coat protein complex II (COPII)-coated vesicles/carriers, which mediate export of proteins from the endoplasmic reticulum (ER), are formed at special ER subdomains in mammals, termed ER exit sites or transitional ER. The COPII coat consists of a small GTPase, Sar1, and two protein complexes, Sec23-Sec24 and Sec13-Sec31. Sec23-Sec24 and Sec13-Sec31 appear to constitute the inner and the outermost layers of the COPII coat, respectively. We previously isolated two mammalian proteins (p125 and p250) that bind to Sec23. p125 was found to be a mammalian-specific, phospholipase A(1)-like protein that participates in the organization of ER exit sites. Here we show that p250 is encoded by the KIAA0310 clone and has sequence similarity to yeast Sec16 protein. Although KIAA0310p was found to be localized at ER exit sites, subcellular fractionation revealed its predominant presence in the cytosol. Cytosolic KIAA0310p was recruited to ER membranes in a manner dependent on Sar1. Depletion of KIAA0310p mildly caused disorganization of ER exit sites and delayed protein transport from the ER, suggesting its implication in membrane traffic out of the ER. Overexpression of KIAA0310p affected ER exit sites in a manner different from that of p125. Binding experiments suggested that KIAA0310p interacts with both the inner and the outermost layer coat complexes, whereas p125 binds principally to the inner layer complex. Our results suggest that KIAA0310p, a mammalian homologue of yeast Sec16, builds up ER exit sites in cooperation with p125 and plays a role in membrane traffic from the ER.     

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.     

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.     

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.     

Vesicle budding: a coat for the COPs

Although vesicular transport in eukaryotic cells involves a number of different carriers, one common feature is that most of them use small GTPases to direct coat assembly at the donor membrane. COPII coated vesicles bud from the endoplasmic reticulum to selectively export secretory cargo en route to the Golgi complex. Vesicle formation involves the stepwise recruitment of the small GTPase Sar1 and two large heterodimeric complexes Sec23-Sec24 and Sec13-Sec31 to the membrane. A new structural study now provides breathtaking molecular insights into the formation of the Sec23-Sec24-Sar1 pre-budding complex and into COPII coat assembly.