Suppression of coatomer mutants by a new protein family with COPI and COPII binding motifs in Saccharomyces cerevisiae

Protein trafficking is achieved by a bidirectional vesicle flow between the various compartments of the eukaryotic cell. COPII coated vesicles mediate anterograde protein transport from the endoplasmic reticulum to the Golgi apparatus, whereas retrograde Golgi-to-endoplasmic reticulum vesicles use the COPI coat. Inactivation of COPI vesicle formation in conditional sec21 (gamma-COP) mutants rapidly blocks transport of certain proteins along the early secretory pathway. We have identified the integral membrane protein Mst27p as a strong suppressor of sec21-3 and ret1-1 mutants. A C-terminal KKXX motif of Mst27p that allows direct binding to the COPI complex is crucial for its suppression ability. Mst27p and its homolog Yar033w (Mst28p) are part of the same complex. Both proteins contain cytoplasmic exposed C termini that have the ability to interact directly with COPI and COPII coat complexes. Site-specific mutations of the COPI binding domain abolished suppression of the sec21 mutants. Our results indicate that overexpression of MST27 provides an increased number of coat binding sites on membranes of the early secretory pathway and thereby promotes vesicle formation. As a consequence, the amount of cargo that can bind COPI might be important for the regulation of the vesicle flow in the early secretory pathway.     

Phosphorylation of the vesicle-tethering protein p115 by a casein kinase II-like enzyme is required for Golgi reassembly from isolated mitotic fragments

Coat protein I (COPI) transport vesicles can be tethered to Golgi membranes by a complex of fibrous, coiled-coil proteins comprising p115, Giantin and GM130. p115 has been postulated to act as a bridge, linking Giantin on the vesicle to GM130 on the Golgi membrane. Here we show that the acidic COOH terminus of p115 mediates binding to both GM130 and Giantin as well as linking the two together. Phosphorylation of serine 941 within this acidic domain enhances the binding as well as the link between them. Phosphorylation is mediated by casein kinase II (CKII) or a CKII-like kinase. Surprisingly, the highly conserved NH(2)-terminal head domain of p115 is not required for the NSF (N-ethylmaleimide-sensitive fusion protein)-catalyzed reassembly of cisternae from mitotic Golgi fragments in a cell-free system. However, the ability of p115 to link GM130 to Giantin and the phosphorylation of p115 at serine 941 are required for NSF-catalyzed cisternal regrowth. p115 phosphorylation may be required for the transition from COPI vesicle tethering to COPI vesicle docking, an event that involves the formation of trans-SNARE [corrected] (trans-soluble NSF attachment protein [SNAP] receptor) complexes.     

A Role for Giantin in Docking COPI Vesicles to Golgi Membranes

We have previously shown that p115, a vesicle docking protein, binds to two proteins (p130 and p400) in detergent extracts of Golgi membranes. p130 was identified as GM130, a Golgi matrix protein, and was shown to act as a membrane receptor for p115. p400 has now been identified as giantin, a Golgi membrane protein with most of its mass projecting into the cytoplasm. Giantin is found on COPI vesicles and pretreatment with antibodies inhibits both the binding of p115 and the docking of these vesicles with Golgi membranes. In contrast, GM130 is depleted from COPI vesicles and inhibition of the GM130 on Golgi membranes, using either antibodies or an NH₂-terminal GM130 peptide, inhibits p115 binding and vesicle docking. Together these results suggest that COPI vesicles are docked by giantin on the COPI vesicles and GM130 on Golgi membranes with p115 providing a bridge.     

Sequential tethering of Golgins and catalysis of SNAREpin assembly by the vesicle-tethering protein p115

p115 tethers coat protein (COP)I vesicles to Golgi membranes. The acidic COOH-terminal domain of p115 links the Golgins, Giantin on COPI vesicles, to GM130 on Golgi membranes. We now show that a SNARE motif-related domain within p115 stimulates the specific assembly of endogenous Golgi SNAREpins containing the t-SNARE, syntaxin 5. p115 catalyzes the construction of a cognate GOS-28-syntaxin-5 (v-/t-SNARE) complex by first linking the SNAREs to promote their direct interaction. These events are essential for NSF-catalyzed reassembly of postmitotic Golgi vesicles and tubules into mature cisternae. Staging experiments reveal that the linking of Golgins precedes SNAREpin assembly. Thus, p115 coordinates sequential tethering and docking of COPI vesicles by first using long tethers (Golgins) and then short tethers (SNAREs).     

Evidence that Golgi structure depends on a p115 activity that is independent of the vesicle tether components giantin and GM130

Inhibition of the putative coatomer protein I (COPI) vesicle tethering complex, giantin-p115-GM130, may contribute to mitotic Golgi breakdown. However, neither this, nor the role of the giantin-p115-GM130 complex in the maintenance of Golgi structure has been demonstrated in vivo. Therefore, we generated antibodies directed against the mapped binding sites in each protein of the complex and injected these into mammalian tissue culture cells. Surprisingly, the injected anti-p115 and antigiantin antibodies caused proteasome-mediated degradation of the corresponding antigens. Reduction of p115 levels below detection led to COPI-dependent Golgi fragmentation and apparent accumulation of Golgi-derived vesicles. In contrast, neither reduction of giantin below detectable levels, nor inhibition of p115 binding to GM130, had any detectable effect on Golgi structure or Golgi reassembly after cell division or brefeldin A washout. These observations indicate that inhibition of p115 can induce a mitotic-like Golgi disassembly, but its essential role in Golgi structure is independent of its Golgi-localized binding partners giantin and GM130.     

ARFGAP1 promotes the formation of COPI vesicles,suggesting function as a component of the coat

The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTPgammaS not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat.     

COPI proteins: A model for their role in vesicle budding

Summary COPI-coated vesicles are involved in intracellular trafficking between the endoplasmic reticulum and the Golgi complex. In the current model for COPI assembly the small GTP-binding protein ADP-ribosylation factor 1 is recruited from the cytoplasm to the Golgi membrane followed by binding of the hetero-oligomeric protein complex coatomer. However, the mechanism of subsequent vesicle budding is discussed controversially. This review summarizes the available experimental data on the COPI coat and discusses a model of how the major coat protein, coatomer, might act in vesicle budding.     

COPI budding within the Golgi stack

The Golgi serves as a hub for intracellular membrane traffic in the eukaryotic cell. Transport within the early secretory pathway, that is within the Golgi and from the Golgi to the endoplasmic reticulum, is mediated by COPI-coated vesicles. The COPI coat shares structural features with the clathrin coat, but differs in the mechanisms of cargo sorting and vesicle formation. The small GTPase Arf1 initiates coating on activation and recruits en bloc the stable heptameric protein complex coatomer that resembles the inner and the outer shells of clathrin-coated vesicles. Different binding sites exist in coatomer for membrane machinery and for the sorting of various classes of cargo proteins. During the budding of a COPI vesicle, lipids are sorted to give a liquid-disordered phase composition. For the release of a COPI-coated vesicle, coatomer and Arf cooperate to mediate membrane separation.     

Structural and Functional Analysis of the Globular Head Domain of p115 Provides Insight into Membrane Tethering

Molecular tethers have a central role in the organization of the complex membrane architecture of eukaryotic cells. p115 is a ubiquitous, essential tether involved in vesicle transport and the structural organization of the exocytic pathway. We describe two crystal structures of the N-terminal domain of p115 at 2.0 Å resolution. The p115 structures show a novel α-solenoid architecture constructed of 12 armadillo-like, tether-repeat, α-helical tripod motifs. We find that the H1 TR binds the Rab1 GTPase involved in endoplasmic reticulum to Golgi transport. Mutation of the H1 motif results in the dominant negative inhibition of endoplasmic reticulum to Golgi trafficking. We propose that the H1 helical tripod contributes to the assembly of Rab-dependent complexes responsible for the tether and SNARE-dependent fusion of membranes.     

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.     

Physical aspects of COPI vesicle formation

Coat proteins orchestrate membrane budding and molecular sorting during the formation of transport intermediates. Coat protein complex I (COPI) vesicles shuttle between the Golgi apparatus and the endoplasmic reticulum and between Golgi stacks. The formation of a COPI vesicle proceeds in four steps: coat self-assembly, membrane deformation into a bud, fission of the coated vesicle and final disassembly of the coat to ensure recycling of coat components. Although some issues are still actively debated, the molecular mechanisms of COPI vesicle formation are now fairly well understood. In this review, we argue that physical parameters are critical regulators of COPI vesicle formation. We focus on recent real-time in vitro assays highlighting the role of membrane tension, membrane composition, membrane curvature and lipid packing in membrane remodelling and fission by the COPI coat.     

Distinct roles for the cytoplasmic tail sequences of Emp24p and Erv25p in transport between the endoplasmic reticulum and Golgi complex

Heteromeric complexes of p24 proteins cycle between early compartments of the secretory pathway and are required for efficient protein sorting. Here we investigated the role of cytoplasmically exposed tail sequences on two p24 proteins, Emp24p and Erv25p, in directing their movement and subcellular location in yeast. Studies on a series of deletion and chimeric Emp24p-Erv25p proteins indicated that the tail sequences impart distinct functional properties that were partially redundant but not entirely interchangeable. Export of an Emp24p-Erv25p complex from the endoplasmic reticulum (ER) did not depend on two other associated p24 proteins, Erp1 and Erp2p. To examine interactions between the Emp24p and Erv25p tail sequences with the COPI and COPII coat proteins, binding experiments with immobilized tail peptides and coat proteins were performed. The Emp24p and Erv25p tail sequences bound the Sec13p/Sec31p subunit of the COPII coat (K(d) approximately 100 microm), and binding depended on a pair of aromatic residues found in both tail sequences. COPI subunits also bound to these Emp24p and Erv25p peptides; however, the Erv25p tail sequence, which contains a dilysine motif, bound COPI more efficiently. These results suggest that both the Emp24p and Erv25p cytoplasmic sequences contain a di-aromatic motif that binds subunits of the COPII coat and promotes export from the ER. The Erv25p tail sequence binds COPI and is responsible for returning this complex to the ER.     

Physical aspects of COPI vesicle formation

Abstract

Coat proteins orchestrate membrane budding and molecular sorting during the formation of transport intermediates. Coat protein complex I (COPI) vesicles shuttle between the Golgi apparatus and the endoplasmic reticulum and between Golgi stacks. The formation of a COPI vesicle proceeds in four steps: coat self-assembly, membrane deformation into a bud, fission of the coated vesicle and final disassembly of the coat to ensure recycling of coat components. Although some issues are still actively debated, the molecular mechanisms of COPI vesicle formation are now fairly well understood. In this review, we argue that physical parameters are critical regulators of COPI vesicle formation. We focus on recent real-time in vitro assays highlighting the role of membrane tension, membrane composition, membrane curvature and lipid packing in membrane remodelling and fission by the COPI coat.     

Structure of the cytoplasmic domain of p23 in solution: implications for the formation of COPI vesicles

Coatomer, the coat protein complex of coat protein (COPI) vesicles, is involved in the budding of these vesicles. Its interaction with the cytoplasmic domains of some p24-family members, type I transmembrane proteins of the Golgi, has been shown to induce a conformational change of coatomer that initiates polymerization of the complex. From stoichiometrical data it is likely that interaction of coatomer with the small tail domains involves an oligomeric form of the p24 proteins. Here we present the structure of peptide analogs of the cytoplasmic domain of p23, a member of the p24 family, as determined by two-dimensional nuclear magnetic resonance spectroscopy in the presence of 2,2,2-trifluoroethanol. An improved strategy for structure calculation revealed that the tail domain peptides form alpha-helices and adopt a tetrameric state. Based on these results we propose an initial model for the binding of coatomer by p23 and the induced conformational change of coatomer that results in its polymerization, curvature of the Golgi membrane to form a bud, and finally a COPI-coated vesicle.     

A conformational change in the alpha-subunit of coatomer induced by ligand binding to gamma-COP revealed by single-pair FRET

Formation of transport vesicles involves polymerization of cytoplasmic coat proteins (COP). In COPI vesicle biogenesis, the heptameric complex coatomer is recruited to donor membranes by the interaction of multiple coatomer subunits with the budding machinery. Specific binding to the trunk domain of γ-COP by the Golgi membrane protein p23 induces a conformational change that causes polymerization of the complex. Using single-pair fluorescence resonance energy transfer, we find that this conformational change takes place in individual coatomer complexes, independent of each other, and that the conformational rearrangement induced in γ-COP is transmitted within the complex to its α-subunit. We suggest that capture of membrane protein machinery triggers cage formation in the COPI system.     

A cryptic Rab1-binding site in the p115 tethering protein

Small GTPases and coiled-coil proteins of the golgin family help to tether COPI vesicles to Golgi membranes. At the cis-side of the Golgi, the Rab1 GTPase binds directly to each of three coiled-coil proteins: p115, GM130, and as now shown, Giantin. Rab1 binds to a coiled-coil region within the tail domain of p115 and this binding is inhibited by the C-terminal, acidic domain of p115. Furthermore, GM130 and Giantin bind to the acidic domain of p115 and stimulate p115 binding to Rab1, suggesting that p115 binding to Rab1 is regulated. Regulation of this interaction by proteins such as GM130 and Giantin may control the membrane recruitment of p115 by Rab1.     

A role for calcium in stabilizing transport vesicle coats.

Calcium has been implicated in regulating vesicle fusion reactions, but its potential role in regulating other aspects of protein transport, such as vesicle assembly, is largely unexplored. We find that treating cells with the membrane-permeable calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), leads to a dramatic redistribution of the vesicle coat protein, coatomer, in the cell. We have used the cell-free reconstitution of coat-protomer I (COPI) vesicle assembly to characterize the mechanisms of this redistribution. We find that the recovery of COPI-coated Golgi vesicles is inhibited by the addition of BAPTA to the cell-free vesicle budding assay. When coatomer-coated membranes are incubated in the presence of calcium chelators, the membranes "uncoat," indicating that calcium is necessary for maintaining the integrity of the coat. This uncoating is reversed by the addition of calcium. Interestingly, BAPTA, a calcium chelator with fast binding kinetics, is more potent at uncoating the coatomer-coated membrane than EGTA, suggesting that a calcium transient or a calcium gradient is important for stabilizing COPI vesicle coat. The primary target for the effects of calcium on coatomer recruitment is a step that occurs after ADP-ribosylation factor binding to the membrane. We suggest that a calcium gradient may serve to regulate the timing of vesicle uncoating.     

ArfGAP1 Activity and COPI Vesicle Biogenesis

Golgi-derived coat protein I (COPI) vesicles mediate transport in the early secretory pathway. The minimal machinery required for COPI vesicle formation from Golgi membranes in vitro consists of (i) the hetero-heptameric protein complex coatomer, (ii) the small guanosine triphosphatase ADP-ribosylation factor 1 (Arf1) and (iii) transmembrane proteins that function as coat receptors, such as p24 proteins. Various and opposing reports exist on a role of ArfGAP1 in COPI vesicle biogenesis. In this study, we show that, in contrast to data in the literature, ArfGAP1 is not required for COPI vesicle formation. To investigate roles of ArfGAP1 in vesicle formation, we titrated the enzyme into a defined reconstitution assay to form and purify COPI vesicles. We find that catalytic amounts of Arf1GAP1 significantly reduce the yield of purified COPI vesicles and that Arf1 rather than ArfGAP1 constitutes a stoichiometric component of the COPI coat. Combining the controversial reports with the results presented in this study, we suggest a novel role for ArfGAP1 in membrane trafficking.     

ARFGAP1 plays a central role in coupling COPI cargo sorting with vesicle formation

Examining how key components of coat protein I (COPI) transport participate in cargo sorting, we find that, instead of ADP ribosylation factor 1 (ARF1), its GTPase-activating protein (GAP) plays a direct role in promoting the binding of cargo proteins by coatomer (the core COPI complex). Activated ARF1 binds selectively to SNARE cargo proteins, with this binding likely to represent at least a mechanism by which activated ARF1 is stabilized on Golgi membrane to propagate its effector functions. We also find that the GAP catalytic activity plays a critical role in the formation of COPI vesicles from Golgi membrane, in contrast to the prevailing view that this activity antagonizes vesicle formation. Together, these findings indicate that GAP plays a central role in coupling cargo sorting and vesicle formation, with implications for simplifying models to describe how these two processes are coupled during COPI transport.     

ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat

In eukaryotic cells, secretion is achieved by vesicular transport. Fusion of such vesicles with the correct target compartment relies on SNARE proteins on both vesicle (v-SNARE) and the target membranes (t-SNARE). At present it is not clear how v-SNAREs are incorporated into transport vesicles. Here, we show that binding of ADP-ribosylation factor (ARF)-GTPase-activating protein (GAP) to ER-Golgi v-SNAREs is an essential step for recruitment of Arf1p and coatomer, proteins that together form the COPI coat. ARF-GAP acts catalytically to recruit COPI components. Inclusion of v-SNAREs into COPI vesicles could be mediated by direct interaction with the coat. The mechanisms by which v-SNAREs interact with COPI and COPII coat proteins seem to be different and may play a key role in determining specificity in vesicle budding.