The COPI system: Molecular mechanisms and function

Transcription

1 FEBS Letters 583 (2009) journal homepage: Review The COPI system: Molecular mechanisms and function R. Beck a,1, M. Ravet b,1, F.T. Wieland c, *, D. Cassel b, * a Yale University School of Medicine, Department of Cell Biology, 333 Cedar Street, SHM C-232, New Haven, CT 06520, USA b Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel c Biochemistry Center, Heidelberg University, Im Neuenheimer Feld 328, D Heidelberg, Germany article info abstract Article history: Received 16 June 2009 Revised 7 July 2009 Accepted 13 July 2009 Available online 22 July 2009 Edited by Lukas Huber Keywords: COPI Golgi Vesicular transport Arf Coatomer Transport of membranes and proteins in eukaryotic cells is mediated by vesicular carriers. Here we review the biogenesis and functions of COPI vesicles, carriers that operate in the early secretory pathway. We focus on mechanisms mediating coat recruitment, uptake of cargo, vesicle budding and fission, and finally dissociation of the coat. In this context, recent findings on the interplay between machinery and auxiliary proteins in COPI vesicle formation and function will be discussed. Specifically, we will weigh the pros and cons of recent data on roles of the small GTP binding protein Arf1, of Arf1GAPs, and lipids during COPI carrier formation. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 1. Introduction * Corresponding authors. address: felix.wieland@bzh.uni-heidelberg.de (F.T. Wieland). 1 These authors contributed equally. Vesicular carriers were first proposed in mammalian cells by Palade and colleagues [1]. In subsequent years, three types of coated carriers have been characterized in detail with regard to their structural components and functions: clathrin-coated vesicles that mediate transport in the late secretory pathway and the endocytic pathway (reviewed in [2]), COPI-coated vesicles that function in the early secretory pathway (reviewed in [3,4]), and COPII vesicles that export proteins from the endoplasmic reticulum (ER) (reviewed in [5]). Some structural and mechanistic similarities that exist between the three systems have been reviewed [6,7]. Here we focus on recent advances in the understanding of the components and mechanisms underlying the formation of COPI vesicles. These carriers were first isolated in a cell-free system, following incubation of Golgi-enriched membranes with cytosol in the presence of a hydrolysis-resistant derivative of GTP, GTPcS [8,9]. Analysis of the coated vesicles initially led to the identification of four large protein subunits (a-, b-, c- and d-cop) [10,11]. A complex containing these and additional subunits was subsequently purified from the cytosol and was termed coatomer (from coat protomer) [12]. The molecular identification of additional coatomer subunits, b 0 -COP [13,14], e-cop [15] and f-cop [16], complemented the set of constituents of a heptameric coat complex. Analysis of the interactions between subunits has led to a model of the organization of the coatomer complex [17,18]. This organization is comparable to that of the heterotetrameric adaptins in complex with clathrin, where the b, c, d and f subunits referred to as the F subcomplex correspond to adaptin subunits. The remaining coatomer subunits a, b 0 and e comprise the B subcomplex [19]. Subunits of the F subcomplex display weak but significant sequence similarities to corresponding adaptin subunits. Functionally, however, the coatomer architecture seems to be more complex, as the binding of different types of membrane proteins involves subunits from both the F- and the B-subcomplexes (see cargo sorting below) [7,20 22]. The process of COPI vesicle biogenesis is controlled by Arf1, a small GTPase of the Ras superfamily, originally identified as a cofactor that is required for the action of cholera toxin in mammalian cells [23]. A guanine nucleotide exchange factor (GEF) containing a Sec7 domain [24] induces the exchange of bound GDP to GTP in Arf1. GBF1, a large Sec7 domain-containing GEF is the major exchange factor involved in COPI vesicle biogenesis [25,26]. Following nucleotide exchange, Arf1 undergoes a conformational change, leading to the exposure of a myristoylated N-terminal amphipathic helix that provides stable membrane anchorage [27 29]. Arf exchange only takes place on membranes as activation in the cytosol would lead to the energetically unfavorable exposure of a hydrophobic patch (the myristoylated amphiphatic helix) to an aqueous surrounding. Following nucleotide exchange, /$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi: /j.febslet

2 2702 R. Beck et al. / FEBS Letters 583 (2009) Arf1-GTP recruits coatomer to the membrane [30 32]; real-time in vitro assays have revealed that Arf1 activation is rate-limiting, and is followed by an almost instantaneous binding of coatomer [33]. Coatomer recruitment to the Golgi also involves members of a family of kd type-i transmembrane proteins (the p24 family) that are sorted into COPI vesicles where they become a major constituent [34,35]. Their short cytoplasmic tails bind directly to coatomer [35,36], thereby increasing the efficiency of vesicle formation [37]. After the formation of a COPI-coated vesicle, the coat must be shed to allow for vesicle fusion with the target membrane. The uncoating reaction depends on GTP hydrolysis by Arf1 [38], catalyzed by Arf1 GTPase-activating proteins (ArfGAPs). In addition to recruiting and releasing coatomer, as described above, an Arf- GEF/GAP mediated cycle of GTP exchange and hydrolysis was proposed to play a role in regulating the uptake of cargo into COPI vesicles [39 43]. 2. Function of COPI in the early secretory system The best characterized function of the COPI system is the retrograde transport of luminal and membrane proteins in the ER-Golgi segment of the secretory pathway [3,44,45]. Soluble cargos such as escaped resident ER glycoproteins are captured by adaptor transmembrane proteins that communicate with the COPI coat at their cytosolic aspect. A major, if not exclusive, adaptor function is provided by KDEL receptors that recognize a four-letter code presented at the carboxy terminus of many ER-resident proteins [46,47]. Membrane protein cargo, e.g., a subunit of the ER-localized oligosaccharide transferase complex, can be retrieved by direct interaction with coatomer [45]. COPI-dependent transport may also play a role in the correct steady-state distribution of proteins within the Golgi stack [48,49], reviewed in [50]. Retrograde transport of Golgi enzymes is a key element in the cisternal maturation mechanism, whereby cisternae mature progressively by receiving processing enzymes from a later cisterna [47,51 53]. According to this mechanism, cargo progression through cisternae would not require anterograde intra-golgi transport, but rather represent bulk flow, a mechanism that had been proposed earlier [54]. While there is also evidence for anterograde transport between Golgi cisternae by COPI vesicles [41,55,56], this mechanism cannot account for the transport of large cargo such as procollagen. A third mechanism for cargo progression through the Golgi involving partitioning and exchange between cisternae has recently been proposed [57]. Retrograde traffic through the COPI system also contributes to an ER quality control mechanism that ensures the correct assembly of certain multisubunit membrane proteins [58]. Such protein assemblies include subunits with a diarginine signal whose interaction with coatomer causes ER retrieval/retention. Upon subunit assembly, the arginine signals become masked, thus allowing for ER exit [59,60]. Evidence for COPI-mediated retrieval of membrane proteins is often based on analysis of their processing by Golgi glycosyltransferases (e.g., [61 63]). Whenever Golgi-type glycosylation cannot be detected, it is difficult to distinguish ER retrieval from ER retention. In one report, a carboxy dilysine signal (KKAA) considered as a canonical signal for ER retrieval through the COPI system was found to cause ER retention rather than retrieval of reporter protein; whether the COPI coat was involved in this process remains unclear [64]. The COPI system also plays a role in the transport of biosynthetic cargo to the Golgi apparatus. Whereas the initial phase of ER exit is mediated by COPII-coated carriers, subsequent transport is carried out by tubulovesicular intermediates (the ERGIC) that are associated with the COPI coat [65 69]. Transition from COPII- to COPI-coated carriers is mediated by the activation of Arf1 by GBF1. Pharmacological agents and genetic manipulations that inhibit Arf1 activation were shown to inhibit ER to Golgi transport [70 75]. It should be noted, however, that inhibition of Arf1 activation may also affect other Arf effectors thought to function in the early secretory system, such as phospholipid-modulating enzymes and tethers [76]. More direct evidence for a role of coatomer in ER to Golgi transport was provided by the demonstration that VSVG transport to the Golgi is inhibited by antibodies directed against the b-cop subunit of coatomer [70,77].InSaccharomyces cerevisiae, a block of anterograde transport is observed in mutants of COPI subunits [78 80]. Two recent studies examined the consequences of reducing b- COP expression in mammalian cells. b-cop depletion in HeLa cells led to the merging of ERGIC, Golgi, TGN and recycling endosomes but not ER, and prevented transport beyond the merged compartment [81]. In another study, b-cop depletion was found to decrease surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) [82]; interestingly, CFTR was found to co-immunoprecipitate with coatomer, indicating that COPI trafficking may play an active role in CFTR transport. However, the interpretation of coatomer disruption studies is complicated by the resulting perturbations of the structure of organelles of the early secretory system, and by possible indirect effects that may result from the inhibition of COPI-dependent recycling of trafficking components. Transport between the ER and the Golgi apparatus involves cytoskeleton-based membrane translocation [83 85]. A possible connection between the coatomer coat and the cytoskeleton is provided by the finding that coatomer interacts with CDC42, which in turn regulates the recruitment of the microtubule motor dynein [86,87]. Further studies should address the precise role of interactions between coat components and cytoskeletal elements in ER to Golgi transport. 3. Cargo sorting 3.1. Sorting of soluble proteins The lumen of the ER contains a variety of soluble proteins that perform essential functions related to protein folding and assembly. Munro and Pelham were the first to have noticed that these ER residents are distinguished from secreted proteins by the presence of a C-terminal KDEL sequence (HDEL in yeast) that prevents their secretion [88]. The finding that KDEL-proteins can be modified by post-er enzymes indicated that these proteins are retrieved from Golgi cisternae [89,90]. Genetic screens in yeast identified ERD2 as the receptor mediating the retrieval of KDEL-proteins (KDELR) [46], and orthologues have since been characterized in vertebrates, including three human isoforms [91 93]. Vectorial transport of KDEL-proteins was suggested to exploit ph differences between the Golgi and the ER [94]. Proteins with a KDEL motif interact with a KDELR in the cis-golgi, and the complex is then transported to the ER. There, the higher ph results in its dissociation, allowing the empty receptor to be recycled and participate in another round of transport. Following the discovery of the KDEL signal, many variants of this sequence have been described and were shown to cause ER localization. In humans, a distinction between the various signals is achieved by differential specificities of the three KDELRs. Although some overlap exists, it appears that each KDEL receptor mediates the retrieval of a subgroup of soluble ER proteins [93].

3 R. Beck et al. / FEBS Letters 583 (2009) Evidence that retrograde transport through the KDELR is COPImediated is based on observations from both yeast and mammalian systems. The KDELR was shown to be present in purified COPI vesicles [95], and to colocalize with coatomer in buds and vesicles of permeabilized cells that have been treated with GTPcS [96]. In contrast to cargo proteins, uptake of the KDELR into COPI vesicles does not depend on the hydrolysis of GTP [41], underlining its role as machinery rather than cargo. Retrieval of HDEL proteins in a yeast-based in vitro system was shown to require the presence of coatomer and Arf1 [97], and ER targeting of endocytosed cholera toxin (which contains the KDEL signal) in Vero cells was inhibited by antibodies directed against b-cop or p23 [98]. The KDELR is a seven-pass membrane protein, a known feature of G protein-coupled receptors. Indeed, some characteristics of the KDELR are reminiscent of signaling receptors. Upon ligand binding the KDELR undergoes oligomerization, followed by interaction with components of the COPI machinery, coatomer and ArfGAP1 [99,100]. These interactions depend on PKA-dependent phosphorylation of a serine residue at the C-terminal cytosolic tail of the KDELR, and phosphorylation is also required for receptor-mediated trafficking [101]. Recent findings suggest that the KDELR may be also involved in a Golgi-based signaling network that senses the load of traffic outbound from the ER. Binding of cargo to the KDELR at the Golgi results in a complex pattern of protein phosphorylation, mediated by Src family kinases required for intra-golgi transport, and may also regulate Golgi to ER retrograde transport [102,103] Sorting of membrane proteins Sorting of membrane proteins into COPI vesicles often depends on cytosolic signals that mediate their binding to coat components. Best characterized is the carboxy-terminal dilysine motif K(X)KXX that is found in some ER-resident proteins [104], and in cycling proteins that function at the ER-Golgi interface such as ERGIC53/ p58, and in p25, a member of the p24 protein family [35, ]. Studies using reporter proteins have indicated that the efficiency of the KKXX signal depends on both the type of the X residues and on the distance of the signal from the membrane [58,108]. The potency of the K(X)KXX motif, in conjunction with the efficiency of ER exit, determines whether at steady state a protein is mainly ER-localized, or whether it distributes between the ER, ERGIC and Golgi compartments. The dilysine motif interacts with coatomer [45], and this interaction is required for the retrieval of tagged proteins to the ER [44]. The Wbp1 dilysine motif was found to interact with a subcomplex of coatomer consisting of the a-, b 0 - and e-cop subunits [45]. Subsequent two-hybrid assays as well as functional analysis showed that binding is mediated by the N-terminal WD40 propeller domains of the a- and b 0 -COP subunits [17]. These experiments also revealed preferential binding of the KKXX and KXKXX signatures to the a- and b 0 -COP propellers, respectively [109]. Another type of coatomer binding signal is presented by members of the p24 membrane protein family (discussed in the next section). The short cytoplasmic tails of these proteins have a signature with the consensus FFXX(KR)(KR)X n, where n P 2. With the exception of p25, these sequences do not conform to the canonical KKXX motif, and the distinction of the p24 signature is further highlighted by the fact that coatomer binding relies more on the diphenylalanine motif than on the two basic residues [19,35,43]. Photo-cross linking studies demonstrated that the p24 motif interacts with the c-cop subunit of coatomer [110], and more recently this interaction was shown to require p24 dimerization and to involve distinct sites in the trunk and appendage domains of c-cop [111]. Arginine (Arg)-based motifs are a class of ER localization signals conforming to the consensus [UWR] R X R (where U/W is an aromatic or bulky hydrophobic residue) [60]. First identified in the invariant chain of the major histocompatibility complex class II, this signal is often found on subunits of multimeric complexes such as cell-surface receptors and ion channels [112,113]. It couples complex assembly to cell-surface transport by maintaining unassembled subunits in the ER; upon correct assembly, the signal is inactivated, allowing for ER export [114,115]. Several mechanisms have been proposed for signal inactivation: steric masking by intersubunit interaction following proper assembly, hindrance of the signal by multivalent binding of proteins, binding of PDZ-domain proteins, and protein phosphorylation [59, ]. Unlike KXKXX signals, the positioning of Arg-based signals is not confined to the extreme C-terminus, and the signal may appear in multiple copies along the cytosolic aspects of membrane proteins. The ability of Arg signals to interact with coatomer suggests that ER localization is due to COPI-mediated retrieval [59,122,124]. Recent studies in yeast identified two highly conserved stretches in the b- and d-cop subunits of coatomer that are required collectively for the binding of Arg-based motifs [60]. Golgi glycosyltransferases are subjected to COPI-mediated retrograde transport within the Golgi; however, these are type II membrane proteins and their short amino terminal tail lack known coatomer-binding motifs. Recent studies demonstrated that the cytosolic vps75p is required for Golgi localization of several glycosyltransferases. Upon oligomerization, vps75p associates with the Golgi where it interacts with a 5-residue motif on the cytosolic tails of the glycosyltransferases, as well as with coatomer, suggesting that vps75p functions as a coat adaptor [48,49]. 4. Molecular mechanisms of COPI-vesicle formation 4.1. Recruitment of Arf1 to the Golgi membrane The key molecule that organizes specific recruitment of the COPI vesicle coat protein coatomer to donor membranes in a GTP-dependent manner is the small GTP binding protein Arf1, which represents a stoichiometric component of the vesicular coat [31,32,35, ]. Both the inactive (GDP-bound) and active (GTP-bound) forms of Arf1 interact with the membrane. Arf1- GDP binds to the cytoplasmic tails of dimeric p23 [128], suggesting a role of the p24 proteins in facilitating the initial recruitment of the small GTPase to the membrane [100,128,129]. More recently, membrane, an early Golgi SNARE, was also proposed to function as an Arf1-GDP receptor [130]. Concomitant with nucleotide exchange, the p23-arf1 complex dissociates [128]. Arf1-GTP then associates with the membrane through its myristoylated amphipathic helix [27 29]. Arf1 activation is stimulated by the Sec7 family of guanine nucleotide exchange factors (GEFs) [131]. GBF1, the only known GEF localized to the cis-golgi, plays an important role in mediating protein trafficking between the ER and the cis-golgi [132,133]. GBF1 is recruited to Golgi membranes and to ER-Golgi intermediate carriers through its interaction with Rab1b and with p115 [ ]. Brefeldin A (BFA), a lactone isolated from fungi, acts as a noncompetitive inhibitor of Arf1 activation by stabilizing a non-productive complex between ARF1-GDP and Sec7 domains of several ArfGEFs [137], including GBF1 [26]. Treatment of mammalian cells with BFA causes rapid but reversible dispersal of the Golgi apparatus in a process whereby the Golgi tubulates and the tubules fuse with the ER [ ]. While the precise mechanism by which BFA causes Golgi dispersal remains controversial, the dramatic effect of BFA on the Golgi suggests a crucial role of coatomer and/or other Arf1 effectors in the maintenance of the identity of this orga-

4 2704 R. Beck et al. / FEBS Letters 583 (2009) nelle. Apart from BFA, several Sec7 domain-specific inhibitors have been introduced, including tyrphostin AG1478 [141], the Coxsackie virus protein 3A [142], and the chemical compound LM11 [143] Coat recruitment Once activated by GTP-exchange, Arf1 can recruit a variety of coats (reviewed in [131]. A direct interaction of Arf1 with coat proteins was first demonstrated in the COPI system, where the switch I region in Arf1 was shown to interact with both the b-cop and c- COP subunits of coatomer [144,145]. In yeast two hybrid studies, b- COP and e-cop were reported to bind Arf1 [17]. Subsequent studies narrowed down the binding to the b- and c- trunk domains of coatomer and revealed that upon this interaction, a novel binding site for Arf1-GTP on d-cop is opened [146]. Acting together through distinct binding sites, oligomeric forms of p24 proteins [111] and the activated Arf1 serve as binding platforms for coatomer [144,145]. The interaction of coatomer with the cytoplasmic tails of p24 protein oligomers induces a conformational change of the coat complex, resulting in coat polymerization and probably membrane deformation [147,148] Arf1 inactivation The uncoating reaction, which is a prerequisite for vesicle fusion, depends on GTP hydrolysis by Arf1 [38], and is catalyzed by Arf-directed GTPase-activating proteins, the ArfGAPs. Members of this family contain a conserved catalytic domain with a characteristic zinc finger motif followed by an invariant arginine (CXXCX 16 CXXCX 4 R), whereas their non-catalytic parts differ between subgroups of the family [149,150]. Two ArfGAPs, Gcs1 and Glo3, have been implicated in COPI-mediated transport in yeast and were shown to function as an essential pair [151]. In mammals, three Golgi-localized ArfGAPs are known, ArfGAP1 and the closely related ArfGAP2/3 [20,152,153]. The mammalian ortholog of Gcs1, ArfGAP1, was the first ArfGAP to be identified [152,154]. Consistent with studies in yeast, triple knockdowns of ArfGAP1/ 2/3 in mammalian cells are lethal, whereas cells can survive when only ArfGAP1 or both Glo3 orthologs, ArfGAP2/3 are silenced [155,156]. Despite functional redundancies in cells, Gcs1-like and Glo3- like ArfGAPs differ in the features of their non-catalytic domains. A distinct feature in the non-catalytic parts of ArfGAP1/Gcs1 is the presence of lipid packaging sensor motives (ALPS) [157,158], elements that are unstructured in solution but form an amphipathic a-helix once bound to poorly packed lipids such as those presented by highly curved membranes. Due to this binding behavior, ArfGAP1 displays curvature-dependent activity in vitro, a mechanism that may be employed in vivo to ensure high efficiency of vesicle uncoating [33,159]. Alternatively, or in addition, since the rims of the Golgi cisternae are highly curved, the ALPS motives could confer specific targeting of ArfGAP1 to the zones of COPI budding in the Golgi. Indeed, the ALPS motifs of ArfGAP1 are required for Golgi targeting in vivo [160,161]. In addition to curvature, ArfGAP1 activity is increased in the presence of liposomes containing diacylglycerols (DAGs), which are also expected to loosen lipid packing due to their cone shape [162]. As will be discussed later, a correlation was observed between cellular DAG levels and ArfGAP1 Golgi association [163,164], but whether the ALPS motifs are involved has not been reported to date. ArfGAP2 and ArfGAP3 both lack an ALPS motif, and their activity is not modulated by membrane curvature in liposome-based assays [165]. Unlike ArfGAP1, the activity of ArfGAP2/3 strictly depends on coatomer, in the presence of which they can become more efficient catalysts than ArfGAP1 [165,166]. A lysine-rich stretch in the middle of the ArfGAP2/3 molecules was identified as a critical determinant mediating coatomer binding and regulation of GAP activity [166,167]. Coatomer was also found to bind to yeast Glo3 and stimulate its activity [17,168], and Golgi localization of ArfGAP2 and ArfGAP3 depends on coatomer interaction [20,166]. The strict regulation by coatomer of ArfGAP2/3 suggests that these proteins are intimately involved in the COPI mechanism; whether these ArfGAPs act as coat components as proposed for Glo3 in yeast [169] or whether their function is primarily related to the regulation of GTP hydrolysis is not quite clear yet. In contrast, ArfGAP1 may play a more general role, acting as a terminator of Arf1 activity. The elimination of activated Arf1 due to ArfGAP1 activity on highly curved membranes was recently proposed to provide a mechanism for tethering flat membranes to vesicles by the Arf1 effector GMAP210 [170]. Contrary to this view of ArfGAP1 function, studies from Hsu s group have suggested the involvement of ArfGAP1 in multiple stages of COPI vesicle formation, including budding, cargo selection and fission (reviewed in [171]). Based on in vitro budding assays, Yang et al. have concluded that ArfGAP1 is an essential and stoichiometric component of the coat of Golgi-derived COPI vesicles [172]. This role is in contrast to reports defining the minimal machinery for the formation of COPI vesicles from liposomal membranes [37,173], or from Golgi membranes in the absence of cytosol [127,165,174,175], and of a role of ARFGAP1 as an uncoating enzyme [159,176]. Furthermore, ArfGAP2/3, but not ArfGAP1, was found on reconstituted COPI vesicles [156], in agreement with earlier findings that Glo3, but not Gsc1 type ArfGAPs interacts with the coat and are found on COPI vesicles in S. cerevisiae [169]. Itisof note that, as mentioned above, individual ArfGAPs, including Arf- GAP1, can be depleted from cells without compromising cell viability, indicating that none of these GAPs is an essential coat component [155,156] Fission Upon exchange of GDP for GTP, Arf1 not only dissociates from p23 and undergoes a conformational change, it also dimerizes on the membrane [174]. A molar ratio of two Arf1 dimers per coatomer complex is estimated to exist in COPI vesicles [32,174]. A mechanistic link between dimerization of Arf1 and vesicle formation comes from the observation that upon activation, the small GTPase generates positive membrane curvature on protein-free liposomes in the absence of any additional factors [174,177,178]. The finding that a dimerization-defective Arf1 mutant retains a considerable capacity to recruit coatomer, but neither generates membrane curvature nor supports COPI vesicle formation [174], indicates that the dimerization of Arf1 is needed for vesicle biogenesis. The exact role of Arf1-mediated curvature formation as a prerequisite for COPI vesicle biogenesis is not yet fully understood. In the mechanistically similar COPII system, the N-terminal amphipathic helix of Sar1, the small GTPase that initiates COPII coat recruitment, was also shown to deform liposomes and was found to be required for vesicle fission [179]; such a scenario seems possible for Arf1 as well. A different line of investigations of the mechanism of COPI vesicle fission was triggered by early studies demonstrating the requirement for acyl-coa for the fission step of COPI vesicle formation in vitro [180]. It was assumed that the transfer of an acyl chain to target proteins and/or lipids was involved in this process [181]. Later studies suggested that the protein BFA-induced ADP-ribosylation substrate (BARS) played a role in the fission of Golgi tubules, and the recombinant protein was reported to have acyltransferase activity [182]. It was shown, however, that this enzymatic activity resulted from a bacterial contamination co-purifying with BARS [183], and that COPI vesicle fission does not depend on this activity [184]. Nevertheless, in vitro and in vivo experiments have provided

5 R. Beck et al. / FEBS Letters 583 (2009) several lines of evidence implicating BARS as a fission factor for COPI vesicles [184]. Cone-shaped lipids, such as phosphatidic acid (PA), and diacylglycerols have the potential of facilitating fission due to their capacity to induce negative membrane curvature [185]. At the Golgi, PA can be generated from phosphatidylcholine (PC) by the action of phospholipase-d (PLD), or by the phosphorylation of DAG by the action of DAG kinase (DAK). In this context, depletion of PLD 2, but not PLD 1 or DAK, was reported to impair the release of COPI vesicles from Golgi membranes [186]. The ability of BARS to deform liposomes, a characteristic that would be expected from a protein involved in fission, was found to depend on the presence of relatively high concentrations of phosphatidic acid, suggesting a mechanism by which PLD 2 and BARS could cooperate to induce membrane fission [186]. Arf1 was shown to activate PLD in a GTP dependent manner [187], and this activation was reported to result in an increased vesicle production from the trans-golgi network [188,189]. More recently, BARS was also found to act as a PLD activator [190]; however, both Arf1 and BARS are effectors of the PLD 1 isozyme but not PLD 2 [191], reviewed in [192]. Together these observations suggest that the two PLD isozymes might function at distinct Golgi sub-compartments, each one in conjunction with its own set of regulators. On the other hand, a role of PA in fission becomes less likely in light of a report that Golgi membrane phosphatidic acid levels do not increase in fact they decrease during cell-free budding reactions [193]. The other kind of cone-shaped lipid, DAG, is generated at the Golgi by several metabolic routes [194].The pathway by which PA, the product of PLD activity, is further converted to DAG by the action of phosphatidate phosphohydrolases (PAPs) was reported to play a role in the COPI mechanism. This was suggested by the findings that the addition of PAP inhibitors to living cells resulted in a decrease in DAG levels at the Golgi and a concomitant inhibition of COPI vesicle formation, as well as retrograde Golgi to ER traffic [163,164]. Interestingly, treatment of cells with the PAP inhibitor propranolol also caused rapid and reversible dissociation of ArfGAP1 from the Golgi [163,164]. While these two studies disagree about the precise stage during budding at which DAG is required, as well as the role of ArfGAP1, these studies along with the study by Yang et al. [186] point to a pathway generating lipid mediators affecting COPI vesicle generation through the action of PLD and PAP. It should be noted, however, that in defined liposomal systems, efficient formation, and thus fission of COPI coated vesicles is achieved without any of the above components [37,173]. 5. Distinct COPI populations defined by their coat composition Genetic studies provided evidence that in higher eukaryotes there are two versions of either both the c- and f-cop subunits [195,196]. This results in four possible combinations of the heptameric complex. Biochemical analysis revealed the presence of coatomer isoforms c1f1, c2f1 and c1f2 at a molar ratio of about 2:1:1, respectively, while the fourth possible combination, c2f2, was estimated below 5% of the total [197]. The distribution within the Golgi apparatus of coatomer complexes shows a statistically significant segregation, with c1f2 COP-containing coatomer restricted to a pre-cis-golgi compartment, the majority of c1f1 in the early Golgi, and c2-cop-containing isoforms localized to the trans-side of the organelle [198]. This differential localization of coatomer isoforms strongly suggests that individual and distinct functions are exerted by the various coat complexes. Different isoforms of coatomer might have binding preferences for isotypic Arf molecules, and/or may exhibit differential interactions with COPI membrane machinery, cargo, and/or auxiliary proteins. In the future, such differential functions will need to be investigated by testing the effect of knockdown of individual isoforms, and by Fig. 1. Individual steps in the formation of a COPI-vesicle (for details see text).

6 2706 R. Beck et al. / FEBS Letters 583 (2009) the isolation of homogenously coated isotypic COPI vesicles and their proteomics analysis. 6. Concluding remarks Despite progress in the understanding of the COPI machinery and the interactions between its structural and regulatory components, much remains to be learned in terms of the precise role of each regulator in vesicle build-up, fission and uncoating. The discovery of coatomer isoforms opens the door to extended functional studies of COPI-mediated trafficking in the ER-Golgi shuttle. A schematic overview of the key players of COPI vesicle formation and their interplay is presented in Fig. 1. Acknowledgements We would like to thank Frank Adolf for providing the figure. Work in this review was supported by the Israel Science Foundation (Grant #171/08), and the German Research Council, SFB 638, Project A10. We would like to apologize to all colleagues whose work could not be cited due to space limitation. References [1] Palade, G. (1975) Intracellular aspects of the process of protein synthesis. Science 189, [2] Robinson, M.S. (2004) Adaptable adaptors for coated vesicles. Trends Cell Biol. 14, [3] Lee, M.C., Miller, E.A., Goldberg, J., Orci, L. and Schekman, R. (2004) Bidirectional protein transport between the ER and Golgi. Annu. Rev. Cell Dev. Biol. 20, [4] Bethune, J., Wieland, F. and Moelleken, J. (2006) COPI-mediated transport. J. Membr. Biol. 211, [5] Hughes, H. and Stephens, D.J. (2008) Assembly, organization, and function of the COPII coat. Histochem. Cell Biol. 129, [6] McMahon, H.T. and Mills, I.G. (2004) COP and clathrin-coated vesicle budding: different pathways, common approaches. Curr. Opin. Cell Biol. 16, [7] Schledzewski, K., Brinkmann, H. and Mendel, R.R. (1999) Phylogenetic analysis of components of the eukaryotic vesicle transport system reveals a common origin of adaptor protein complexes 1, 2, and 3 and the F subcomplex of the coatomer COPI. J. Mol. Evol. 48, [8] Malhotra, V., Serafini, T., Orci, L., Shepherd, J.C. and Rothman, J.E. (1989) Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack. Cell 58, [9] Melancon, P., Glick, B.S., Malhotra, V., Weidman, P.J., Serafini, T., Gleason, M.L., Orci, L. and Rothman, J.E. (1987) Involvement of GTP-binding G proteins in transport through the Golgi stack. Cell 51, [10] Serafini, T., Stenbeck, G., Brecht, A., Lottspeich, F., Orci, L., Rothman, J.E. and Wieland, F.T. (1991) A coat subunit of Golgi-derived non-clathrin-coated vesicles with homology to the clathrin-coated vesicle coat protein betaadaptin. Nature 349, [11] Duden, R., Griffiths, G., Frank, R., Argos, P. and Kreis, T.E. (1991) Beta-COP, a 110 kd protein associated with non-clathrin-coated vesicles and the Golgi complex, shows homology to beta-adaptin. Cell 64, [12] Waters, M.G., Serafini, T. and Rothman, J.E. (1991) Coatomer : a cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles. Nature 349, [13] Stenbeck, G., Harter, C., Brecht, A., Herrmann, D., Lottspeich, F., Orci, L. and Wieland, F.T. (1993) Beta 0 -COP, a novel subunit of coatomer. EMBO J. 12, [14] Harrison-Lavoie, K.J., Lewis, V.A., Hynes, G.M., Collison, K.S., Nutland, E. and Willison, K.R. (1993) A 102 kda subunit of a Golgi-associated particle has homology to beta subunits of trimeric G proteins. EMBO J. 12, [15] Hara-Kuge, S., Kuge, O., Orci, L., Amherdt, M., Ravazzola, M., Wieland, F.T. and Rothman, J.E. (1994) En bloc incorporation of coatomer subunits during the assembly of COP-coated vesicles. J. Cell Biol. 124, [16] Kuge, O., Hara-Kuge, S., Orci, L., Ravazzola, M., Amherdt, M., Tanigawa, G., Wieland, F.T. and Rothman, J.E. (1993) Zeta-COP, a subunit of coatomer, is required for COP-coated vesicle assembly. J. Cell Biol. 123, [17] Eugster, A., Frigerio, G., Dale, M. and Duden, R. (2000) COP I domains required for coatomer integrity, and novel interactions with ARF and ARF-GAP. Embo J. 19, [18] Faulstich, D. et al. (1996) Architecture of coatomer: molecular characterization of delta-cop and protein interactions within the complex. J. Cell Biol. 135, [19] Fiedler, K., Veit, M., Stamnes, M.A. and Rothman, J.E. (1996) Bimodal interaction of coatomer with the p24 family of putative cargo receptors. Science 273, [20] Watson, P.J., Frigerio, G., Collins, B.M., Duden, R. and Owen, D.J. (2004) Gamma-COP appendage domain structure and function. Traffic 5, [21] Hoffman, G.R., Rahl, P.B., Collins, R.N. and Cerione, R.A. (2003) Conserved structural motifs in intracellular trafficking pathways: structure of the gammacop appendage domain. Mol. Cell 12, [22] Yu, W., Lin, J., Jin, C. and Xia, B. (2009) Solution structure of human zeta-cop: direct evidences for structural similarity between COP I and clathrin-adaptor coats. J. Mol. Biol. 386, [23] Kahn, R.A. and Gilman, A.G. (1984) Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin. J. Biol. Chem. 259, [24] Chardin, P., Paris, S., Antonny, B., Robineau, S., Beraud-Dufour, S., Jackson, C.L. and Chabre, M. (1996) A human exchange factor for ARF contains Sec7- and pleckstrin-homology domains. Nature 384, [25] Zhao, X., Lasell, T.K. and Melancon, P. (2002) Localization of large ADPribosylation factor-guanine nucleotide exchange factors to different Golgi compartments: evidence for distinct functions in protein traffic. Mol. Biol. Cell 13, [26] Niu, T.K., Pfeifer, A.C., Lippincott-Schwartz, J. and Jackson, C.L. (2005) Dynamics of GBF1, a Brefeldin A-sensitive Arf1 exchange factor at the Golgi. Mol. Biol. Cell 16, [27] Antonny, B., Beraud-Dufour, S., Chardin, P. and Chabre, M. (1997) N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange. Biochemistry 36, [28] Franco, M., Chardin, P., Chabre, M. and Paris, S. (1996) Myristoylationfacilitated binding of the G protein ARF1GDP to membrane phospholipids is required for its activation by a soluble nucleotide exchange factor. J. Biol. Chem. 271, [29] Liu, Y., Kahn, R.A. and Prestegard, J.H. (2009) Structure and membrane interaction of myristoylated ARF1. Structure 17, [30] Donaldson, J.G., Cassel, D., Kahn, R.A. and Klausner, R.D. (1992) ADPribosylation factor, a small GTP-binding protein, is required for binding of the coatomer protein beta-cop to Golgi membranes. Proc. Natl. Acad. Sci. USA 89, [31] Palmer, D.J., Helms, J.B., Beckers, C.J., Orci, L. and Rothman, J.E. (1993) Binding of coatomer to Golgi membranes requires ADP-ribosylation factor. J. Biol. Chem. 268, [32] Serafini, T., Orci, L., Amherdt, M., Brunner, M., Kahn, R.A. and Rothman, J.E. (1991) ADP-ribosylation factor is a subunit of the coat of Golgi-derived COPcoated vesicles: a novel role for a GTP-binding protein. Cell 67, [33] Bigay, J., Gounon, P., Robineau, S. and Antonny, B. (2003) Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature. Nature 426, [34] Stamnes, M.A., Craighead, M.W., Hoe, M.H., Lampen, N., Geromanos, S., Tempst, P. and Rothman, J.E. (1995) An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding. Proc. Natl. Acad. Sci. USA 92, [35] Sohn, K. et al. (1996) A major transmembrane protein of Golgi-derived COPIcoated vesicles involved in coatomer binding. J. Cell Biol. 135, [36] Nickel, W., Sohn, K., Bunning, C. and Wieland, F.T. (1997) P23, a major COPIvesicle membrane protein, constitutively cycles through the early secretory pathway. Proc. Natl. Acad. Sci. USA 94, [37] Bremser, M. et al. (1999) Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors. Cell 96, [38] Tanigawa, G., Orci, L., Amherdt, M., Ravazzola, M., Helms, J.B. and Rothman, J.E. (1993) Hydrolysis of bound GTP by ARF protein triggers uncoating of Golgi-derived COP-coated vesicles. J. Cell Biol. 123, [39] Lanoix, J., Ouwendijk, J., Stark, A., Szafer, E., Cassel, D., Dejgaard, K., Weiss, M. and Nilsson, T. (2001) Sorting of Golgi resident proteins into different subpopulations of COPI vesicles: a role for ArfGAP1. J. Cell Biol. 155, [40] Malsam, J., Gommel, D., Wieland, F.T. and Nickel, W. (1999) A role for ADP ribosylation factor in the control of cargo uptake during COPI-coated vesicle biogenesis. FEBS Lett. 462, [41] Nickel, W., Malsam, J., Gorgas, K., Ravazzola, M., Jenne, N., Helms, J.B. and Wieland, F.T. (1998) Uptake by COPI-coated vesicles of both anterograde and retrograde cargo is inhibited by GTPgammaS in vitro. J. Cell Sci. 111 (Pt 20), [42] Pepperkok, R., Whitney, J.A., Gomez, M. and Kreis, T.E. (2000) COPI vesicles accumulating in the presence of a GTP restricted arf1 mutant are depleted of anterograde and retrograde cargo. J. Cell Sci. 113 (Pt 1), [43] Goldberg, J. (2000) Decoding of sorting signals by coatomer through a GTPase switch in the COPI coat complex. Cell 100, [44] Letourneur, F., Gaynor, E.C., Hennecke, S., Demolliere, C., Duden, R., Emr, S.D., Riezman, H. and Cosson, P. (1994) Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum. Cell 79, [45] Cosson, P. and Letourneur, F. (1994) Coatomer interaction with di-lysine endoplasmic reticulum retention motifs. Science 263, [46] Semenza, J.C., Hardwick, K.G., Dean, N. and Pelham, H.R. (1990) ERD2, a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway. Cell 61,

7 R. Beck et al. / FEBS Letters 583 (2009) [47] Lewis, M.J. and Pelham, H.R. (1992) Ligand-induced redistribution of a human KDEL receptor from the Golgi complex to the endoplasmic reticulum. Cell 68, [48] Tu, L., Tai, W.C., Chen, L. and Banfield, D.K. (2008) Signal-mediated dynamic retention of glycosyltransferases in the Golgi. Science 321, [49] Schmitz, K.R., Liu, J., Li, S., Setty, T.G., Wood, C.S., Burd, C.G. and Ferguson, K.M. (2008) Golgi localization of glycosyltransferases requires a Vps74p oligomer. Dev. Cell 14, [50] Rabouille, C. and Klumperman, J. (2005) Opinion: the maturing role of COPI vesicles in intra-golgi transport. Nat. Rev. Mol. Cell Biol. 6, [51] Glick, B.S., Elston, T. and Oster, G. (1997) A cisternal maturation mechanism can explain the asymmetry of the Golgi stack. FEBS Lett. 414, [52] Bonfanti, L. et al. (1998) Procollagen traverses the Golgi stack without leaving the lumen of cisternae: evidence for cisternal maturation. Cell 95, [53] Matsuura-Tokita, K., Takeuchi, M., Ichihara, A., Mikuriya, K. and Nakano, A. (2006) Live imaging of yeast Golgi cisternal maturation. Nature 441, [54] Wieland, F.T., Gleason, M.L., Serafini, T.A. and Rothman, J.E. (1987) The rate of bulk flow from the endoplasmic reticulum to the cell surface. Cell 50, [55] Orci, L. et al. (2000) Anterograde flow of cargo across the golgi stack potentially mediated via bidirectional percolating COPI vesicles. Proc. Natl. Acad. Sci. USA 97, [56] Orci, L., Stamnes, M., Ravazzola, M., Amherdt, M., Perrelet, A., Sollner, T.H. and Rothman, J.E. (1997) Bidirectional transport by distinct populations of COPIcoated vesicles. Cell 90, [57] Patterson, G.H., Hirschberg, K., Polishchuk, R.S., Gerlich, D., Phair, R.D. and Lippincott-Schwartz, J. (2008) Transport through the Golgi apparatus by rapid partitioning within a two-phase membrane system. Cell 133, [58] Zerangue, N., Malan, M.J., Fried, S.R., Dazin, P.F., Jan, Y.N., Jan, L.Y. and Schwappach, B. (2001) Analysis of endoplasmic reticulum trafficking signals by combinatorial screening in mammalian cells. Proc. Natl. Acad. Sci. USA 98, [59] Yuan, H., Michelsen, K. and Schwappach, B. (2003) Dimers probe the assembly status of multimeric membrane proteins. Curr. Biol. 13, [60] Michelsen, K., Yuan, H. and Schwappach, B. (2005) Hide and run. Argininebased endoplasmic-reticulum-sorting motifs in the assembly of heteromultimeric membrane proteins. EMBO Rep. 6, [61] Itin, C., Schindler, R. and Hauri, H.P. (1995) Targeting of protein ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway. J. Cell Biol. 131, [62] Gaynor, E.C., te Heesen, S., Graham, T.R., Aebi, M. and Emr, S.D. (1994) Signalmediated retrieval of a membrane protein from the Golgi to the ER in yeast. J. Cell Biol. 127, [63] Jackson, M.R., Nilsson, T. and Peterson, P.A. (1993) Retrieval of transmembrane proteins to the endoplasmic reticulum. J. Cell Biol. 121, [64] Andersson, H., Kappeler, F. and Hauri, H.P. (1999) Protein targeting to endoplasmic reticulum by dilysine signals involves direct retention in addition to retrieval. J. Biol. Chem. 274, [65] Appenzeller-Herzog, C. and Hauri, H.P. (2006) The ER-Golgi intermediate compartment (ERGIC): in search of its identity and function. J. Cell Sci. 119, [66] Aridor, M., Bannykh, S.I., Rowe, T. and Balch, W.E. (1995) Sequential coupling between COPII and COPI vesicle coats in endoplasmic reticulum to Golgi transport. J. Cell Biol. 131, [67] Stephens, D.J., Lin-Marq, N., Pagano, A., Pepperkok, R. and Paccaud, J.P. (2000) COPI-coated ER-to-Golgi transport complexes segregate from COPII in close proximity to ER exit sites. J. Cell Sci. 113 (Pt 12), [68] Scales, S.J., Pepperkok, R. and Kreis, T.E. (1997) Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COPII and COPI. Cell 90, [69] Shima, D.T., Scales, S.J., Kreis, T.E. and Pepperkok, R. (1999) Segregation of COPI-rich and anterograde-cargo-rich domains in endoplasmic-reticulum-to- Golgi transport complexes. Curr. Biol. 9, [70] Peter, F., Plutner, H., Zhu, H., Kreis, T.E. and Balch, W.E. (1993) Beta-COP is essential for transport of protein from the endoplasmic reticulum to the Golgi in vitro. J. Cell Biol. 122, [71] Feng, Y. et al. (2003) Exo1: a new chemical inhibitor of the exocytic pathway. Proc. Natl. Acad. Sci. USA 100, [72] Szul, T., Grabski, R., Lyons, S., Morohashi, Y., Shestopal, S., Lowe, M. and Sztul, E. (2007) Dissecting the role of the ARF guanine nucleotide exchange factor GBF1 in Golgi biogenesis and protein trafficking. J. Cell Sci. 120, [73] Zhao, X., Claude, A., Chun, J., Shields, D.J., Presley, J.F. and Melancon, P. (2006) GBF1, a cis-golgi and VTCs-localized ARF-GEF, is implicated in ER-to-Golgi protein traffic. J. Cell Sci. 119, [74] Dascher, C. and Balch, W.E. (1994) Dominant inhibitory mutants of ARF1 block endoplasmic reticulum to Golgi transport and trigger disassembly of the Golgi apparatus. J. Biol. Chem. 269, [75] Saraste, J. and Svensson, K. (1991) Distribution of the intermediate elements operating in ER to Golgi transport. J. Cell Sci. 100 (Pt 3), [76] Donaldson, J.G., Honda, A. and Weigert, R. (2005) Multiple activities for Arf1 at the Golgi complex. Biochim. Biophys. Acta 1744, [77] Pepperkok, R., Scheel, J., Horstmann, H., Hauri, H.P., Griffiths, G. and Kreis, T.E. (1993) Beta-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo. Cell 74, [78] Hosobuchi, M., Kreis, T. and Schekman, R. (1992) SEC21 is a gene required for ER to Golgi protein transport that encodes a subunit of a yeast coatomer. Nature 360, [79] Duden, R., Hosobuchi, M., Hamamoto, S., Winey, M., Byers, B. and Schekman, R. (1994) Yeast beta- and beta 0 -coat proteins (COP). Two coatomer subunits essential for endoplasmic reticulum-to-golgi protein traffic. J. Biol. Chem. 269, [80] Sutterlin, C., Doering, T.L., Schimmoller, F., Schroder, S. and Riezman, H. (1997) Specific requirements for the ER to Golgi transport of GPI-anchored proteins in yeast. J. Cell Sci. 110, [81] Styers, M.L., O Connor, A.K., Grabski, R., Cormet-Boyaka, E. and Sztul, E. (2008) Depletion of beta-cop reveals a role for COP-I in compartmentalization of secretory compartments and in biosynthetic transport of caveolin-1. Am. J. Physiol. Cell Physiol. 294, C1485 C1498. [82] Rennolds, J. et al. (2008) Cystic fibrosis transmembrane conductance regulator trafficking is mediated by the COPI coat in epithelial cells. J. Biol. Chem. 283, [83] Hehnly, H. and Stamnes, M. (2007) Regulating cytoskeleton-based vesicle motility. FEBS Lett. 581, [84] Myers, K.R. and Casanova, J.E. (2008) Regulation of actin cytoskeleton dynamics by Arf-family GTPases. Trends Cell Biol. 18, [85] Palmer, K.J., Watson, P. and Stephens, D.J. (2005) The role of microtubules in transport between the endoplasmic reticulum and Golgi apparatus in mammalian cells. Biochem. Soc. Symp. 1, 13. [86] Wu, W.J., Erickson, J.W., Lin, R. and Cerione, R.A. (2000) The gamma-subunit of the coatomer complex binds Cdc42 to mediate transformation. Nature 405, [87] Chen, J.L., Fucini, R.V., Lacomis, L., Erdjument-Bromage, H., Tempst, P. and Stamnes, M. (2005) Coatomer-bound Cdc42 regulates dynein recruitment to COPI vesicles. J. Cell Biol. 169, [88] Munro, S. and Pelham, H.R. (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48, [89] Pelham, H.R. (1988) Evidence that luminal ER proteins are sorted from secreted proteins in a post-er compartment. Embo J. 7, [90] Dean, N. and Pelham, H.R. (1990) Recycling of proteins from the Golgi compartment to the ER in yeast. J. Cell Biol. 111, [91] Lewis, M.J. and Pelham, H.R. (1990) A human homologue of the yeast HDEL receptor. Nature 348, [92] Lewis, M.J. and Pelham, H.R. (1992) Sequence of a second human KDEL receptor. J. Mol. Biol. 226, [93] Raykhel, I., Alanen, H., Salo, K., Jurvansuu, J., Nguyen, V.D., Latva-Ranta, M. and Ruddock, L. (2007) A molecular specificity code for the three mammalian KDEL receptors. J. Cell Biol. 179, [94] Wilson, D.W., Lewis, M.J. and Pelham, H.R. (1993) PH-dependent binding of KDEL to its receptor in vitro. J. Biol. Chem. 268, [95] Sonnichsen, B., Watson, R., Clausen, H., Misteli, T. and Warren, G. (1996) Sorting by COP I-coated vesicles under interphase and mitotic conditions. J. Cell Biol. 134, [96] Griffiths, G. et al. (1994) Localization of the Lys, Asp, Glu, Leu tetrapeptide receptor to the Golgi complex and the intermediate compartment in mammalian cells. J. Cell Biol. 127, [97] Spang, A. and Schekman, R. (1998) Reconstitution of retrograde transport from the Golgi to the ER in vitro. J. Cell Biol. 143, [98] Majoul, I., Sohn, K., Wieland, F.T., Pepperkok, R., Pizza, M., Hillemann, J. and Soling, H.D. (1998) KDEL receptor (Erd2p)-mediated retrograde transport of the cholera toxin A subunit from the Golgi involves COPI, p23, and the COOH terminus of Erd2p. J. Cell Biol. 143, [99] Aoe, T., Cukierman, E., Lee, A., Cassel, D., Peters, P.J. and Hsu, V.W. (1997) The KDEL receptor, ERD2, regulates intracellular traffic by recruiting a GTPaseactivating protein for ARF1. Embo J. 16, [100] Majoul, I., Straub, M., Hell, S.W., Duden, R. and Soling, H.D. (2001) KDEL-cargo regulates interactions between proteins involved in COPI vesicle traffic: measurements in living cells using FRET. Dev. Cell 1, [101] Cabrera, M., Muniz, M., Hidalgo, J., Vega, L., Martin, M.E. and Velasco, A. (2003) The retrieval function of the KDEL receptor requires PKA phosphorylation of its C-terminus. Mol. Biol. Cell 14, [102] Bard, F., Mazelin, L., Pechoux-Longin, C., Malhotra, V. and Jurdic, P. (2003) Src regulates Golgi structure and KDEL receptor-dependent retrograde transport to the endoplasmic reticulum. J. Biol. Chem. 278, [103] Pulvirenti, T. et al. (2008) A traffic-activated Golgi-based signalling circuit coordinates the secretory pathway. Nat. Cell Biol. 10, [104] Jackson, M.R., Nilsson, T. and Peterson, P.A. (1990) Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum. Embo J. 9, [105] Hauri, H.P., Kappeler, F., Andersson, H. and Appenzeller, C. (2000) ERGIC-53 and traffic in the secretory pathway. J. Cell Sci. 113 (Pt 4), [106] Dominguez, M. et al. (1998) Gp25L/emp24/p24 protein family members of the cis-golgi network bind both COP I and II coatomer. J. Cell Biol. 140, [107] Gommel, D. et al. (1999) p24 and p23, the major transmembrane proteins of COPI-coated transport vesicles, form hetero-oligomeric complexes and cycle between the organelles of the early secretory pathway. FEBS Lett. 447, [108] Vincent, M.J., Martin, A.S. and Compans, R.W. (1998) Function of the KKXX motif in endoplasmic reticulum retrieval of a transmembrane protein