ADP-ribosylation factor and coatomer couple fusion to vesicle budding

The coat proteins required for budding COP-coated vesicles from Golgi membranes, coatomer and ADP-ribosylation factor (ARF) protein, are shown to be required to reconstitute the orderly process of transport between Golgi cisternae in which fusion of transport vesicles begins only after budding ends. When either coat protein is omitted, fusion is uncoupled from budding-donor and acceptor compartments pair directly without an intervening vesicle. Coupling may therefore results from the sequestration of fusogenic membrane proteins into assembling coated vesicles that are only exposed when the coat is removed after budding is complete. This mechanism of coupling explains the phenomenon of "retrograde transport" triggered by uncouplers such as the drug brefeldin A.     

Coatomer vesicles are not required for inhibition of Golgi transport by G-protein activators

The G-protein activators guanosine 5'-O-(3-thiodiphosphate) (GTPΓS) and aluminum fluoride (AlF) are thought to inhibit transport between Golgi cisternae by causing the accumulation of nonfunctional coatomer-coated transport vesicles on the Golgi. Although GTPΓS and AlF inhibit transport in cell-free intra-Golgi transport systems, blocking coatomer vesicle formation does not. We therefore determined whether inhibition of in vitro Golgi transport by these agents requires coatomer vesicle formation. Depletion of coatomer was found to completely block coated vesicle formation on Golgi cisternae without affecting inhibition of in vitro transport by either GTPΓS or AlF. Depletion of ADP-ribosylation factor (ARF) prevented inhibition of transport by GTPΓS, but not by AlF, suggesting that the AlF-sensitive component in transport may not be a GTP-binding protein. Surprisingly, depletion of cytosolic ARF did not prevent the GTPΓS-induced formation of Golgi-coated vesicles, whereas ARF was required for AlF-induced vesicle formation. Although ARF or coatomer depletion caused an increase in the fenestration of cisternae, no other utrastructural changes were observed that might explain the inhibition of transport by GTPΓS or AlF. These findings suggest that ARF-GTPΓS and AlF act by distinct and coatomer-independent mechanisms to inhibit membrane fusion in cell-free intra-Golgi transport.     

The G protein-activating peptide, mastoparan, and the synthetic NH2- terminal ARF peptide, ARFp13, inhibit in vitro Golgi transport by irreversibly damaging membranes

Mastoparan is a cationic amphipathetic peptide that activates trimeric G proteins, and increases binding of the coat protein beta-COP to Golgi membranes. ARFp13 is a cationic amphipathic peptide that is a putative specific inhibitor of ARF function, and inhibits coat protein binding to Golgi membranes. Using a combination of high resolution, three- dimensional electron microscopy and cell-free Golgi transport assays, we show that both of these peptides inhibit in vitro Golgi transport, not by interfering in the normal functioning of GTP-binding proteins, but by damaging membranes. Inhibition of transport is correlated with inhibition of nucleotide sugar uptake and protein glycoslation, a decrease in the fraction of Golgi cisternae exhibiting normal morphology, and a decrease in the density of Golgi-coated buds and vesicles. At peptide concentrations near the IC50 for transport, those cisternae with apparently normal morphology had a higher steady state level of coated buds and vesicles. Kinetic analysis suggests that this increase in density was due to a decrease in the rate of vesicle fission. Pertussis toxin treatment of the membranes appeared to increase the rate of vesicle formation, but did not prevent the membrane damage induced by mastoparan. We conclude that ARFp13 is not a specific inhibitor of ARF function, as originally proposed, and that surface active peptides, such as mastoparan, have the potential for introducing artifacts that complicate the analysis of trimeric G protein involvement in regulation of Golgi vesicle dynamics.     

Activated ADP-ribosylation factor assembles distinct pools of actin on golgi membranes

The small GTP-binding protein ADP-ribosylation factor (ARF) has been shown to regulate the interaction of actin and actin-binding proteins with the Golgi apparatus. Here we report that ARF activation stimulates the assembly of distinct pools of actin on Golgi membranes. One pool of actin cofractionates with coatomer (COPI)- coated vesicles and is sensitive to salt extraction and the plus end actin-binding toxin cytochalasin D. A second ARF-dependent actin pool remains on the Golgi membranes following vesicle extraction and is insensitive to cytochalasin D. Isolation of the salt-extractable ARF-dependent actin from the Golgi reveals that it is bound to a distinct repertoire of actin-binding proteins. The two abundant actin-binding proteins of the ARF-dependent actin complex are identified as spectrin and drebrin. We show that drebrin is a specific component of the cytochalasin D-sensitive, ARF-dependent actin pool on the Golgi. Finally, we show that depolymerization of this actin pool with cytochalasin D increases the extent of the salt-dependent release of COPI-coated vesicles from the Golgi following cell-free budding reactions. Together these data suggest that regulation of the actin-based cytoskeleton may play an important role during ARF-mediated transport vesicle assembly or release on the Golgi.     

Cytosolic ARFs are required for vesicle formation but not for cell-free intra-Golgi transport: evidence for coated vesicle-independent transport.

We investigated the role of ADP-ribosylation factors (ARFs) in Golgi function using biochemical and morphological cell-free assays. An ARF-free cytosol produced from soluble Chinese hamster ovary (CHO) extracts supports intra-Golgi transport by a mechanism that is biochemically indistinguishable from control transport reactions: ARF-free transport reactions are NSF-dependent, remain sensitive to the donor Golgi-specific inhibitor ilimaquinone, and exhibit kinetics that are identical to that of control reactions containing ARFs. In contrast, ARF-free cytosol does not support the formation of coated vesicles on Golgi cisternae. However, vesicle formation is reconstituted upon the addition of ARF1. These data suggest that neither soluble ARFs nor coated vesicle formation are essential for transport. We conclude that cell-free intra-Golgi transport proceeds via a coated vesicle-independent mechanism regardless of vesicle formation on Golgi cisternae.     

Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack

We describe a scheme for the purification of the nonclathrin-coated vesicles that mediate transport of proteins between Golgi cisternae and probably from ER to Golgi. These "Golgi-derived coated vesicles" accumulate when Golgi membranes are incubated with ATP and cytosol in the presence of GTP gamma S, a compound that blocks vesicle fusion. The coated vesicles dissociate from the Golgi cisternae in high salt and can then be purified by employing differential and density gradient centrifugation. Golgi-derived coated vesicles have a putative polypeptide composition that is distinct from both cytosol and Golgi membranes, as well as from that of clathrin-coated vesicles.     

ARFGAP2 and ARFGAP3 Are Essential for COPI Coat Assembly on the Golgi Membrane of Living Cells

Coat protein complex I (COPI) vesicles play a central role in the recycling of proteins in the early secretory pathway and transport of proteins within the Golgi stack. Vesicle formation is initiated by the exchange of GDP for GTP on ARF1 (ADP-ribosylation factor 1), which, in turn, recruits the coat protein coatomer to the membrane for selection of cargo and membrane deformation. ARFGAP1 (ARF1 GTPase-activating protein 1) regulates the dynamic cycling of ARF1 on the membrane that results in both cargo concentration and uncoating for the generation of a fusion-competent vesicle. Two human orthologues of the yeast ARFGAP Glo3p, termed ARFGAP2 and ARFGAP3, have been demonstrated to be present on COPI vesicles generated in vitro in the presence of guanosine 5′-3-O-(thio)triphosphate. Here, we investigate the function of these two proteins in living cells and compare it with that of ARFGAP1. We find that ARFGAP2 and ARFGAP3 follow the dynamic behavior of coatomer upon stimulation of vesicle budding in vivo more closely than does ARFGAP1. Electron microscopy of ARFGAP2 and ARFGAP3 knockdowns indicated Golgi unstacking and cisternal shortening similarly to conditions where vesicle uncoating was blocked. Furthermore, the knockdown of both ARFGAP2 and ARFGAP3 prevents proper assembly of the COPI coat lattice for which ARFGAP1 does not seem to play a major role. This suggests that ARFGAP2 and ARFGAP3 are key components of the COPI coat lattice and are necessary for proper vesicle formation.     

Biosynthetic protein transport through the early secretory pathway

 Newly synthesized proteins destined for delivery to the cell surface are inserted cotranslationally into the endoplasmic reticulum (ER) and, after their correct folding, are transported out of the ER. During their transport to the cell surface, cargo proteins pass through the various cisternae of the Golgi apparatus and, in the trans-most cisternae of the stack, are sorted into constitutive secretory vesicles that fuse with the plasma membrane. Simultaneously with anterograde protein transport, retrograde protein transport occurs within the Golgi complex as well as from the Golgi back to the ER. Vesicular transport within the early secretory pathway is mediated by two types of non-clathrin coated vesicles: COPI- and COPII-coated vesicles. The formation of these carrier vesicles depends on the recruitment of cytosolic coat proteins that are thought to act as a mechanical device to shape a flattened donor membrane into a spherical vesicle. A general molecular machinery that mediates targeting and fusion of carrier vesicles has been identified as well. Beside a general overview of the various coat structures known today, we will discuss issues specifically related to the biogenesis of COPI-coated vesicles: (1) a possible role of phospholipase D in the formation of COPI-coated vesicles; (2) a functional role of a novel family of transmembrane proteins, the p24 family, in the initiation of COPI assembly; and (3) the direction COPI-coated vesicles may take within the early secretory pathway. Moreover, we will consider two alternative mechanisms of protein transport through the Golgi stack: vesicular transport versus cisternal maturation. Accepted: 24 October 1997     

A major transmembrane protein of Golgi-derived COPI-coated vesicles involved in coatomer binding

Formation of non-clathrin-coated vesicles requires the recruitment of several cytosolic factors to the Golgi membrane. To identify membrane proteins involved in this budding process, a highly abundant type I transmembrane protein (p23) was isolated from mammalian Golgi-derived COPI-coated vesicles, and its cDNA was cloned and sequenced. It belongs to the p24 family of proteins involved in the budding of transport vesicles (Stamnes, M.A., M.W. Craighead, M.H. Hoe, N. Lampen, S. Geromanos, P. Tempst, and J.E. Rothman. 1995. Proc. Natl. Acad. Sci. USA. 92:8011-8015). p23 consists of a large NH2-terminal luminal domain and a short COOH-terminal cytoplasmic tail (-LRRFFKAKKLIE-CO2-) that shows similarity, but not identity, with the sequence motif-KKXX-CO2-, known as a signal for retrieval of escaped ER-resident membrane proteins (Jackson, M.R., T. Nilsson, and P.A. Peterson. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:3153-3162; Nilsson, T., M. Jackson, and P.A. Peterson. 1989. Cell. 58:707-718). The cytoplasmic tail of p23 binds to coatomer with similar efficiency as known KKXX motifs. However, the p23 tail differs from the KKXX motif in having an additional motif needed for binding of coatomer. p23 is localized to Golgi cisternae and, during vesicle formation, it concentrates into COPI-coated buds and vesicles. Biochemical analysis revealed that p23 is enriched in vesicles by a factor of approximately 20, as compared with the donor Golgi fraction, and is present in amounts stoichiometric to the small GTP-binding protein ADP-ribosylation factor (ARF) and coatomer. From these data we conclude that p23 represents a Golgi- specific receptor for coatomer involved in the formation of COPI-coated vesicles.     

Phospholipase D Stimulates Release of Nascent Secretory Vesicles from the trans-Golgi Network

Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited PLD-catalyzed formation of phosphatidic acid. Furthermore, ARF-1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.     

Dissection of a single round of vesicular transport: sequential intermediates for intercisternal movement in the Golgi stack

We take advantage of a cell-free system that reconstitutes essentially a single round of transport of the VSV-encoded G protein between Golgi cisternae to identify discrete stages in the maturation of carrier vesicles. Using GTP gamma S and N-ethylmaleimide (NEM) as selective inhibitors to accumulate coated and uncoated vesicles, respectively, we find these to be successive and obligatory transport intermediates. We find that the coated and uncoated vesicles that accumulate when transport is blocked have already transferred from donor to acceptor stacks but not yet fused. Similar coated and uncoated vesicles accumulate in appropriately treated whole cells. Our studies imply that a coated bud (pit)-coated vesicle-uncoated vesicle system analogous to that responsible for receptor-mediated endocytosis carries biosynthetic protein transport across the Golgi stack. However, "Golgi"-coated buds do not contain clathrin and seem to act as bulk carriers, whereas endocytic clathrin-coated pits carry a highly selective cargo.     

真核细胞内膜泡运输的分子机制

真核细胞内一些蛋白质需靠膜泡进行定向运输,膜泡是在外衣蛋白的作用下形成的,根据外衣蛋白的不同,膜泡分为笼蛋白,COPⅠ和COPⅡ外衣膜泡,这些外衣膜泡分别在细胞内不同供膜（donor membrane)处形成,因为被运输蛋白具有分选信号可与供膜上相应的受体结合,所以能被包裹在特异的膜泡之中,在膜泡形成过程中,外衣蛋白在“芽生”膜泡的细胞质侧组装成笼状外衣,帮助“芽生”膜泡从供膜处脱落,一旦笼状外衣膜泡脱离供膜,笼状外衣蛋白便发生解聚而成为无衣膜泡,无衣膜泡在Rab蛋白的调控下可定向运输蛋白质,而解聚后的外衣蛋白可重新介导新的外衣膜泡形成。     

A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack

Isolated Golgi membranes incubated in the presence of ATP and a cytosolic protein fraction form a population of coated buds or vesicles from the Golgi cisternae. The coats do not have the characteristic hexagonal-pentagonal basketwork of clathrin, and do not react with anti-clathrin polyclonal antibody. The conditions that produce these apparently nonclathrin-coated buds also reconstitute protein transport between compartments of the Golgi stack. The membrane of the buds contains the glycoprotein in transit through these Golgi stacks (VSV-encoded G protein). This suggests that protein transport through the Golgi stack is mediated by a new type of coated vesicle that does not contain clathrin. The concentration of G protein in the coated buds reflects the local concentration of G protein in the cisternae, raising the possibility that the Golgi coated vesicles may be "bulk" membrane carriers.     

The yeast p24 complex is required for the formation of COPI retrograde transport vesicles from the Golgi apparatus

The p24 family members are transmembrane proteins assembled into heteromeric complexes that continuously cycle between the ER and the Golgi apparatus. These cargo proteins were assumed to play a structural role in COPI budding because of their major presence in mammalian COPI vesicles. However, this putative function has not been proved conclusively so far. Furthermore, deletion of all eight yeast p24 family members does not produce severe transport phenotypes, suggesting that the p24 complex is not essential for COPI function. In this paper we provide direct evidence that the yeast p24 complex plays an active role in retrograde transport from Golgi to ER by facilitating the formation of COPI-coated vesicles. Therefore, our results demonstrate that p24 proteins are important for vesicle formation instead of simply being a passive traveler, supporting the model in which cargo together with a small GTPase of the ARF superfamily and coat subunits act as primer for vesicle formation.     

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.     

Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors

COPI-coated vesicle budding from lipid bilayers whose composition resembles mammalian Golgi membranes requires coatomer, ARF, GTP, and cytoplasmic tails of putative cargo receptors (p24 family proteins) or membrane cargo proteins (containing the KKXX retrieval signal) emanating from the bilayer surface. Liposome-derived COPI-coated vesicles are similar to their native counterparts with respect to diameter, buoyant density, morphology, and the requirement for an elevated temperature for budding. These results suggest that a bivalent interaction of coatomer with membrane-bound ARF[GTP] and with the cytoplasmic tails of cargo or putative cargo receptors is the molecular basis of COPI coat assembly and provide a simple mechanism to couple uptake of cargo to transport vesicle formation.     

Active ADP-ribosylation factor-1 (ARF1) is required for mitotic Golgi fragmentation

In mammalian cells the Golgi apparatus undergoes an extensive disassembly process at the onset of mitosis that is believed to facilitate equal partitioning of this organelle into the two daughter cells. However, the underlying mechanisms for this fragmentation process are so far unclear. Here we have investigated the role of the ADP-ribosylation factor-1 (ARF1) in this process to determine whether Golgi fragmentation in mitosis is mediated by vesicle budding. ARF1 is a small GTPase that is required for COPI vesicle formation from the Golgi membranes. Treatment of Golgi membranes with mitotic cytosol or with purified coatomer together with wild type ARF1 or its constitutive active form, but not the inactive mutant, converted the Golgi membranes into COPI vesicles. ARF1-depleted mitotic cytosol failed to fragment Golgi membranes. ARF1 is associated with Golgi vesicles generated in vitro and with vesicles in mitotic cells. In addition, microinjection of constitutive active ARF1 did not affect mitotic Golgi fragmentation or cell progression through mitosis. Our results show that ARF1 is active during mitosis and that this activity is required for mitotic Golgi fragmentation.     

Fatty acyl-coenzyme A is required for budding of transport vesicles from Golgi cisternae

We describe a new role for fatty acylation. Conditions were established under which vesicular transport from the cis to the medial Golgi compartment in vitro depends strongly upon the addition of a fatty acyl-coenzyme A, e.g., palmitoyl-CoA. Using an inhibitor of long-chain acyl-CoA synthetase, we demonstrate that the fatty acid has to be activated by CoA to stimulate transport. A nonhydrolyzable analog of palmitoyl-CoA competitively inhibits transport. Electron microscopy and biochemical studies show that fatty acyl-CoA is required for budding of (non-clathrin-) coated transport vesicles from Golgi cisternae and that budding is inhibited by the nonhydrolyzable analog.     

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.     

The amino terminus of ADP-ribosylation factor (ARF) is a critical determinant of ARF activities and is a potent and specific inhibitor of protein transport.

Deletion of the amino-terminal 17 residues from human ADP-ribosylation factor (ARF) resulted in a protein ([delta 1-17]mARF1p) devoid of ARF activity but which retained the ability to bind guanine nucleotides with high affinity. Unlike the wild type, the binding of guanine nucleotides to this deletion mutant was found to be independent of added phospholipids. A chimeric protein was produced, consisting of 10% (the amino-terminal 17 amino acids) human ARF1p and 90% ARL1p, an ARF-like protein (55% identical protein sequence) from Drosophila. This chimera was found to have ARF activity, lacking in the parental ARL1 protein. Thus, the amino terminus of ARF1p was shown to be a critical component of ARF activity. A synthetic peptide, derived from the amino terminus of ARF1p, has no ARF activity. Rather, the peptide was found to be a specific inhibitor of ARF activities. This peptide was also found to be a potent and specific inhibitor of both an in vitro intra-Golgi transport assay and the guanosine 5'-3-O-(thio)triphosphate-stimulated accumulation of coated vesicles and buds from Golgi preparations. We conclude that ARF is required for the budding of coated vesicles from the Golgi stacks and serves a regulatory role in protein secretion through the Golgi in eukaryotic cells.