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.     

ARFGAP1 promotes AP-2-dependent endocytosis

COPI (coat protein I) and the clathrin-AP-2 (adaptor protein 2) complex are well-characterized coat proteins, but a component that is common to these two coats has not been identified. The GTPase-activating protein (GAP) for ADP-ribosylation factor 1 (ARF1), ARFGAP1, is a known component of the COPI complex. Here, we show that distinct regions of ARFGAP1 interact with AP-2 and coatomer (components of the COPI complex). Selectively disrupting the interaction of ARFGAP1 with either of these two coat proteins leads to selective inhibition in the corresponding transport pathway. The role of ARFGAP1 in AP-2-regulated endocytosis has mechanistic parallels with its roles in COPI transport, as both its GAP activity and coat function contribute to promoting AP-2 transport.     

Members of a mammalian SNARE complex interact in the endoplasmic reticulum in vivo and are found in COPI vesicles

Retrograde traffic between the Golgi apparatus and the endoplasmic reticulum (ER) is largely mediated by COPI-coated transport vesicles. In mammalian cells, retrograde traffic can pass through an intermediate compartment. Here, we report that the mammalian soluble N-ethylmaleimide-sensitive factor (NSF) attachment receptor (SNARE) proteins mSec22b, mUse1/D12, mSec20/BNIP1, and syntaxin 18 form a quaternary SNARE complex. Fluorescence resonance energy transfer (FRET) experiments prove that these interactions occur in the ER of living cells. In addition, mUse1/D12 and mSec20/BNIP1 form homo-oligomers in vivo. Furthermore, we show that mSec22b, mUse1/D12, mSec20/BNIP1, and syntaxin 18 are recruited into COPI-coated vesicles formed in vitro. Immunogold electron microscopy confirmed that these SNARE proteins colocalize with the KDEL receptor ERD2 in COPI-coated vesicles. Moreover, both FRET and immunoprecipitation experiments reveal interactions of these SNAREs with both ERD2 and COPI subunits. We conclude that the SNAREs described here are sorted via interaction with components of the COPI-dependent budding complex into Golgi-to-ER retrograde COPI vesicles and function in retrograde transport from the Golgi to the ER Golgi intermediate compartment (ERGIC) or the ER.     

The KDEL receptor, ERD2, regulates intracellular traffic by recruiting a GTPase-activating protein for ARF1.

The small GTPase ADP-ribosylation factor 1 (ARF1) is a key regulator of intracellular membrane traffic. Regulators of ARF1, its GTPase-activating protein (GAP) and its guanine nucleotide exchange factor have been identified recently. However, it remains uncertain whether these regulators drive the GTPase cycle of ARF1 autonomously or whether their activities can be regulated by other proteins. Here, we demonstrate that the intracellular KDEL receptor, ERD2, self-oligomerizes and interacts with ARF1 GAP, and thereby regulates the recruitment of cytosolic ARF1 GAP to membranes. Because ERD2 overexpression enhances the recruitment of GAP to membranes and results in a phenotype that reflects ARF1 inactivation, our findings suggest that ERD2 regulates ARF1 GAP, and thus regulates ARF1-mediated transport.     

Plant ERD2s self-interact and interact with GTPase-activating proteins and ADP-ribosylation factor 1

ERD2s (ER luminal protein receptors)-mediated retrograde transport is one of the most substantial processes to maintain the endoplasmic reticulum (ER) homeostasis. It is completed by the recognition of the escaped ER luminal proteins, the gathering into COP I vesicle, and the fusion and releasing into the ER. ERD2s can recognize HDEL/KDEL motifs at the C-terminal of the escaped ER luminal proteins at the Golgi to initiate the retrograde transport. However, these mechanisms remain largely unknown in plants. We recently found that two Nicotiana benthamiana homologs, ERD2a and ERD2b, functioned as ER luminal protein receptors, were required for both HDEL/KDEL motifs-mediated ER retrieval and participated in cell death triggered by ER stress and nonhost pathogens. Here, we provide a set of new data that ERD2a/2b can form homo- or hetero-oligomerization and interact with both the ADP-ribosylation factor 1 (ARF1) and its potential GTPase-activating proteins (GAP) indicated by the firefly luciferase complementation imaging assay (LCI). These evidences further support the ER luminal protein receptor function of ERD2a/2b in plants and suggest their evolutionarily conserved mechanism during the retrograde trafficking. We also analyze the characteristics of ERD2s within a species and among different species.     

Key components of the fission machinery are interchangeable

Brefeldin-A ADP-ribosylated substrate (BARS) and dynamin function in membrane fission in distinct intracellular transport pathways, but whether their functions are mechanistically similar is unclear. Here, we show that ARFGAP1, a GTPase-activating protein (GAP) for ADP-ribosylation factor 1 (ARF1), couples to either BARS or endophilin B for vesicle formation by the coat protein I (COPI) complex - a finding that reveals an unanticipated mechanistic flexibility in mammalian COPI transport. Because dynamin is coupled to endophilin A in vesicle formation by the clathrin-coat complex, our finding also predicts that dynamin and ARF GAPs are likely to be functional counterparts in membrane fission among different transport pathways that connect intracellular membrane compartments.     

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.     

Structural and functional analysis of the ARF1-ARFGAP complex reveals a role for coatomer in GTP hydrolysis

The crystal structure of the complex of ARF1 GTPase bound to GDP and the catalytic domain of ARF GTPase-activating protein (ARFGAP) has been determined at 1.95 A resolution. The ARFGAP molecule binds to switch 2 and helix alpha3 to orient ARF1 residues for catalysis, but it supplies neither arginine nor other amino acid side chains to the GTPase active site. In the complex, the effector-binding region appears to be unobstructed, suggesting that ARFGAP could stimulate GTP hydrolysis while ARF1 maintains an interaction with its effector, the coatomer complex of COPI-coated vesicles. Biochemical experiments show that coatomer directly participates in the GTPase reaction, accelerating GTP hydrolysis a further 1000-fold in an ARFGAP-dependent manner. Thus, a tripartite complex controls the GTP hydrolysis reaction triggering disassembly of COPI vesicle coats.     

Spatiotemporal dynamics of the COPI vesicle machinery

Assembly of the coat protein I (COPI) vesicle coat is controlled by the small GTPase ADP ribosylation factor 1 (ARF1) and its GTPase-activating protein, ARFGAP1. Here, we investigate the diffusional behaviours of coatomer, the main component of the coat, and also those of ARF1 and ARFGAP1. Using fluorescence-correlation spectroscopy, we found that most ARF1 and ARFGAP1 molecules are highly mobile in the cytosol (diffusion constant D ≈ 15 μm² s⁻¹), whereas coatomer diffuses 5–10 times more slowly than expected (D ≈ 1 μm² s⁻¹). This slow diffusion causes diffusion-limited binding kinetics to Golgi membranes, which, in FRAP (fluorescence recovery after photobleaching) experiments, translates into a twofold slower binding rate. The addition of aluminium fluoride locks coatomer onto Golgi membranes and also decreases the binding kinetics of both ARF1 and ARFGAP1, suggesting that these proteins function in concert to mediate sorting and vesicle formation.     

ARFGAP1 Is Dynamically Associated with Lipid Droplets in Hepatocytes

The ARF GTPase Activating Protein 1 (ARFGAP1) associates mainly with the cytosolic side of Golgi cisternal membranes where it participates in the formation of both COPI and clathrin-coated vesicles. In this study, we show that ARFGAP1 associates transiently with lipid droplets upon addition of oleate in cultured cells. Also, that addition of cyclic AMP shifts ARFGAP1 from lipid droplets to the Golgi apparatus and that overexpression and knockdown of ARFGAP1 affect lipid droplet formation. Examination of human liver tissue reveals that ARFGAP1 is found associated with lipid droplets at steady state in some but not all hepatocytes.     

Recruitment to Golgi membranes of ADP-ribosylation factor 1 is mediated by the cytoplasmic domain of p23.

Binding to Golgi membranes of ADP ribosylation factor 1 (ARF1) is the first event in the initiation of COPI coat assembly. Based on binding studies, a proteinaceous receptor has been proposed to be critical for this process. We now report that p23, a member of the p24 family of Golgi-resident transmembrane proteins, is involved in ARF1 binding to membranes. Using a cross-link approach based on a photolabile peptide corresponding to the cytoplasmic domain of p23, the GDP form of ARF1 (ARF1-GDP) is shown to interact with p23 whereas ARF1-GTP has no detectable affinity to p23. The p23 binding is shown to localize specifically to a 22 amino acid C-terminal fragment of ARF1. While a monomeric form of a non-photolabile p23 peptide does not significantly inhibit formation of the cross-link product, the corresponding dimeric form does compete efficiently for this interaction. Consistently, the dimeric p23 peptide strongly inhibits ARF1 binding to native Golgi membranes suggesting that an oligomeric form of p23 acts as a receptor for ARF1 before nucleotide exchange takes place.     

Diacylglycerol is required for the formation of COPI vesicles in the Golgi-to-ER transport pathway

Diacylglycerol is necessary for trans-Golgi network (TGN) to cell surface transport, but its functional relevance in the early secretory pathway is unclear. Although depletion of diacylglycerol did not affect ER-to-Golgi transport, it led to a redistribution of the KDEL receptor to the Golgi, indicating that Golgi-to-ER transport was perturbed. Electron microscopy revealed an accumulation of COPI-coated membrane profiles close to the Golgi cisternae. Electron tomography showed that the majority of these membrane profiles originate from coated buds, indicating a block in membrane fission. Under these conditions the Golgi-associated pool of ARFGAP1 was reduced, but there was no effect on the binding of coatomer or the membrane fission protein CtBP3/BARS to the Golgi. The addition of 1,2-dioctanoyl-sn-glycerol or the diacylglycerol analogue phorbol 12,13-dibutyrate reversed the effects of endogenous diacylglycerol depletion. Our findings implicate diacylglycerol in the retrograde transport of proteins from Golgi to the ER and suggest that it plays a critical role at a late stage of COPI vesicle formation.     

A kinetic proof-reading mechanism for protein sorting

Resident proteins of the exocytic pathway are maintained at various levels through coatomer protein I (COPI)-mediated recycling. Sorting of cargo by COPI requires GTP hydrolysis by ADP-ribosylation factor 1 (ARF-1). This small GTPase recruits coatomer onto Golgi membranes and upon hydrolysis, is thought to release coatomer back into the cytosol. This step requires the activating protein, ARFGAP1. By coupling sorting to a cargo-induced sequestering of ARFGAP1, we have formulated a kinetic proof-reading model that explains how a GTP hydrolysis-driven coat release can yield an active sorting event. The sorting scheme predicts a dependency on the amount of ARFGAP1 and explains the recent experimental findings that ARF-1 and COPI detach with different time constants from the Golgi membrane in vivo .     

人源细胞内物质转运调节分子ARFGAP1对细胞分泌功能的影响

 ARF GAP是重要的细胞内物质转运调节分子 .最近 ,在人胎肝 c DNA文库中发现一种新基因 ,其编码的氨基酸序列与大鼠的 ARF1 GAP有 32 %同源性 ,故将其命名为“ARFGAP1”.对ARFGAP1进行功能研究 ,利用分子克隆技术构建绿色荧光蛋白 (GFP) - ARFGAP1融合基因表达质粒 (p EGFP- C1 - ARFGAP1 ) ,经脂质体转染将其导入 COS- 7细胞瞬时表达 ,利用绿色荧光确定ARFGAP1的亚细胞定位 .结果显示 ,ARFGAP1位于细胞质部分 ,表达量高时 ,在核周高尔基体区聚集呈团块状或颗粒状 .构建真核表达质粒 pc DNA3.1 /myc- His- ARFGAP1 ,在 COS- 7细胞中表达 ,并用 ARFGAP1和分泌型碱性磷酸酶 (SEAP)真核表达质粒共同转染 COS- 7细胞 ,发现ARFGAP1在细胞中过表达能部分抑制 SEAP的分泌 .结果证明 ,ARFGAP1对细胞的物质转运和分泌功能有调节作用 .     

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.     

Functional characterization of novel human ARFGAP3

ADP ribosylation factors (ARFs) are critical in the vesicular trafficking pathway. ARF activity is controlled by GTPase-activating proteins (GAPs). We have identified recently a novel tentative ARF GAP derived from human fetal liver, ARFGAP3 (originally named as ARFGAP1). In the present study, we demonstrated that ARFGAP3 had GAP activity in vitro and remarked that the GAP activity of ARFGAP3 was regulated by phospholipids, i.e. phosphatidylinositol 4,5-diphosphate as agonist and phosphatidylcholine as antagonist. ARFGAP3 is a predominantly cytosolic protein, and concentrated in the perinuclear region. Its transient ectopic overexpression in cultured mammalian cells reduced the constitutive secretion of secreted alkaline phosphatase, indicating that ectopic overexpression of ARFGAP3 inhibits the early secretory pathway of proteins in vivo. These results demonstrated that ARFGAP3 is a novel GAP for ARF1 and might be involved in intracellular traffic of proteins and vesicular transport as predicted.     

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.     

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.     

COPI-mediated blood meal digestion in vector mosquitoes is independent of midgut ARF-GEF and ARF-GAP regulatory activities

We have previously shown that defects in COPI coatomer proteins cause 80% mortality in blood fed Aedes aegypti mosquitoes by 96 h post-feeding. In this study we show that similar deficiencies in COPII and clathrin mediated vesicle transport do not disrupt blood meal digestion and are not lethal, even though both COPII and clathrin functions are required for ovarian development. Since COPI vesicle transport is controlled in mammalian cells by upstream G proteins and associated regulatory factors, we investigated the function of the orthologous ADP-ribosylation factor 1 (ARF1) and ARF4 proteins in mosquito tissues. We found that both ARF1 and ARF4 function upstream of COPI vesicle transport in blood fed mosquitoes given that an ARF1/ARF4 double deficiency is required to phenocopy the feeding-induced mortality observed in COPI coatomer deficient mosquitoes. Small molecule inhibitors of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) are often transitory, and therefore, we investigated the role of five Ae. aegypti ARF-GEF and ARF-GAP proteins in blood meal digestion using RNA interference. Surprisingly, we found that ARF-GEF and ARF-GAP functions are not required for blood meal digestion, even though both vitellogenesis and ovarian development in Ae. aegypti are dependent on GBF1 (ARF-GEF) and GAP1/GAP2 (ARF-GAPs) proteins. Moreover, deficiencies in orthologous COPI regulating genes in Anopheles stephensi mosquitoes had similar phenotypes, indicating conserved functions in these two mosquito species. We propose that based on the need for rapid initiation of protein digestion and peritrophic membrane formation, COPI vesicle transport in midgut epithelial cells is not dependent on ARF-GEF and ARF-GAP regulatory proteins to mediate vesicle recycling within the first 48 h post-feeding.     

Sorting signals in the cytosolic tail of membrane proteins involved in the interaction with plant ARF1 and coatomer

In mammals and yeast, a cytosolic dilysine motif is critical for endoplasmic reticulum (ER) localization of type I membrane proteins. Retrograde transport of type I membrane proteins containing dilysine motifs at their cytoplasmic carboxy (C)-terminal tail involves the interaction of these motifs with the COPI coat. The C-terminal dilysine motif has also been shown to confer ER localization to type I membrane proteins in plant cells. Using in vitro binding assays, we have analyzed sorting motifs in the cytosolic tail of membrane proteins, which may be involved in the interaction with components of the COPI coat in plant cells. We show that a dilysine motif in the -3,-4 position (relative to the cytosolic C-terminus) recruits in a very specific manner all the subunits of the plant coatomer complex. Lysines cannot be replaced by arginines or histidines to bind plant coatomer. A diphenylalanine motif in the -7,-8 position, which by itself has a low ability to bind plant coatomer, shows a clear cooperativity with the dilysine motif. Both dilysine and diphenylalanine motifs are present in the cytosolic tail of several proteins of the p24 family of putative cargo receptors, which has several members in plant cells. The cytosolic tail of a plant p24 protein is shown to recruit not only coatomer but also ADP ribosylation factor 1 (ARF1), a process which depends on both dilysine and diphenylalanine motifs. ARF1 binding increases twofold upon treatment with brefeldin A (BFA) and is completely abolished upon treatment with GTPgammaS, suggesting that ARF1 can only interact with the cytosolic tail of p24 proteins in its GDP-bound form.