Several ADP-ribosylation factor (Arf) isoforms support COPI vesicle formation

Newly synthesized proteins and lipids are transported in vesicular carriers along the secretory pathway. Arfs (ADP-ribosylation factors), a family of highly conserved GTPases within the Ras superfamily, control recruitment of molecular coats to membranes, the initial step of coated vesicle biogenesis. Arf1 and coatomer constitute the minimal cytosolic machinery leading to COPI vesicle formation from Golgi membranes. Although some functional redundancies have been suggested, other Arf isoforms have been poorly analyzed in this context. In this study, we found that Arf1, Arf4, and Arf5, but not Arf3 and Arf6, associate with COPI vesicles generated in vitro from Golgi membranes and purified cytosol. Using recombinant myristoylated proteins, we show that Arf1, Arf4, and Arf5 each support COPI vesicle formation individually. Unexpectedly, we found that Arf3 could also mediate vesicle biogenesis. However, Arf3 was excluded from the vesicle fraction in the presence of the other isoforms, highlighting a functional competition between the different Arf members.     

A COPI coat subunit interacts directly with an early‐Golgi localized Arf exchange factor

Arf (ADP‐ribosylation factor) family small G proteins are crucial regulators of intracellular transport. The active GTP‐bound form of Arf interacts with a set of proteins—effectors—which mediate the downstream signalling events of Arf activation. A well‐studied class of Arf1 effectors comprises the coat complexes, such as the cis‐Golgi‐localized COPI (coat protein complex I) coat, and trans‐Golgi network‐endosomal clathrin coats. At least five different coats require Arf1‐GTP to localize to organelle membranes. How a single Arf protein recruits different coat complexes to distinct membrane sites raises the question of how specificity is achieved. Here, we propose a molecular mechanism of this specificity for the COPI coat by showing a direct and specific interaction between a COPI subunit and a cis‐Golgi localized subfamily of Arf guanine nucleotide exchange factors (GEFs) that takes place independently of Arf1 activation. In this way, a specific output on Arf1 activation can be programmed before the exchange reaction by the GEF itself.     

Multiple activities for Arf1 at the Golgi complex

The Arf family of GTPases regulates membrane traffic and organelle structure. At the Golgi complex, Arf proteins facilitate membrane recruitment of many cytoplasmic coat proteins to allow sorting of membrane proteins for transport, stimulate the activity of enzymes that modulate the lipid composition of the Golgi, and assemble a cytoskeletal scaffold on the Golgi. Arf1 is the Arf family member most closely studied for its function at the Golgi complex. A number of regulators that activate and inactivate Arf1 on the Golgi have been described that localize to different regions of the organelle. This spatial distribution of Arf regulators may facilitate the recruitment of the coat proteins and other Arf effectors to different regions of the Golgi complex.     

Membrane recruitment of effector proteins by Arf and Rab GTPases

In their GTP-bound form, Arf and Rab family GTPases associate with distinct organelle membranes, to which they recruit specific sets of effector proteins that regulate vesicular transport. The Arf GTPases are involved in the formation of coated carrier vesicles by recruiting coat proteins. On the other hand, the Rab GTPases are involved in the tethering, docking and fusion of transport vesicles with target organelles, acting in concert with the tethering and fusion machineries. Recent structural studies of the Arf1-GGA and Rab5-Rabaptin-5 complexes, as well as other effector structures in complex with the Arf and Rab GTPases, have shed light on the mechanisms underlying the GTP-dependent membrane recruitment of these effector proteins.     

Site-specific photocrosslinking to probe interactions of Arf1 with proteins involved in budding of COPI vesicles

ADP-ribosylation factor 1 (Arf1) plays an important role in early and intra-Golgi protein trafficking. During this process, Arf1 interacts with many different proteins and other molecules that regulate its state of activation or are involved in its intracellular function. To determine which of these proteins interact directly with Arf1 during coat protein type I (COPI) vesicle biogenesis, we probed the molecular environment of Arf1 by use of site-specific photocrosslinking. This method was first used successfully in the field of protein trafficking to study the mechanisms involved in protein translocation across the endoplasmic reticulum during protein synthesis. In such a hydrophobic environment, crosslink yields of up to 30% have been observed. We have now applied this method to study the mechanism of vesicle budding from the cytosolic face of the Golgi apparatus, an aqueous environment. Although the crosslink yield is significantly lower under these conditions, due to predominant reaction of the photolabile probes with water, a specific interaction of Arf1 with subunits of coatomer, the major coat protein of COPI vesicles, could readily be identified.     

ArfGAP1 Activity and COPI Vesicle Biogenesis

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

Peroxisome biogenesis: where Arf and coatomer might be involved

The present review summarizes recent observations on binding of Arf and COPI coat to isolated rat liver peroxisomes. The general structural and functional features of both Arf and coatomer were considered along with the requirements and dependencies of peroxisomal Arf and coatomer recruitment. Studies on the expression of mammalian Pex11 proteins, mainly Pex11alpha and Pex11beta, intimately related to the process of peroxisome proliferation, revealed a sequence of individual steps including organelle elongation/tubulation, formation of membrane and matrix protein patches segregating distinct proteins from each other, development of membrane constrictions and final membrane fission. Based on the similarities of the processes leading to cargo selection and concentration on Golgi membranes on the one hand and to the formation of peroxisomal protein patches on the other hand, an implication of Arf and COPI in distinct processes of peroxisomal proliferation is hypothesized. Alternatively, peroxisomal Arf/COPI might facilitate the formation of COPI-coated peroxisomal vesicles functioning in cargo transport and retrieval from peroxisomes to the ER. Recent observations suggesting transport of Pex3 and Pex19 during early steps of peroxisome biogenesis from the ER to peroxisomes inevitably propose such a retrieval mechanism, provided the ER to peroxisome pathway is based on transporting vesicles.     

GBF1 and Arf1 function in vesicular trafficking,lipid homoeostasis and organelle dynamics

The ADP‐ribosylation factor (Arf) small G proteins act as molecular switches to coordinate multiple downstream pathways that regulate membrane dynamics. Their activation is spatially and temporally controlled by the guanine nucleotide exchange factors (GEFs). Members of the evolutionarily conserved GBF/Gea family of Arf GEFs are well known for their roles in formation of coat protein complex I (COPI) vesicles, essential for maintaining the structure and function of the Golgi apparatus. However, studies over the past 10 years have found new functions for these GEFs, along with their substrate Arf1, in lipid droplet metabolism, clathrin‐independent endocytosis, signalling at the plasma membrane, mitochondrial dynamics and transport along microtubules. Here, we describe these different functions, focussing in particular on the emerging theme of GFB1 and Arf1 regulation of organelle movement on microtubules.     

Arf regulates interaction of GGA with mannose-6-phosphate receptor

The role of ADP-ribosylation factor (Arf) in Golgi associated, γ-adaptin homologous, Arf-interacting protein (GGA)-mediated membrane traffic was examined. GGA is a clathrin adaptor protein that binds Arf through its GAT domain and the mannose-6-phosphate receptor through its VHS domain. The GAT and VHS domains interacted such that Arf and mannose-6-phosphate receptor binding to GGA were mutually exclusive. In vivo , GGA bound membranes through either Arf or mannose-6-phosphate receptor. However, mannose-6-phosphate receptor excluded Arf from GGA-containing structures outside of the Golgi. These data are inconsistent with predictions based on the model for Arf's role in COPI veside coat function. We propose that Arf recruits GGA to a membrane and then, different from the current model, 'hands-off' GGA to mannose-6-phosphate receptor. GGA and mannose-6-phosphate receptor are then incorporated into a transport intermediate that excludes Arf .     

Modeling the dynamic behaviors of the COPI vesicle formation regulators,the small GTPase Arf1 and its activating Sec7 guanine nucleotide exchange factor GBF1 on Golgi membranes

The components and subprocesses underlying the formation of COPI-coated vesicles at the Golgi are well understood. The coating cascade is initiated after the small GTPase Arf1 is activated by the Sec7 domain–containing guanine nucleotide exchange factor GBF1 (Golgi brefeldin A resistant guanine nucleotide exchange factor 1). This causes a conformational shift within Arf1 that facilitates stable association of Arf1 with the membrane, a process required for subsequent recruitment of the COPI coat. Although we have atomic-level knowledge of Arf1 activation by Sec7 domain–containing GEFs, our understanding of the biophysical processes regulating Arf1 and GBF1 dynamics is limited. We used fluorescence recovery after photobleaching data and kinetic Monte Carlo simulation to assess the behavior of Arf1 and GBF1 during COPI vesicle formation in live cells. Our analyses suggest that Arf1 and GBF1 associate with Golgi membranes independently, with an excess of GBF1 relative to Arf1. Furthermore, the GBF1-mediated Arf1 activation is much faster than GBF1 cycling on/off the membrane, suggesting that GBF1 is regulated by processes other than its interactions Arf1. Interestingly, modeling the behavior of the catalytically inactive GBF1/E794K mutant stabilized on the membrane is inconsistent with the formation of a stable complex between it and an endogenous Arf1 and suggests that GBF1/E794K is stabilized on the membrane independently of complex formation.     

The small G-protein Arf6GTP recruits the AP-2 adaptor complex to membranes

The small GTP-binding protein ADP-ribosylation factor 6 (Arf6) is involved in plasma membrane/endosomes trafficking. However, precisely how the activation of Arf6 regulates vesicular transport is still unclear. Here, we show that, in vitro, recombinant Arf6GTP recruits purified clathrin-adaptor complex AP-2 (but not AP-1) onto phospholipid liposomes in the absence of phosphoinositides. We also show that phosphoinositides and Arf6 tightly cooperate to translocate AP-2 to the membrane. In vivo, Arf6GTP (but not Arf6GDP) was found associated to AP-2. The expression of the GTP-locked mutant of Arf6 leads to the plasma membrane redistribution of AP-2 in Arf6GTP-enriched areas. Finally, we demonstrated that the expression of the GTP-locked mutant of Arf6 inhibits transferrin receptor internalization without affecting its recycling. Altogether, our results demonstrated that Arf6GTP interacts specifically with AP-2 and promotes its membrane recruitment. These findings strongly suggest that Arf6 plays a major role in clathrin-mediated endocytosis by directly controlling the assembly of the AP-2/clathrin coat.     

ArfGAP1 generates an Arf1 gradient on continuous lipid membranes displaying flat and curved regions

ArfGAP1, which promotes GTP hydrolysis on the small G protein Arf1 on Golgi membranes, interacts preferentially with positively curved membranes through its amphipathic lipid packing sensor (ALPS) motifs. This should influence the distribution of Arf1‐GTP when flat and curved regions coexist on a continuous membrane, notably during COPI vesicle budding. To test this, we pulled tubes from giant vesicles using molecular motors or optical tweezers. Arf1‐GTP distributed on the giant vesicles and on the tubes, whereas ArfGAP1 bound exclusively to the tubes. Decreasing the tube radius revealed a threshold of R≈35 nm for the binding of ArfGAP1 ALPS motifs. Mixing catalytic amounts of ArfGAP1 with Arf1‐GTP induced a smooth Arf1 gradient along the tube. This reflects that Arf1 molecules leaving the tube on GTP hydrolysis are replaced by new Arf1‐GTP molecules diffusing from the giant vesicle. The characteristic length of the gradient is two orders of magnitude larger than a COPI bud, suggesting that Arf1‐GTP diffusion can readily compensate for the localized loss of Arf1 during budding and contribute to the stability of the coat until fission.     

ArfGAP1 dynamics and its role in COPI coat assembly on Golgi membranes of living cells

Secretory protein trafficking relies on the COPI coat, which by assembling into a lattice on Golgi membranes concentrates cargo at specific sites and deforms the membranes at these sites into coated buds and carriers. The GTPase-activating protein (GAP) responsible for catalyzing Arf1 GTP hydrolysis is an important part of this system, but the mechanism whereby ArfGAP is recruited to the coat, its stability within the coat, and its role in maintenance of the coat are unclear. Here, we use FRAP to monitor the membrane turnover of GFP-tagged versions of ArfGAP1, Arf1, and coatomer in living cells. ArfGAP1 underwent fast cytosol/Golgi exchange with approximately 40% of the exchange dependent on engagement of ArfGAP1 with coatomer and Arf1, and affected by secretory cargo load. Permanent activation of Arf1 resulted in ArfGAP1 being trapped on the Golgi in a coatomer-dependent manner. These data suggest that ArfGAP1, coatomer and Arf1 play interdependent roles in the assembly-disassembly cycle of the COPI coat in vivo.     

Multiple and stepwise interactions between coatomer and ADP-ribosylation factor-1 (Arf1)-GTP

The small GTPase ADP-ribosylation factor-1 (Arf1) plays a key role in the formation of coat protein I (COP I)-coated vesicles. Upon recruitment to the donor Golgi membrane by interaction with dimeric p24 proteins, Arf1's GDP is exchanged for GTP. Arf1-GTP then dissociates from p24, and together with other Golgi membrane proteins, it recruits coatomer, the heptameric coat protein complex of COP I vesicles, from the cytosol. In this process, Arf1 was shown to specifically interact with the coatomer beta and gamma-COP subunits through its switch I region, and with epsilon-COP. Here, we mapped the interaction of the Arf1-GTP switch I region to the trunk domains of beta and gamma-COP. Site-directed photolabeling at position 167 in the C-terminal helix of Arf1 revealed a novel interaction with coatomer via a putative longin domain of delta-COP. Thus, coatomer is linked to the Golgi through multiple interfaces with membrane-bound Arf1-GTP. These interactions are located within the core, adaptor-like domain of coatomer, indicating an organizational similarity between the COP I coat and clathrin adaptor complexes.     

In tobacco leaf epidermal cells, the integrity of protein export from the endoplasmic reticulum and of ER export sites depends on active COPI machinery

Trafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on coat protein complexes I (COPI) and II (COPII) machineries. To date, full characterization of the distribution and dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences COPII-mediated protein transport from the ER in plant cells are largely uncharacterized. Here we dissect the dynamics of COPI in intact cells using live-cell imaging and fluorescence recovery after photobleaching analyses to provide insights into the distribution of COPI and COPII machineries and the mechanisms by which COPI influences COPII-mediated protein export from the ER. We found that Arf1 and coatomer are dynamically associated with the Golgi apparatus and that the COPII coat proteins Sec24 and Sec23 localize at ER export sites that track with the Golgi apparatus in tobacco leaf epidermal cells. Arf1 is also localized at additional structures that originate from the Golgi apparatus but that lack coatomer, supporting the model that Arf1 also has a coatomer-independent role for post-Golgi protein transport in plants. When ER to Golgi protein transport is inhibited by mutations that hamper Arf1-GTPase activity without directly disrupting the COPII machinery for ER protein export, Golgi markers are localized in the ER and the punctate distribution of Sec24 and Sec23 at the ER export sites is lost. These findings suggest that Golgi membrane protein distribution is maintained by the balanced action of COPI and COPII systems, and that Arf1-coatomer is most likely indirectly required for forward trafficking out of the ER due to its role in recycling components that are essential for differentiation of the ER export domains formed by the Sar1-COPII system.     

Overexpression of an ADP-ribosylation factor-guanine nucleotide exchange factor, BIG2, uncouples brefeldin A-induced adaptor protein-1 coat dissociation and membrane tubulation.

BIG2 is a guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and adaptor protein (AP)-1 coat protein complexes. A fungal metabolite, brefeldin A (BFA), inhibits ARF-GEFs and leads to redistribution of coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). To investigate the function of BIG2, we examined the effects of BIG2-overexpression on the BFA-induced redistribution of ARF, coat proteins, and organelle markers. The BIG2 overexpression blocked BFA-induced redistribution from membranes of ARF1 and the AP-1 complex but not that of the COPI complex. These observations indicate that BIG2 is implicated in membrane association of AP-1, but not that of COPI, through activating ARF. Furthermore, not only BIG2 but also ARF1 and AP-1 were found as queues of spherical swellings along the BFA-induced membrane tubules emanating from the TGN. These observations indicate that BFA-induced AP-1 dissociation from TGN membranes and tubulation of TGN membranes are not coupled events and suggest that a BFA target other than ARF-GEFs exists in the cell.     

Kinetic studies of the Arf activator Arno on model membranes in the presence of Arf effectors suggest control by a positive feedback loop

Proteins of the cytohesin/Arno/Grp1 family of Arf activators are positive regulators of the insulin-signaling pathway and control various remodeling events at the plasma membrane. Arno has a catalytic Sec7 domain, which promotes GDP to GTP exchange on Arf, followed by a pleckstrin homology (PH) domain. Previous studies have revealed two functions of the PH domain: inhibition of the Sec7 domain and membrane targeting. Interestingly, the Arno PH domain interacts not only with a phosphoinositide (phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate) but also with an activating Arf family member, such as Arf6 or Arl4. Using the full-length membrane-bound forms of Arf1 and Arf6 instead of soluble forms, we show here that the membrane environment dramatically affects the mechanism of Arno activation. First, Arf6-GTP stimulates Arno at nanomolar concentrations on liposomes compared with micromolar concentrations in solution. Second, mutations in the PH domain that abolish interaction with Arf6-GTP render Arno completely inactive when exchange reactions are reconstituted on liposomes but have no effect on Arno activity in solution. Third, Arno is activated by its own product Arf1-GTP in addition to a distinct activating Arf isoform. Consequently, Arno activity is strongly modulated by competition with Arf effectors. These results show that Arno behaves as a bistable switch, having an absolute requirement for activation by an Arf protein but, once triggered, becoming highly active through the positive feedback effect of Arf1-GTP. This property of Arno might provide an explanation for its function in signaling pathways that, once triggered, must move forward decisively.     

Intra-endosomal pH-sensitive recruitment of the Arf-nucleotide exchange factor ARNO and Arf6 from cytoplasm to proximal tubule endosomes

Kidney proximal tubule epithelial cells have an extensive apical endocytotic apparatus that is critical for the reabsorption and degradation of proteins that traverse the glomerular filtration barrier and that is also involved in the extensive recycling of functionally important apical plasma membrane transporters. We show here that an Arf-nucleotide exchange factor, ARNO (ADP-ribosylation factor nucleotide site opener) as well as Arf6 and Arf1 small GTPases are located in the kidney proximal tubule receptor-mediated endocytosis pathway, and that ARNO and Arf6 recruitment from cytosol to endosomes is pH-dependent. In proximal tubules in situ, ARNO and Arf6 partially co-localized with the V-ATPase in apical endosomes in proximal tubules. Arf1 was localized both at the apical pole of proximal tubule epithelial cells, but also in the Golgi. By Western blot analysis ARNO, Arf6, and Arf1 were detected both in purified endosomes and in proximal tubule cytosol. A translocation assay showed that ATP-driven endosomal acidification triggered the recruitment of ARNO and Arf6 from proximal tubule cytosol to endosomal membranes. The translocation of both ARNO and Arf6 was reversed by V-type ATPase inhibitors and by uncouplers of endosomal intralumenal pH, and was correlated with the magnitude of intra-endosomal acidification. Our data suggest that V-type ATPase-dependent acidification stimulates the selective recruitment of ARNO and Arf6 to proximal tubule early endosomes. This mechanism may play an important role in the pH-dependent regulation of receptor-mediated endocytosis in proximal tubules in situ.     

COPI budding within the Golgi stack

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

Two lipid-packing sensor motifs contribute to the sensitivity of ArfGAP1 to membrane curvature

ArfGAP1 (Arf GTPase activating protein 1) controls the cycling of the COPI coat on Golgi membranes by catalyzing GTP hydrolysis in the small G protein Arf1. ArfGAP1 contains a central motif named ALPS (ArfGAP1 lipid-packing sensor) that adsorbs preferentially onto highly curved membranes. This motif allows coupling of the rate of GTP hydrolysis in Arf1 with membrane curvature induced by the COPI coat. Upon membrane adsorption, the ALPS motif folds into an amphipathic alpha-helix. This helix contrasts from a classical membrane-adsorbing helix in the abundance of S and T residues and the paucity of charged residues in its polar face. We show here that ArfGAP1 contains a second motif with similar physicochemical properties. This motif, ALPS2, also forms an amphipathic alpha-helix at the surface of small vesicles and contributes to the Golgi localization of ArfGAP1 in vivo. Using several quantitative assays, we determined the relative contribution of the two ALPS motifs in the recognition of liposomes of defined curvature and composition. Our results show that ALPS1 is the primary determinant of the interaction of ArfGAP1 with lipid membranes and that ALPS2 reinforces this interaction 40-fold. Furthermore, our results suggest that depending on the engagement of one or two functional ALPS motifs, ArfGAP1 can respond to a wide range of membrane curvature and can adapt to lipid membranes of various acyl chain compositions.