Differential roles of ArfGAP1, ArfGAP2, and ArfGAP3 in COPI trafficking

The formation of coat protein complex I (COPI)–coated vesicles is regulated by the small guanosine triphosphatase (GTPase) adenosine diphosphate ribosylation factor 1 (Arf1), which in its GTP-bound form recruits coatomer to the Golgi membrane. Arf GTPase-activating protein (GAP) catalyzed GTP hydrolysis in Arf1 triggers uncoating and is required for uptake of cargo molecules into vesicles. Three mammalian ArfGAPs are involved in COPI vesicle trafficking; however, their individual functions remain obscure. ArfGAP1 binds to membranes depending on their curvature. In this study, we show that ArfGAP2 and ArfGAP3 do not bind directly to membranes but are recruited via interactions with coatomer. In the presence of coatomer, ArfGAP2 and ArfGAP3 activities are comparable with or even higher than ArfGAP1 activity. Although previously speculated, our results now demonstrate a function for coatomer in ArfGAP-catalyzed GTP hydrolysis by Arf1. We suggest that ArfGAP2 and ArfGAP3 are coat protein–dependent ArfGAPs, whereas ArfGAP1 has a more general function.     

The ArfGAP Glo3 is required for the generation of COPI vesicles

The small GTPase Arf and coatomer (COPI) are required for the generation of retrograde transport vesicles. Arf activity is regulated by guanine exchange factors (ArfGEF) and GTPase-activating proteins (ArfGAPs). The ArfGAPs Gcs1 and Glo3 provide essential overlapping function for retrograde vesicular transport from the Golgi to the endoplasmic reticulum. We have identified Glo3 as a component of COPI vesicles. Furthermore, we find that a mutant version of the Glo3 protein exerts a negative effect on retrograde transport, even in the presence of the ArfGAP Gcs1. Finally, we present evidence supporting a role for ArfGAP protein in the generation of COPI retrograde transport vesicles.     

ACAP-A/B Are ArfGAP Homologs in Dictyostelium Involved in Sporulation but Not in Chemotaxis

Arfs and Arf GTPase-activating proteins (ArfGAPs) are regulators of membrane trafficking and actin dynamics in mammalian cells. In this study, we identified a primordial Arf, ArfA, and two ArfGAPs (ACAP-A/B) containing BAR, PH, ArfGAP and Ankyrin repeat domains in the eukaryote Dictyostelium discoideum. In vitro, ArfA has similar nucleotide binding properties as mammalian Arfs and, with GTP bound, is a substrate for ACAP-A and B. We also investigated the physiological functions of ACAP-A/B by characterizing cells lacking both ACAP-A and B. Although ACAP-A/B knockout cells showed no defects in cell growth, migration or chemotaxis, they exhibited abnormal actin protrusions and ∼50% reduction in spore yield. We conclude that while ACAP-A/B have a conserved biochemical mechanism and effect on actin organization, their role in migration is not conserved. The absence of an effect on Dictyostelium migration may be due to a specific requirement for ACAPs in mesenchymal migration, which is observed in epithelial cancer cells where most studies of mammalian ArfGAPs were performed.     

The carboxy-terminal region of SMAP2 directs subcellular localization as well as Arf protein specificity

Small G proteins play a central role in the organization of secretory and endocytotic pathways. The recruitment of some effectors, including vesicle coat proteins, is mediated by the ADP-ribosylation factor (Arf) family. Arf proteins have distinct subcellular localizations. ArfGAPs (Arf GTPase-activating proteins) regulate Arf GTPase activity. Thus, each ArfGAP is distinctly localized to allow it to maintain a specific interaction with its target Arf(s). However, the domains that regulate the subcellular localization of ArfGAPs and the way in which these subcellular localizations affect the target specificities of ArfGAPs remain unclear. Recently, we identified two novel ArfGAPs, SMAP1 (Small ArfGAP protein 1) and SMAP2. In the current study, we identified sequences in the carboxy-terminal region of SMAP2 that are critical for its specific subcellular localization and its specificity for Arf proteins.     

The yeast Arf GTPase-activating protein Age1 is regulated by phospholipase D for post-Golgi vesicular transport

Vesicular transport shuttles cargo among intracellular compartments. Several stages of vesicular transport are mediated by the small GTPase Arf, which is controlled in a cycle of GTP binding and hydrolysis by Arf guanine-nucleotide exchange factors and Arf GTPase-activating proteins (ArfGAPs), respectively. In budding yeast the Age2 + Gcs1 ArfGAP pair facilitates post-Golgi transport. We have found the AGE1 gene, encoding another ArfGAP, can in high gene-copy number alleviate the temperature sensitivity of cells carrying mutations affecting the Age2 + Gcs1 ArfGAP pair. Moreover, increased AGE1 gene dosage compensates for the complete absence of the otherwise essential Age2 + Gcs1 ArfGAP pair. Increased dosage of SFH2, encoding a phosphatidylinositol transfer protein, also allows cell growth in the absence of the Age2 + Gcs1 pair, but good growth in this situation requires Age1. The ability of Age1 to overcome the need for Age2 + Gcs1 depends on phospholipase D activity that regulates lipid composition. We show by direct assessment of Age1 ArfGAP activity that Age1 is regulated by lipid composition and can provide ArfGAP function for post-Golgi transport.     

Cell migration: GAPs between membrane traffic and the cytoskeleton

During cell migration, coordination between membrane traffic, cell substrate adhesion and actin reorganization is required for protrusive activity to occur at the leading edge. Actin organization is regulated by Rho family GTPases and, with a contribution from the endocytic cycle, serves to extend the cell front. The details of the molecular mechanisms that direct membrane traffic at sites of adhesion and rearrange actin at the cell edge are still unknown. However, recent findings show that a number of multi-domain proteins characterized by an ArfGAP domain interact with both actin-regulating and integrin-binding proteins, as well as affecting Rac-mediated protrusive activity and cell migration. Some of these proteins have been shown to localize to endocytic compartments and to have a role in regulating endocytosis. Given the participation of Arf proteins in regulating membrane traffic, one appealing hypothesis is that the ArfGAPs act as molecular devices that coordinate membrane traffic and cytoskeletal reorganization during cell motility.     

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.     

Systematic structure-function analysis of the small GTPase Arf1 in yeast

Members of the ADP-ribosylation factor (Arf) family of small GTPases are implicated in vesicle traffic in the secretory pathway, although their precise function remains unclear. We generated a series of 23 clustered charge-to-alanine mutations in the Arf1 protein of Saccharomyces cerevisiae to determine the portions of this protein important for its function in cells. These mutants display a number of phenotypes, including conditional lethality at high or low temperature, defects in glycosylation of invertase, dominant lethality, fluoride sensitivity, and synthetic lethality with the arf2 null mutation. All mutations were mapped onto the available crystal structures for Arf1p: Arf1p bound to GDP, to GTP, and complexed with the regulatory proteins ArfGEF and ArfGAP. From this systematic structure-function analysis we demonstrate that all essential mutations studied map to one hemisphere of the protein and provide strong evidence in support of the proposed ArfGEF contact site on Arf1p but minimal evidence in support of the proposed ArfGAP-binding site. In addition, we describe the isolation of a spatially distant intragenic suppressor of a dominant lethal mutation in the guanine nucleotide-binding region of Arf1p.     

Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18p/NSF

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are key components of the fusion machinery in vesicular transport and in homotypic membrane fusion. We previously found that ADP-ribosylation factor GTPase activating proteins (ArfGAPs) promoted a conformational change on SNAREs that allowed recruitment of the small GTPase Arf1p in stoichiometric amounts. Here, we show that the ArfGAP Gcs1p accelerates vesicle (v)-target membrane (t)-SNARE complex formation in vitro, indicating that ArfGAPs may act as folding chaperones. These SNARE complexes were resolved in the presence of ATP by the yeast homologues of alpha-soluble N-ethylmaleimide-sensitive factor attachment protein and N-ethylmaleimide-sensitive factor, Sec17p and Sec18p, respectively. In addition, Sec18p and Sec17p also recognized the "activated" SNAREs even when they were not engaged in v-t-SNARE complexes. Here again, the induction of a conformational change by ArfGAPs was essential. Surprisingly, recruitment of Sec18p to SNAREs did not require Sec17p or ATP hydrolysis. Moreover, Sec18p displaced prebound Arf1p from SNAREs, indicating that Sec18p may have more than one function: first, to ensure that all vesicle coat proteins are removed from the SNAREs before the engagement in a trans-SNARE complex; and second, to resolve cis-SNARE complexes after fusion has occurred.     

Kinetic analysis of Arf GAP1 indicates a regulatory role for coatomer

Arf GAPs are a family of enzymes that catalyze the hydrolysis of GTP bound to Arf. Arf GAP1 is one member of the family that has a critical role in membrane traffic at the Golgi apparatus. Two distinct models for the regulation of Arf GAP1 in membrane traffic have been proposed. In one model, Arf GAP1 functions in a ternary complex with coat proteins and is inhibited by cargo proteins. In another model, Arf GAP1 is recruited to a membrane surface that has defects created by the increased membrane curvature that accompanies transport vesicle formation. Here we have used kinetic and mutational analysis to test predictions of models of regulation of Arf GAP1. We found that Arf GAP1 has a similar affinity for Arf1.GTP as another Arf GAP, ASAP1, but the catalytic rate is approximately 0.5% that of ASAP1. Coatomer stimulated Arf GAP1 activity; however, different from that predicted from the current model, coatomer affected the K(m) and not the k(cat) values. Effects of most mutations in Arf GAP1 paralleled those in ASAP1. Mutation of an arginine that aligned with an arginine presumed to be catalytic in ASAP1 abrogated activity. Peptide from the cytoplasmic tail of cargo proteins inhibited Arf GAP1; however, the unrelated Arf GAP ASAP1 was also inhibited. The curvature of the lipid bilayer had a small effect on activity of Arf GAP1 under the conditions of our experiments. We conclude that coatomer is an allosteric regulator of Arf GAP1. The relevance of the results to the two models of Arf GAP1-mediated regulation of Arf1 is discussed.     

ArfGAP1 interacts with coat proteins through tryptophan-based motifs

The Arf1 GTPase-activating protein ArfGAP1 regulates vesicular traffic through the COPI system. This protein consists of N-terminal catalytic domain, lipid packing sensors (the ALPS motifs) in the central region, and a carboxy part of unknown function. The carboxy part contains several diaromatic sequences that are reminiscent of motifs known to interact with clathrin adaptors. In pull-down experiments using GST-fused peptides from rat ArfGAP1, a peptide containing a ³²⁹WETF sequence interacted strongly with clathrin adaptors AP1 and AP2, whereas a major coatomer-binding determinant was identified within the extreme carboxy terminal peptide (⁴⁰⁵AADEGWDNQNW). Mutagenesis and peptide competition experiments revealed that this determinant is required for coatomer binding to full-length ArfGAP1, and that interaction is mediated through the δ-subunit of the coatomer adaptor-like subcomplex. Evidence for a role of the carboxy motif in ArfGAP1-coatomer interaction in vivo is provided by means of a reporter fusion assay. Our findings point to mechanistic differences between ArfGAP1 and the other ArfGAPs known to function in the COPI system.     

Discrete Determinants in ArfGAP2/3 Conferring Golgi Localization and Regulation by the COPI Coat

From yeast to mammals, two types of GTPase-activating proteins, ArfGAP1 and ArfGAP2/3, control guanosine triphosphate (GTP) hydrolysis on the small G protein ADP-ribosylation factor (Arf) 1 at the Golgi apparatus. Although functionally interchangeable, they display little similarity outside the catalytic GTPase-activating protein (GAP) domain, suggesting differential regulation. ArfGAP1 is controlled by membrane curvature through its amphipathic lipid packing sensor motifs, whereas Golgi targeting of ArfGAP2 depends on coatomer, the building block of the COPI coat. Using a reporter fusion approach and in vitro assays, we identified several functional elements in ArfGAP2/3. We show that the Golgi localization of ArfGAP3 depends on both a central basic stretch and a carboxy-amphipathic motif. The basic stretch interacts directly with coatomer, which we found essential for the catalytic activity of ArfGAP3 on Arf1-GTP, whereas the carboxy-amphipathic motif interacts directly with lipid membranes but has minor role in the regulation of ArfGAP3 activity. Our findings indicate that the two types of ArfGAP proteins that reside at the Golgi use a different combination of protein–protein and protein–lipid interactions to promote GTP hydrolysis in Arf1-GTP.     

Role for a Cindr-Arf6 axis in patterning emerging epithelia

Patterning of the Drosophila pupal eye is characterized by precise cell movements. In this paper, we demonstrate that these movements require an Arf regulatory cycle that connects surface receptors to actin-based movement. dArf6 activity-regulated by the Arf GTPase-activating proteins (ArfGAPs) dAsap and dArfGAP3 and the Arf GTP exchange factors Schizo and dPsd-promoted large cellular extensions; time-lapse microscopy indicated that these extensions presage cell rearrangements into correct epithelial niches. During this process, the Drosophila eye also requires interactions between surface Neph1/nephrin adhesion receptors Roughest and Hibris, which bind the adaptor protein Cindr (CD2AP). We provide evidence that Cindr forms a physical complex with dArfGAP3 and dAsap. Our data suggest this interaction sequesters ArfGAP function to liberate active dArf6 elsewhere in the cell. We propose that a Neph1/nephrin-Cindr/ArfGAP complex accumulates to limit local Arf6 activity and stabilize adherens junctions. Our model therefore links surface adhesion via an Arf6 regulatory cascade to dynamic modeling of the cytoskeleton, accounting for precise cell movements that organize the functional retinal field. Further, we demonstrate a similar relationship between the mammalian Cindr orthologue CD2AP and Arf6 activity in cell motility assays. We propose that this Cindr/CD2AP-mediated regulation of Arf6 is a widely used mechanism in emerging epithelia.     

Distinct role of subcomplexes of the COPI coat in the regulation of ArfGAP2 activity

COPI vesicles serve for transport of proteins and membrane lipids in the early secretory pathway. Their coat protein (coatomer) is a heptameric complex that is recruited to the Golgi by the small GTPase Arf1. Although recruited en bloc, coatomer can be viewed as a stable assembly of an adaptin‐like tetrameric subcomplex (CM4) and a trimeric ‘cage’ subcomplex (CM3). Following recruitment, coatomer stimulates ArfGAP‐dependent GTP hydrolysis on Arf1. Here, we employed recombinant coatomer subcomplexes to study the role of coatomer components in the regulation of ArfGAP2, an ArfGAP whose activity is strictly coatomer‐dependent. Within CM4, we define a novel hydrophobic pocket for ArfGAP2 interaction on the appendage domain of γ₁‐COP. The CM4 subcomplex (but not CM3) is recruited to membranes through Arf1 and can subsequently recruit ArfGAP2. Neither CM3 nor CM4 in itself is effective in stimulating ArfGAP2 activity, but stimulation is regained when both subcomplexes are present. Our findings point to a distinct role of each of the two coatomer subcomplexes in the regulation of ArfGAP2‐dependent GTP hydrolysis on Arf1, where the CM4 subcomplex functions in GAP recruitment, while, similarly to the COPII system, the cage‐like CM3 subcomplex stimulates the catalytic reaction.     

Oligomerization and dissociation of AP-1 adaptors are regulated by cargo signals and by ArfGAP1-induced GTP hydrolysis

The mechanism of AP-1/clathrin coat formation was analyzed using purified adaptor proteins and synthetic liposomes presenting tyrosine sorting signals. AP-1 adaptors recruited in the presence of Arf1.GTP and sorting signals were found to oligomerize to high-molecular-weight complexes even in the absence of clathrin. The appendage domains of the AP-1 adaptins were not required for oligomerization. On GTP hydrolysis induced by the GTPase-activating protein ArfGAP1, the complexes were disassembled and AP-1 dissociated from the membrane. AP-1 stimulated ArfGAP1 activity, suggesting a role of AP-1 in the regulation of the Arf1 "GTPase timer." In the presence of cytosol, AP-1 could be recruited to liposomes without sorting signals, consistent with the existence of docking factors in the cytosol. Under these conditions, however, AP-1 remained monomeric, and recruitment in the presence of GTP was short-lived. Sorting signals allowed stable recruitment and oligomerization also in the presence of cytosol. These results suggest a mechanism whereby initial assembly of AP-1 with Arf1.GTP and ArfGAP1 on the membrane stimulates Arf1 GTPase activity, whereas interaction with cargo induces oligomerization and reduces the rate of GTP hydrolysis, thus contributing to efficient cargo sorting.     

Modifications to the C‐Terminus of Arf1 Alter Cell Functions and Protein Interactions

Arf family proteins are ≈21‐kDa GTP‐binding proteins that are critical regulators of membrane traffic and the actin cytoskeleton. Studies examining the complex signaling pathways underlying Arf action have relied on recombinant proteins comprised of Arf fused to epitope tags or proteins, such as glutathione S‐transferase or green fluorescent protein, for both cell‐based mammalian cell studies and bacterially expressed recombinant proteins for biochemical assays. However, the effects of such protein fusions on the biochemical properties relevant to the cellular function have been only incompletely studied at best. Here, we have characterized the effect of C‐terminal tagging of Arf1 on (i) function in Saccharomyces cerevisiae, (ii) in vitro nucleotide exchange and (iii) interaction with guanine nucleotide exchange factors and GTPase‐activating proteins. We found that the tagged Arfs were substantially impaired or altered in each assay, compared with the wild‐type protein, and these changes are certain to alter actions in cells. We discuss the results related to the interpretation of experiments using these reagents and we propose that authors and editors consistently adopt a few simple rules for describing and discussing results obtained with Arf family members that can be readily applied to other proteins.     

A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor

BACKGROUND: Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. RESULTS: ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. CONCLUSIONS: The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.     

Internalization of exogenous ADP-ribosylation factor 6 (Arf6) proteins into cells

Endogenous Arf6 is a myristoylated protein mainly involved in endosomal membrane traffic and structural organization at the plasma membrane. It has been shown that Arf6 mediates cancer cell invasion and shedding of plasma membrane microvesicles derived from tumor cells. In this article, we determined that Arf6 proteins both in the GDP and GTPγS bound forms can enter cells when simply added in the cell culture medium without requiring the myristoyl group. The GTPγS bound can enter cells at a faster rate than the GDP-bound Arf6. Despite the role of the endogenous Arf6 in endocytosis and membrane trafficking, the internalization of exogenous Arf6 may involve non-endocytic processes. As protein therapeutics is becoming important in medicine, we examined the effect of the uptake of Arf6 proteins on cellular functions and determined that exogenous Arf6 inhibits proliferation, invasion, and migration of cells. Future studies of the internalization of Arf6 mutants will reveal key residues that play a role in the internalization of Arf6 and its interaction and possible structural conformations bound to the plasma membrane.     

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.