Topology of amphipathic motifs mediating Golgi localization in ArfGAP1 and its splice isoforms

The interaction of the Arf1-directed GTPase-activating protein ArfGAP1 with the Golgi apparatus depends on motifs in its noncatalytic part that are unstructured in solution but are capable of folding into amphipathic helices in vitro upon interaction with poorly packed lipids. In previous studies a few hydrophobic residues that are critical for lipid binding and Golgi localization were identified, but the precise topology of the amphipathic motifs has not been determined. Here we present a detailed analysis of the Golgi targeting and in vitro folding features of the region encompassing the amphipathic motifs (residues 199-294). Point mutation analysis revealed that most hydrophobic residues within this region contribute to Golgi localization, whereas analysis by proline replacements and alanine insertions revealed that Golgi interaction depends on folding into two amphipathic helices with a short interrupting sequence. Analysis of splice isoforms containing 10-residue in-frame insertions within their first amphipathic motifs revealed that the insertion causes a truncation of the amphipathic helix that does not extend beyond the insertion sequence. Lastly, a lysine replacement mutant recently reported to bind to negatively charged liposomes in a curvature-independent manner showed normal cellular distribution, suggesting that Golgi targeting of Arf-GAP1 may involve factors other than sensing lipid packing.     

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.     

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.     

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.     

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.     

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.     

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.     

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 responds to membrane curvature through the folding of a lipid packing sensor motif

ArfGAP1 promotes GTP hydrolysis in Arf1, a small G protein that interacts with lipid membranes and drives the assembly of the COPI coat in a GTP-dependent manner. The activity of ArfGAP1 increases with membrane curvature, suggesting a negative feedback loop in which COPI-induced membrane deformation determines the timing and location of GTP hydrolysis within a coated bud. Here we show that a central sequence of about 40 amino acids in ArfGAP1 acts as a lipid-packing sensor. This ALPS motif (ArfGAP1 Lipid Packing Sensor) is also found in the yeast homologue Gcs1p and is necessary for coupling ArfGAP1 activity with membrane curvature. The ALPS motif binds avidly to small liposomes and shows the same hypersensitivity on liposome radius as full-length ArfGAP1. Site-directed mutagenesis, limited proteolysis and circular dichroism experiments suggest that the ALPS motif, which is unstructured in solution, inserts bulky hydrophobic residues between loosely packed lipids and forms an amphipathic helix on highly curved membranes. This helix differs from classical amphipathic helices by the abundance of serine and threonine residues on its polar face.     

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.     

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.     

The Sec7 Arf-GEF is recruited to the trans-Golgi network by positive feedback

Arf GTPases are key regulators of both retrograde and anterograde traffic at the Golgi complex. The Golgi-localized Arf activators, Arf-GEFs (guanine exchange factor) of the BIG/GBF family, are poorly understood in terms of both their regulatory and localization mechanisms. We have performed a detailed kinetic characterization of a functional Golgi Arf-GEF, the trans-Golgi network (TGN)-localized Sec7 protein from yeast. We demonstrate that Sec7 is regulated by both autoinhibition and positive feedback. We show that positive feedback arises through the stable recruitment of Sec7 to membranes via its HDS1 domain by interaction with its product, activated Arf1. This interaction mediates localization of Sec7 to the TGN, because deletion of the HDS1 domain or mutation of the HDS1 domain in combination with deletion of Arf1 significantly increases cytoplasmic localization of Sec7. Our results lead us to propose a model in which Arf-GEF recruitment is linked to Golgi maturation via Arf1 activation.     

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.     

A common peptide stretch among enzymes localized to the Golgi apparatus: structural similarity of Golgi-associated glycosyltransferases

A common peptide motif has been discovered among a series of Golgi-localized glycosyltransferases. The peptide stretch, (Ser/Thr)-X-(Glu/Gln)-(Arg/Lys), always occurs near a hydrophobic domain close to the N-terminus of these enzymes which is believed to anchor them to the membrane lipid bilayer (Paulson and Colley, J. Biol. Chem., 264, 17615-17618, 1989). The finding that this similar peptide motif is not associated with catalytic activity of these enzymes, and its presence near the hydrophobic domain suggest that the stretch may be involved in localization of these enzymes to the Golgi apparatus.     

The GAP Domain and the SNARE, Coatomer and Cargo Interaction Region of the ArfGAP2/3 Glo3 are Sufficient for Glo3 Function

The ArfGAP Glo3 is required for coat protein I vesicle generation in the Golgi–endoplasmic reticulum (ER) shuttle. The best-understood role of Glo3 is the stimulation of the GTPase activity of Arf1. In this study, we characterized functional domains of the ArfGAP Glo3 and identified an interaction interface for coatomer, SNAREs and cargo in the central region of Glo3 (BoCCS region). The GAP domain together with the BoCCS region is necessary and sufficient for all vital Glo3 functions. Expression of a truncated Glo3 lacking the GAP domain results in a dominant negative growth phenotype in glo3 Δ cells at 37°C. This phenotype was alleviated by mutating either the BoCCS region or the Glo3 regulatory motif (GRM), or by overexpression of ER–Golgi SNAREs or the ArfGAP Gcs1. The GRM is not essential for Glo3 function; it may act as an intrinsic sensor coupling GAP activity to SNARE binding to avoid dead-end complex formation at the Golgi membrane. Our data suggest that membrane-interaction modules and cargo-sensing regions have evolved independently in ArfGAP1s versus ArfGAP2/3s.     

Mutational analysis of the Arf1*GTP/Arf GAP interface reveals an Arf1 mutant that selectively affects the Arf GAP ASAP1

Arf1 is a GTP binding protein that functions at a number of cellular sites to control membrane traffic and actin remodeling. Arf1 is regulated by site-specific GTPase-activating proteins (GAPs). The combined results of crystallographic and biochemical studies have led to the proposal that Arf1 GAPs differ in the specific interface formed with Arf1. To test this hypothesis, we have used mutagenesis to examine the interaction of three Arf GAPs (ASAP1, AGAP1, and ArfGAP1) with switch 1, switch 2, and alpha helix3 of Arf1. The GAPs were similar in being affected by mutations in switch 1 and 2. However, effects of a mutation within alpha helix3 and specific mutations within switch 1 and 2 differed among the GAPs. The largest differences were observed with a change of isoleucine 46 to aspartate ([I46D]Arf1), which reduced ASAP1-induced catalysis by approximately 10,000-fold but had a 3-fold effect on AGAP1. The reduction was due to an isolated effect on the catalytic rate, k(cat). In vivo [I46D]Arf1 had no detectable effect on the Golgi apparatus but, instead, functioned as a constitutively active mutant in the cell periphery, affecting the localization of ASAP1 and paxillin. Based on our results, we conclude that the contribution of specific residues within switch 1 of Arf to binding and achieving a transition state toward GTP hydrolysis differs among Arf GAPs.     

Sorting of Golgi resident proteins into different subpopulations of COPI vesicles: a role for ArfGAP1.

We present evidence for two subpopulations of coatomer protein I vesicles, both containing high amounts of Golgi resident proteins but only minor amounts of anterograde cargo. Early Golgi proteins p24alpha2, beta1, delta1, and gamma3 are shown to be sorted together into vesicles that are distinct from those containing mannosidase II, a glycosidase of the medial Golgi stack, and GS28, a SNARE protein of the Golgi stack. Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride. Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes. We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.     

ArfGAP1 restricts Mycobacterium tuberculosis entry by controlling the actin cytoskeleton

The interaction of Mycobacterium tuberculosis (Mtb) with pulmonary epithelial cells is critical for early stages of bacillus colonization and during the progression of tuberculosis. Entry of Mtb into epithelial cells has been shown to depend on F‐actin polymerization, though the molecular mechanisms are still unclear. Here, we demonstrate that mycobacterial uptake into epithelial cells requires rearrangements of the actin cytoskeleton, which are regulated by ADP‐ribosylation factor 1 (Arf1) and phospholipase D1 (PLD1), and is dependent on the M3 muscarinic receptor (M₃R). We show that this pathway is controlled by Arf GTPase‐activating protein 1 (ArfGAP1), as its silencing has an impact on actin cytoskeleton reorganization leading to uncontrolled uptake and replication of Mtb. Furthermore, we provide evidence that this pathway is critical for mycobacterial entry, while the cellular infection with other pathogens, such as Shigella flexneri and Yersinia pseudotuberculosis, is not affected. Altogether, these results reveal how cortical actin plays the role of a barrier to prevent mycobacterial entry into epithelial cells and indicate a novel role for ArfGAP1 as a restriction factor of host–pathogen interactions.     

ArfGAP1 function in COPI mediated membrane traffic: currently debated models and comparison to other coat-binding ArfGAPs

The ArfGAPs are a family of proteins containing an ArfGAP catalytic domain that induces the hydrolysis of GTP bound to the small guanine nucleotide binding-protein ADP-ribosylation factor (Arf). Functional models for Arfs, which are regulators of membrane traffic, are based on the idea that guanine nucleotide-binding proteins function as switches: Arf with GTP bound is active and binds to effector proteins; the conversion of GTP to GDP inactivates Arf. The cellular activities of ArfGAPs have been examined primarily as regulatory proteins that inactivate Arf; however, Arf function in membrane traffic does not strictly adhere to the concept of a simple switch, adding complexity to models explaining the role of ArfGAPs. Here, we review the literature addressing the function Arf and ArfGAP1 in COPI mediated transport, focusing on two critical and integrated functions of membrane traffic, cargo sorting and vesicle coat polymerization. We briefly discuss other ArfGAPs that may have similar function in Arf-dependent membrane traffic outside the ER-Golgi.     

ArfGAP1 inhibits mTORC1 lysosomal localization and activation

The mammalian target of rapamycin complex 1 (mTORC1) integrates nutrients, growth factors, stress, and energy status to regulate cell growth and metabolism. Amino acids promote mTORC1 lysosomal localization and subsequent activation. However, the subcellular location or interacting proteins of mTORC1 under amino acid‐deficient conditions is not completely understood. Here, we identify ADP‐ribosylation factor GTPase‐activating protein 1 (ArfGAP1) as a crucial regulator of mTORC1. ArfGAP1 interacts with mTORC1 in the absence of amino acids and inhibits mTORC1 lysosomal localization and activation. Mechanistically, the membrane curvature‐sensing amphipathic lipid packing sensor (ALPS) motifs that bind to vesicle membranes are crucial for ArfGAP1 to interact with and regulate mTORC1 activity. Importantly, ArfGAP1 represses cell growth through mTORC1 and is an independent prognostic factor for the overall survival of pancreatic cancer patients. Our study identifies ArfGAP1 as a critical regulator of mTORC1 that functions by preventing the lysosomal transport and activation of mTORC1, with potential for cancer therapeutics.