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.     

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.     

The HOPS/Class C Vps Complex Tethers High‐Curvature Membranes via a Direct Protein–Membrane Interaction

Membrane tethering is a physical association of two membranes before their fusion. Many membrane tethering factors have been identified, but the interactions that mediate inter‐membrane associations remain largely a matter of conjecture. Previously, we reported that the homotypic fusion and protein sorting/Class C vacuolar protein sorting (HOPS/Class C Vps) complex, which has two binding sites for the yeast vacuolar Rab GTPase Ypt7p, can tether two low‐curvature liposomes when both membranes bear Ypt7p. Here, we show that HOPS tethers highly curved liposomes to Ypt7p‐bearing low‐curvature liposomes even when the high‐curvature liposomes are protein‐free. Phosphorylation of the curvature‐sensing amphipathic lipid‐packing sensor (ALPS) motif from the Vps41p HOPS subunit abrogates tethering of high‐curvature liposomes. A HOPS complex without its Vps39p subunit, which contains one of the Ypt7p binding sites in HOPS, lacks tethering activity, though it binds high‐curvature liposomes and Ypt7p‐bearing low‐curvature liposomes. Thus, HOPS tethers highly curved membranes via a direct protein–membrane interaction. Such high‐curvature membranes are found at the sites of vacuole tethering and fusion. There, vacuole membranes bend sharply, generating large areas of vacuole‐vacuole contact. We propose that HOPS localizes via the Vps41p ALPS motif to these high‐curvature regions. There, HOPS binds via Vps39p to Ypt7p in an apposed vacuole membrane.      

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.     

Arf1 and Membrane Curvature Cooperate to Recruit Arfaptin2 to Liposomes

Arfaptin2 contains a Bin/Amphiphysin/Rvs (BAR) domain and directly interacts with proteins of the Arf/Arl family in their active GTP-bound state. It has been proposed that BAR domains are able to sense membrane curvature and to induce membrane tubulation. We report here that active Arf1 is required for the recruitment of Arfaptin2 to artificial liposomes mimicking the Golgi apparatus lipid composition. The Arf1-dependent recruitment of Arfaptin2 increases with membrane curvature, while the recruitment of Arf1 itself is not sensitive to curvature. At high protein concentrations, the binding of Arfaptin2 induces membrane tubulation. Finally, membrane-bound Arfaptin2 is released from the liposome when ArfGAP1 catalyzes the hydrolysis of GTP to GDP in Arf1. These results show that both Arf1 activation and high membrane curvature are required for efficient recruitment of Arfaptin2 to membranes.     

Sar1p N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle

Secretory proteins traffic from the ER to the Golgi via COPII-coated transport vesicles. The five core COPII proteins (Sar1p, Sec23/24p, and Sec13/31p) act in concert to capture cargo proteins and sculpt the ER membrane into vesicles of defined geometry. The molecular details of how the coat proteins deform the lipid bilayer into vesicles are not known. Here we show that the small GTPase Sar1p directly initiates membrane curvature during vesicle biogenesis. Upon GTP binding by Sar1p, membrane insertion of the N-terminal amphipathic alpha helix deforms synthetic liposomes into narrow tubules. Replacement of bulky hydrophobic residues in the alpha helix with alanine yields Sar1p mutants that are unable to generate highly curved membranes and are defective in vesicle formation from native ER membranes despite normal recruitment of coat and cargo proteins. Thus, the initiation of vesicle budding by Sar1p couples the generation of membrane curvature with coat-protein assembly and cargo capture.     

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.     

Mechanisms for the modulation of membrane bilayer properties by amphipathic helical peptides

The amphipathic helix, in which hydrophobia and hydrophilic residues are grouped on opposing faces, is a structural mot if found in many peptides and proteins that bind to membranes. One of the physical properties of membranes that can be altered by the binding of amphipathic helices is membrane monolayer curvature strain. Class A amphipathic helices, which are present in exchangeable plasma lipoproteins, can stabilize membranes by reducing negative monolayer curvature strain; proline-punctuated class A amphipathic helical segments are particularly effective in this regard. This property is suggested to be associated with some of the beneficial biological effects of this protein. On the other hand, lytic amphipathic helical peptides can act by increasing negative curvature strain or by forming pores composed of helical clusters. Thus, different amphipathic helical peptides can be membrane stabilizing or be lytic to membranes, depending on the structural motif of the helix, which in turn determines the nature of its association with membranes. Features of these peptides that are responsible for their specific properties are discussed. © 1994 John Wiley & Sons, Inc.     

Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor

Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation.     

Sensing Membrane Stresses by Protein Insertions

Protein domains shallowly inserting into the membrane matrix are ubiquitous in peripheral membrane proteins involved in various processes of intracellular membrane shaping and remodeling. It has been suggested that these domains sense membrane curvature through their preferable binding to strongly curved membranes, the binding mechanism being mediated by lipid packing defects. Here we make an alternative statement that shallow protein insertions are universal sensors of the intra-membrane stresses existing in the region of the insertion embedding rather than sensors of the curvature per se. We substantiate this proposal computationally by considering different independent ways of the membrane stress generation among which some include changes of the membrane curvature whereas others do not alter the membrane shape. Our computations show that the membrane-binding coefficient of shallow protein insertions is determined by the resultant stress independently of the way this stress has been produced. By contrast, consideration of the correlation between the insertion binding and the membrane curvature demonstrates that the binding coefficient either increases or decreases with curvature depending on the factors leading to the curvature generation. To validate our computational model, we treat quantitatively the experimental results on membrane binding by ALPS1 and ALPS2 motifs of ArfGAP1.     

Mechanism of endophilin N-BAR domain-mediated membrane curvature

Endophilin-A1 is a BAR domain-containing protein enriched at synapses and is implicated in synaptic vesicle endocytosis. It binds to dynamin and synaptojanin via a C-terminal SH3 domain. We examine the mechanism by which the BAR domain and an N-terminal amphipathic helix, which folds upon membrane binding, work as a functional unit (the N-BAR domain) to promote dimerisation and membrane curvature generation. By electron paramagnetic resonance spectroscopy, we show that this amphipathic helix is peripherally bound in the plane of the membrane, with the midpoint of insertion aligned with the phosphate level of headgroups. This places the helix in an optimal position to effect membrane curvature generation. We solved the crystal structure of rat endophilin-A1 BAR domain and examined a distinctive insert protruding from the membrane interaction face. This insert is predicted to form an additional amphipathic helix and is important for curvature generation. Its presence defines an endophilin/nadrin subclass of BAR domains. We propose that N-BAR domains function as low-affinity dimers regulating binding partner recruitment to areas of high membrane curvature.     

The hydrophobic insertion mechanism of membrane curvature generation by proteins

A wide spectrum of intracellular processes is dependent on the ability of cells to dynamically regulate membrane shape. Membrane bending by proteins is necessary for the generation of intracellular transport carriers and for the maintenance of otherwise intrinsically unstable regions of high membrane curvature in cell organelles. Understanding the mechanisms by which proteins curve membranes is therefore of primary importance. Here we suggest, for the first time to our knowledge, a quantitative mechanism of lipid membrane bending by hydrophobic or amphipathic rodlike inclusions which simulate amphipathic α-helices—structures shown to sculpt membranes. Considering the lipid monolayer matrix as an anisotropic elastic material, we compute the intramembrane stresses and strains generated by the embedded inclusions, determine the resulting membrane shapes, and the accumulated elastic energy. We characterize the ability of an inclusion to bend membranes by an effective spontaneous curvature, and show that shallow rodlike inclusions are more effective in membrane shaping than are lipids having a high propensity for curvature. Our computations provide experimentally testable predictions on the protein amounts needed to generate intracellular membrane shapes for various insertion depths and membrane thicknesses. We also predict that the ability of N-BAR domains to produce membrane tubules in vivo can be ascribed solely to insertion of their amphipathic helices.     

Golgi localization determinants in ArfGAP1 and in new tissue-specific ArfGAP1 isoforms

The Arf1-directed GTPase-activating protein ArfGAP1 is a Golgi-localized protein that controls the dynamics of the COPI coat of carriers that mediate transport in the endoplasmic reticulum-Golgi shuttle. Previously the interaction of ArfGAP1 with the Golgi was allocated to a portion of the non-catalytic, carboxyl part of the protein, but the mechanism of this interaction has not been established. In this study we identify a short stretch in the non-catalytic part of ArfGAP1 (residues 204-214) in which several hydrophobic residues contribute to Golgi localization. Even single alanine replacement of two of these residues (Leu-207 and Trp-211) strongly diminished Golgi localization. Mutations in the hydrophobic residues also diminished the in vitro activity of ArfGAP1 on Arf1 bound to Golgi membranes. The stretch containing the hydrophobic residues was recently shown to mediate the binding of ArfGAP1 to loosely packed lipids of highly curved liposomes (Bigay, J., Casella, J. F., Drin, G., Mesmin, B., and Antonny, B. (2005) EMBO J. 24, 2244-2253). Whereas short fragments containing the hydrophobic stretch were not Golgi-localized, a proximal 10-residue in-frame insertion that is present in new ArfGAP1 isoforms that we identified in brain and heart tissues could confer Golgi localization on these fragments. This localization was abrogated by alanine replacement of residues Phe-240 or Trp-241 of the insertion sequence but not by their replacement with leucines. Our findings indicate that ArfGAP1 interacts with the Golgi through multiple hydrophobic motifs and that alternative modes of interaction may exist in tissue-specific ArfGAP1 isoforms.     

Amphipathic-Lipid-Packing-Sensor interactions with lipids assessed by atomistic molecular dynamics

The Amphipathic-Lipid-Packing-Sensor (ALPS) motif targets the protein ArfGAP1 to curved membranes during vesicle formation in the Golgi apparatus. ALPS specifically recognizes lipid packing defects due to the positive curvature of budding vesicles. In this work we assessed the microscopic interactions between ALPS and two phospholipid membranes at different degrees of lipid packing by explicit molecular dynamics (MD). Simulations were performed within loosely packed membranes composed of a mixture of dioleoylphosphatidylcholine (DOPC)/dioleoylglycerol (DOG) at a molar ratio 85:15. Some other simulations were performed in pure DOPC for which lipid packing is tighter. We show that the presence of DOG causes packing defects at the phosphate level and thereby modifies some properties of the bilayer. This leads to a higher hydration of the lipid headgroups. When embedded in a membrane with such defects, ALPS displays a higher degree of conformational flexibility than in a more packed membrane. We propose that lipid packing sensing by ALPS may have an entropic origin and that its flexibility is a key feature.     

Factors influencing local membrane curvature induction by N-BAR domains as revealed by molecular dynamics simulations

N-BAR domains are protein modules that bind to and induce curvature in membranes via a charged concave surface and N-terminal amphipathic helices. Recently, molecular dynamics simulations have demonstrated that the N-BAR domain can induce a strong local curvature that matches the curvature of the BAR domain surface facing the bilayer. Here we present further molecular dynamics simulations that examine in greater detail the roles of the concave surface and amphipathic helices in driving local membrane curvature. We find that the strong curvature induction observed in our previous simulations requires the stable presentation of the charged concave surface to the membrane and is not driven by the membrane-embedded amphipathic helices. Nevertheless, without these amphipathic helices embedded in the membrane, the N-BAR domain does not maintain a close association with the bilayer, and fails to drive membrane curvature. Increasing the membrane negative charge through the addition of PIP₂ facilitates closer association with the membrane in the absence of embedded helices. At sufficiently high concentrations, amphipathic helices embedded in the membrane drive membrane curvature independently of the BAR domain.     

The lamin B receptor of the inner nuclear membrane undergoes mitosis-specific phosphorylation and is a substrate for p34cdc2-type protein kinase.

The lamin B receptor (LBR) is an integral protein of the inner nuclear membrane that interacts with lamin B in vitro. If contains a 204-amino acid nucleoplasmic amino-terminal domain and a hydrophobic carboxyl-terminal domain with eight putative transmembrane segments. We found cell cycle-dependent phosphorylation of LBR using phosphoamino acid analysis and phosphopeptide mapping of in vivo 32P-labeled LBR immunoprecipitated from chicken cells in interphase and arrested in mitosis. LBR was phosphorylated only on serine residues in interphase and on serine and threonine residues in mitosis. Some serine residues phosphorylated in interphase were not phosphorylated in mitosis. To identify a threonine residue specifically phosphorylated in mitosis and the responsible protein kinase, wild-type and mutant LBR nucleoplasmic domain fusion proteins were phosphorylated in vitro by p34cdc2-type protein kinase. Comparisons of phosphopeptide maps to those of in vivo 32P-labeled mitotic LBR showed that Thr188 is likely to be phosphorylated by this enzyme during mitosis. These phosphorylation/dephosphorylation events may be responsible for some of the changes in the interaction between the nuclear lamina and the inner nuclear membrane that occur during mitosis.     

Roles of Amphipathic Helices and the Bin/Amphiphysin/Rvs (BAR) Domain of Endophilin in Membrane Curvature Generation

Control of membrane curvature is required in many important cellular processes, including endocytosis and vesicular trafficking. Endophilin is a bin/amphiphysin/rvs (BAR) domain protein that induces vesicle formation by promotion of membrane curvature through membrane binding as a dimer. Using site-directed spin labeling and EPR spectroscopy, we show that the overall BAR domain structure of the rat endophilin A1 dimer determined crystallographically is maintained under predominantly vesiculating conditions. Spin-labeled side chains on the concave surface of the BAR domain do not penetrate into the acyl chain interior, indicating that the BAR domain interacts only peripherally with the surface of a curved bilayer. Using a combination of EPR data and computational refinement, we determined the structure of residues 63–86, a region that is disordered in the crystal structure of rat endophilin A1. Upon membrane binding, residues 63–75 in each subunit of the endophilin dimer form a slightly tilted, amphipathic α-helix that directly interacts with the membrane. In their predominant conformation, these helices are located orthogonal to the long axis of the BAR domain. In this conformation, the amphipathic helices are positioned to act as molecular wedges that induce membrane curvature along the concave surface of the BAR domain.     

IFITM3 requires an amphipathic helix for antiviral activity

Interferon‐induced transmembrane protein 3 (IFITM3) is a cellular factor that blocks virus fusion with cell membranes. IFITM3 has been suggested to alter membrane curvature and fluidity, though its exact mechanism of action is unclear. Using a bioinformatic approach, we predict IFITM3 secondary structures and identify a highly conserved, short amphipathic helix within a hydrophobic region of IFITM3 previously thought to be a transmembrane domain. Consistent with the known ability of amphipathic helices to alter membrane properties, we show that this helix and its amphipathicity are required for the IFITM3‐dependent inhibition of influenza virus, Zika virus, vesicular stomatitis virus, Ebola virus, and human immunodeficiency virus infections. The homologous amphipathic helix within IFITM1 is also required for the inhibition of infection, indicating that IFITM proteins possess a conserved mechanism of antiviral action. We further demonstrate that the amphipathic helix of IFITM3 is required to block influenza virus hemagglutinin‐mediated membrane fusion. Overall, our results provide evidence that IFITM proteins utilize an amphipathic helix for inhibiting virus fusion.