Two Lipid-Packing Sensor Motifs Contribute to the Sensitivity of ArfGAP1 to Membrane Curvature

Abstract

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.

A sub-nanometre view of how membrane curvature and composition modulate lipid packing and protein recruitment

“…3a, where NBD-labelled ALPS1-ALPS2 was incubated with POPC liposomes of various radii. In agreement with previous observations 13,14,27,38 , membrane partitioning of this motif depended acutely on membrane curvature, with almost no binding on large (R4100 nm; curvatureo0.02 nm À 1 ) liposomes and complete binding on small (R ¼ 25 nm; curvature ¼ 0.08 nm À 1 ) liposomes. The curvature threshold for the binding of the ALPS1-ALPS2 tandem was lower than that observed for simple ALPS motifs (0.02 and 0.05-0.06 nm À 1 , respectively; Supplementary Fig.…”

“…Our model protein for the detection of lipid-packing defects is ArfGAP1, a protein that controls the lifetime of the small G protein Arf1 at the Golgi apparatus. Numerous in vitro studies suggest that ArfGAP1 recognizes membrane curvature through the adsorption of two amphipathic lipid-packing sensor motifs (ALPS) [13][14][15]27 . ALPS are short amphipathic helices that display a peculiar chemistry: their polar face is scarce in charged residues and abundant in serines and threonines, while their hydrophobic face mostly contains bulky hydrophobic residues.…”

“…2f and Supplementary Figs 2 and 3). The ALPS1-ALPS2 motif peptide of ArfGAP1 was covalently labelled with the environment-sensitive probe NBD (N,N 0 -dimethyl-N-(iodoacetyl)-N 0 -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine) and the increase in NBD fluorescence was used as an index of ALPS1-ALPS2 binding to large unilamellar vesicles 14,27 (see also Supplementary Figs 2 and 3). As shown in Fig.…”

“…The NBD labelled peptides were prepared as previously described 27 . Briefly, the ALPS1 and ALPS1-ALPS2 peptides (residues 192-257 and 192-304 of ArfGAP1, respectively) carrying the A236C mutation were expressed in Escherichia coli as glutathione S-transferase (GST) fusions.…”

Régulation du transport vésiculaire par la courbure membranaire

“…Comment ArfGAP1 reconnaît-il la courbure membranaire ? Nous avons identifié dans ArfGAP1 deux motifs d'une trentaine d'acides aminés que nous avons appelés ALPS (amphipathic lipid packing sensor) qui se structurent en hélice amphipathique et interagissent avec une membrane courbée [14,15]. À l'inverse de celles d'Arf1 et de Sar1, ces hélices n'induisent pas de courbure positive, mais la détectent.…”