ArfGAP1 responds to membrane curvature through the folding of a lipid packing sensor motif

Abstract

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 COPIinduced 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. Sitedirected 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.

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.…”

“…In turn, this stress triggers a response characterized by the upregulation of a fatty acid desaturase (Ole1 in yeast) and the arrest of cholesterol synthesis [9][10][11][12] . In addition, several peripheral proteins involved in various functions (lipid transfer, autophagy, nuclear pore formation, vesicle tethering and protein coat dynamics) seem to recognize lipid-packing defects by adsorbing preferentially to positively curved membranes through the insertion of long amphipathic sequences 4,[13][14][15][16][17] .…”

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.…”

COPI Budding within the Golgi Stack

“…This sensitivity is based on the presence of a motif termed ALPS (for ArfGAP1 Lipid Packing Sensor) that on membrane contact forms an amphipathic helix that inserts bulky hydrophobic side chains between the loosely packed lipid head-groups of the outer leaflet of a positively curved membrane (Bigay et al 2005).…”