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. 2008 Sep 1;414(2):189-94.
doi: 10.1042/BJ20081237.

Arf family GTP loading is activated by, and generates, positive membrane curvature

Richard Lundmark  1 , Gary J Doherty, Yvonne Vallis, Brian J Peter, Harvey T McMahon
Affiliations

Arf family GTP loading is activated by, and generates, positive membrane curvature

Richard Lundmark et al. Biochem J. .

Abstract

Small G-proteins belonging to the Arf (ADP-ribosylation factor) family serve as regulatory proteins for numerous cellular processes through GTP-dependent recruitment of effector molecules. In the present study we demonstrate that proteins in this family regulate, and are regulated by, membrane curvature. Arf1 and Arf6 were shown to load GTP in a membrane-curvature-dependent manner and stabilize, or further facilitate, changes in membrane curvature through the insertion of an amphipathic helix.

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Figures

Figure 1
Figure 1. Arfs induce membrane curvature in a GTP-dependent manner
(A) Helical-wheel representation of amino acids (a.a.) 2–13 in Arf1 (top wheel) and 2–11 in Arf6 (bottom wheel). Hydrophobic and charged amino acids are highlighted by dark grey circles or light grey circles respectively. Myristoylation of the second residue (glycine) is depicted by the long oval containing the word ‘myristoyl’. (B) Sequence alignment of the N-terminal amino acids of Arf/Arl family members. Hydrophobic amino acids are highlighted in dark grey. (C) Liposome co-sedimentation assays. Liposomes generated from brain-derived lipids (Folch fraction I) were incubated with the indicated proteins and GTP or GDP before centrifugation and analysis of supernatants (S) and pellets (P) by SDS/PAGE and Coomassie Blue staining. A control experiment where protein was incubated in the absence of liposomes is shown. The proteins used were myristoylated Arf1 purified from Sf9 insect cells [myr-Arf1 (Sf9)], Arf1 purified from bacteria [Arf1 (bacteria)], myristoylated Arf1 purified from bacteria co-expressing N-myristoyltransferase [myr-Arf1 (bacteria)] and N-terminally truncated Arf1 purified from bacteria [ΔN-term Arf1 (bacteria)]. (D) Electron micrographs of negatively stained 800 nm liposomes incubated in the presence of equal concentrations of proteins (10 μM) and nucleotides (100 μM) as indicated. Arrowheads indicate tubules that are approx. 45 nm in diameter and asterisks indicate tubules that are 13–17 nm wide. Scale bars represent 100 nm. Abbreviation: myr, myristoylated.
Figure 2
Figure 2. Arf-family proteins are activated by high positive membrane curvature
(A)–(C) Indicated proteins were incubated together with or without liposomes (−lip) of the indicated diameters and GTP. GTP loading was analysed in real time by measuring tryptophan fluorescence (see the Experimental section). Fluorescence is presented as a percentage of the value at 1000 s for 50 nm liposomes and Arf1 and Arf6 respectively. Insets in (A) and (B) shows liposome binding assays of Arf1 (A) and Arf6 (B) in the absence or presence of liposomes of different diameters. S, supernatant; P, pellet. Results shown in (A)–(C) are representative of three independent experiments with the same set of proteins and lipids. (D) Electron micrographs of negatively stained liposomes incubated together with GTP-loaded Arf6 (left) and Arf6 K3E (right). The scale bar represents 100 nm. The asterisks and arrowheads have the same meaning as in Figure 1. myr, myristoylated. (E) Epifluorescent micrograph of a HeLa cell expressing a myc-tagged constitutively active Arf6 mutant (Arf6 Q67L-myc). Arrowheads indicate tubular localization. The scale bar represents 5 μm. (F) Schematic model of the GTP-dependent role of Arfs as curvature sensors at the rim of a budding vesicle. Arfs loaded with GDP (ARF–GDP) display a transient membrane binding while Arfs bound to GTP (ARF–GTP) stay membrane-associated within the diffusion barrier generated by positive membrane curvature.
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