Membrane curvature induced by Arf1-GTP is essential for vesicle formation - PubMed (original) (raw)

Membrane curvature induced by Arf1-GTP is essential for vesicle formation

Rainer Beck et al. Proc Natl Acad Sci U S A. 2008.

Abstract

The GTPase Arf1 is considered as a molecular switch that regulates binding and release of coat proteins that polymerize on membranes to form transport vesicles. Here, we show that Arf1-GTP induces positive membrane curvature and find that the small GTPase can dimerize dependent on GTP. Investigating a possible link between Arf dimerization and curvature formation, we isolated an Arf1 mutant that cannot dimerize. Although it was capable of exerting the classical role of Arf1 as a coat receptor, it could not mediate the formation of COPI vesicles from Golgi-membranes and was lethal when expressed in yeast. Strikingly, this mutant was not able to deform membranes, suggesting that GTP-induced dimerization of Arf1 is a critical step inducing membrane curvature during the formation of coated vesicles.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Arf1-mediated tubulation of synthetic lipid sheets. Lipids containing p23 lipopeptide were spotted on a glass surface and hydrated with buffer containing either GTP or GTP and the exchange factor ARNO (50 nM). After addition of myristoylated Arf1-GDP (1 μM), the lipid surface was observed by light microscopy. Shown are the reactions in the presence of Arf1-wt with GTP (A), Arf1-wt with GDP (B), and Arf1-Y35A with GTP (C). Scale bars, 5 μm.

Fig. 2.

Fig. 2.

Arf1 dimerizes on protein-free liposomes. Protein-free liposomes were incubated with full-length myristoylated Arf1, GTP, and the cross-linkers EMCS (lane 2) or BMH (lane 4) as indicated. After centrifugation, the liposome-bound material was analyzed by Western blotting with an anti-Arf1 antibody.

Fig. 3.

Fig. 3.

Biochemical characterization and modeling of an Arf1-GTP dimer. (A) The distance between the cysteines in an Arf1 dimer is optimal at 16Å. Protein-free liposomes were incubated with full-length myristoylated Arf1, GTPγS, and homobifunctional thiol-reactive cross-linkers of increasing spacer length as indicated (see Materials and Methods). The liposome-bound material was analyzed by Western blotting with an anti-Arf1 antibody. (B) Model of an Arf1 dimer. Shown are models of the Arf1-GTP dimer in a view toward the membrane (Upper) and from the membrane plane (Lower). The model is based on the crystal structure of monomeric Arf1-GTP (PDB ID code 1O3Y) and the constraints as described in the text. The binding site of the Arf-GAP1 catalytic domain and mapped coatomer interactions are shown schematically for one monomer. The two missing cross-linked peptides from the mass spectrometry experiment are colored in yellow (–36) and orange (152–178), and the critical tyrosyl residue in position 35, whose mutation to alanin results in a strong dimerization phenotype, is indicated. The cross-linked residues are connected by dashed lines, and their distances are indicated (the distance between Cys-159 and Lys-36 in the model is 10 Å, in accordance with the distance of the functional groups within the cross-linker EMCS). The interface coincides with the binding site of the N-terminal helix in Arf1-GDP and includes an intermolecular β-sheet completion formed by the exposed interswitch regions of Arf1-GTP.

Fig. 4.

Fig. 4.

Dimerization assay on Golgi membranes. Golgi-enriched membranes from rat liver were incubated with full-length myristoylated Arf1-wt, Arf1-Y35A, and GTPγS, and after recovery of membranes by centrifugation, the cross-linker BMH was added as described in Materials and Methods. Membrane-bound material was analyzed by Western blotting with an anti-Arf1 antibody.

Fig. 5.

Fig. 5.

The non dimerizing mutant Arf1-Y35A is lethal in yeast and does not support COPI-vesicle formation in vitro. (A) In vivo analysis of Arf1 mutants in yeast. Yeast strain NYY539 (_arf1_Δ, _arf2_Δ, pURA3-Arf1) was transformed with pRS315-Arf1, pRS315-arf1-S162A, and pRS315-arf1-Y35A. Transformants were spotted in 10−1 dilution series on SDC+FOA. Pictures were taken after 2 days or 5 days of incubation at 30°C. (B) In vitro formation of COPI vesicles in the presence of monomeric or dimeric Arf1. Golgi-enriched membranes were pretreated with 250 mM KCl and incubated with purified constituents of the COPI machinery, such as Arf1-wt or the monomeric mutant Y35A, and rabbit liver coatomer and GTP. COPI-coated vesicles were purified by sucrose density gradient centrifugation. For each sample, 5% of total input (I), and 50% of the purified vesicles (V) were analyzed by SDS/PAGE and Western blotting using antibodies against the coatomer subunit δ-COP and the transmembrane protein transferrin receptor (TfR). (C) Quantification of vesicle formation in the presence of monomeric or dimeric Arf1. Vesicles were generated either as described in Materials and Methods or after isopycnic density gradient centrifugation (33), followed by negative staining electron microscopy. Twenty meshes each of the samples were randomly chosen and the number of vesicles counted.

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