Myristoylation-facilitated Binding of the G Protein ARF1 to Membrane Phospholipids Is Required for Its Activation by a Soluble Nucleotide Exchange Factor

doi:10.1074/jbc.271.3.1573

Myristoylation-facilitated Binding of the G Protein ARF1 to Membrane Phospholipids Is Required for Its Activation by a Soluble Nucleotide Exchange Factor (*)

From the From CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France

**To whom correspondence should be addressed. Tel.: 33-93-95-77-71; Fax: 33-93-95-77-10.

Abstract

We have investigated the role of N-myristoylation in the activation of bovine ADP-ribosylation factor 1 (ARF1). We previously showed that myristoylation allows some spontaneous GDP-to-GTP exchange to occur on ARF1 at physiological Mg levels in the presence of phospholipid vesicles (Franco, M., Chardin, P., Chabre, M., and Paris, S.(1995) J. Biol. Chem. 270, 1337-1341). Here, we report that this basal nucleotide exchange can be accelerated (by up to 5-fold) by addition of a soluble fraction obtained from bovine retinas. This acceleration is totally abolished by brefeldin A (IC = 2 μM) and by trypsin treatment of the retinal extract, as expected for an ARF-specific guanine nucleotide exchange factor. To accelerate GDP release from ARF1, this soluble exchange factor absolutely requires myristoylation of ARF1 and the presence of phospholipid vesicles. The retinal extract also stimulates guanosine 5′-3-O-(thio)triphosphate (GTPS) release from ARF1 in the presence of phospholipids, but in this case myristoylation of ARF is not required. These observations, together with our previous findings that both myristoylated and nonmyristoylated forms of ARF but only the myristoylated form of ARF bind to membrane phospholipids, suggest that (i) the retinal exchange factor acts only on membrane-bound ARF, (ii) the myristate is not involved in the protein-protein interaction between ARF1 and the exchange factor, and (iii) N-myristoylation facilitates both spontaneous and catalyzed GDP-to-GTP exchange on ARF1 simply by facilitating the binding of ARF to membrane phospholipids.

Footnotes

↵^§ Supported by the Association pour la Recherche sur le Cancer (ARC).
↵^¶ Supported by INSERM.
↵* This work was supported by a grant from the Ministère de l'Enseignement Supérieur et de la Recherche (action “Physico-Chimie des Membranes Biologiques”). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ARF

ADP-ribosylation factor

PAGE

polyacrylamide gel electrophoresis

GTPS

5′-3-O-(thio)triphosphate

BFA

brefeldin A

ROS

rod outer segments

RIE

retinal isotonic extract.
- Received October 10, 1995.