Myristoylation-facilitated binding of the G protein ARF1GDP to membrane phospholipids is required for its activation by a soluble nucleotide exchange factor.
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 Mg2+ 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 (IC50 = 2 microM) 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 (GTP gamma S) 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 non-myristoylated forms of ARF GTP-gamma S but only the myristoylated form of ARFGDP 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 ARFGDP to membrane phospholipids.
Franco
M
M
CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France.
Chardin
P
P
Chabre
M
M
Paris
S
S
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000818:Animals; D020126:Brefeldin A; D002352:Carrier Proteins / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003517:Cyclopentanes / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D008274:Magnesium / Q000494:pharmacology; D008563:Membrane Lipids / Q000378:metabolism*; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D002352:Carrier Proteins; D003517:Cyclopentanes; D008081:Liposomes; D008563:Membrane Lipids; D009227:Myristic Acids; D010743:Phospholipids; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D019814:Myristic Acid; D006153:Guanosine Diphosphate; D020126:Brefeldin A; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D014357:Trypsin; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D008274:Magnesium