Myristoylation of ADP-ribosylation factor 1 facilitates nucleotide exchange at physiological Mg2+ levels.
Recombinant N-myristoylated bovine ADP-ribosylation factor 1 (myr-rARF1) has been expressed in bacteria and purified to near homogeneity with a high (85%) myristoylation efficiency. Myr-rARF1 and nonmyristoylated rARF1 have been compared with respect to their kinetics of guanine nucleotide exchange and their interactions with phospholipids. Myristoylation is shown to allow the release of bound GDP at physiological (mM) concentrations of Mg2+. GDP dissociation is slow in the absence of phospholipids but is accelerated 2-fold in the presence of phospholipid vesicles. On the contrary, myristoylation decreases 10-fold the rate of dissociation of GTP or guanosine 5'-O-(thiotriphosphate) (GTP gamma S) in the presence of phospholipids. As a result, myr-ARF1 can be spontaneously activated by GTP or GTP gamma S (t1/2 approximately 30 min at 37 degrees C) at 1 mM Mg2+, in the sole presence of phospholipid membranes without the need for a nucleotide exchange factor. In contrast to the nonacylated protein, the GDP-bound form of myr-ARF1 interacts with phospholipids, as demonstrated by its cosedimentation with phospholipid vesicles and its comigration with phospholipid/cholate micelles on gel filtration. The interaction is, however, weaker than for the GTP-bound form, suggesting that only the myristate in myr-ARF1GDP interacts with phospholipids, whereas both the myristate and the amino-terminal hydrophobic residues in myr-ARF1GTP bind to phospholipids.
CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France.
M
M
Franco
P
P
Chardin
M
M
Chabre
S
S
Paris
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D019826:Cholic Acid; D002793:Cholic Acids / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008823:Micelles; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D011485:Protein Binding
D002793:Cholic Acids; D008823:Micelles; D009227:Myristic Acids; D009838:Oligodeoxyribonucleotides; D019814:Myristic Acid; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D019826:Cholic Acid; D008274:Magnesium; D004134:Dimyristoylphosphatidylcholine