Myristoylation of an inhibitory GTP-binding protein alpha subunit is essential for its membrane attachment.
We transfected COS cells with cDNAs for the alpha subunits of stimulatory and inhibitory GTP-binding proteins, alpha s and alpha i1, respectively, and immunoprecipitated the metabolically labeled products with specific peptide antibodies. Cells were separated into particulate and soluble fractions before immunoprecipitation; [35S]methionine-labeled alpha s and alpha i were both found primarily in the particulate fraction. [3H]Myristate was incorporated into endogenous and transfected alpha i but could not be detected in alpha s even when it was overexpressed. We converted the second residue, glycine, of alpha i1 into alanine by site-directed mutagenesis. Upon transfection of the mutant alpha i1 into COS cells, the [35S]methionine-labeled product was localized primarily to the soluble fraction, and, also unlike normal alpha i1, the mutant failed to incorporate [3H]myristate. The unmyristoylated mutant alpha i1 could still interact with the beta-gamma complex, since purified beta gamma subunits promoted pertussis toxin-catalyzed ADP-ribosylation of both the normal and mutant alpha i1 subunits. These results indicate that myristoylation is critical for membrane attachment of alpha i but not alpha s subunits.
Jones
T L
TL
Molecular Pathophysiology Branch, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, MD 20892.
Simonds
W F
WF
Merendino
J J
JJ
Brann
M R
MR
Spiegel
A M
AM
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D037342:Pertussis Toxin; D011499:Protein Processing, Post-Translational; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000378:metabolism
D006898:Hydroxylamines; D046911:Macromolecular Substances; D009227:Myristic Acids; D010566:Virulence Factors, Bordetella; D019814:Myristic Acid; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D019811:Hydroxylamine; D004247:DNA; D008715:Methionine; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins