Differential isoprenylation of carboxy-terminal mutants of an inhibitory G-protein alpha-subunit: neither farnesylation nor geranylgeranylation is sufficient for membrane attachment.
To determine the effect of protein isoprenylation with farnesyl vs geranylgeranyl groups on membrane association in vivo, COS cells were transfected with cDNAs encoding the wild-type G-protein alpha i1 (WT) subunit, the soluble nonmyristoylated G-protein alpha i1 glycine to alanine mutant (GA), a double mutant in which the carboxy-terminal residues CGLF of GA were mutated to CVLS (GA-CVLS), and a double mutant in which the carboxy terminus of GA was mutated to CALL (GA-CALL). As opposed to the WT and GA proteins, the GA-CVLS and GA-CALL proteins were not pertussis toxin substrates nor were they recognized by antibodies that recognize the nonmutated alpha i1 carboxy terminus. Only the GA-CVLS and GA-CALL proteins incorporated [3H]mevalonate in the form of a farnesyl and a geranylgeranyl moiety, respectively. Subcellular localization, as assessed by immunoblotting and immunoprecipitation, revealed that the WT protein localizes almost exclusively to the membrane fraction, whereas the GA, GA-CVLS, and GA-CALL proteins localize predominantly to the soluble fraction. The soluble GA-CVLS and GA-CALL proteins were not carboxyl methylated, but the small amount localized to the membrane was partially carboxyl methylated. These results indicate that neither farnesylation nor geranylgeranylation is sufficient alone to lead to membrane association.
Butrynski
J E
JE
Molecular Pathophysiology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Mental Health, Bethesda, Maryland 20892.
Jones
T L
TL
Backlund
P S
PS
Spiegel
A M
AM
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry / Q000235:genetics; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D008745:Methylation; D008798:Mevalonic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D012717:Sesquiterpenes; D014162:Transfection
D046911:Macromolecular Substances; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D004247:DNA; D019204:GTP-Binding Proteins; C002963:geranylgeranyl pyrophosphate; D008798:Mevalonic Acid