Isoprenylation of an inhibitory G protein α subunit occurs only upon mutagenesis of the carboxyl terminus

We transfected COS cells with expression vectors for the wild-type G proteins α(i1) subunit (pWT) and for mutated α(i1) subunits, including the nonmyristylated glycine 2 to alanine mutant (pGA) and mutants in which the carboxyl termini of pWT and pGA were changed from CGLF to CVLS (pCVLS and pGA-CVLS, respectively). Immunoblot analysis of transfected COS cells with an antibody to residues 159-168 of the α(i1) protein indicated that all four proteins were expressed. Unlike the WT and GA proteins, both CVLS mutant proteins failed to react with an antibody specific for the carboxyl terminus and failed to undergo pertussis toxin-catalyzed ADP-ribosylation. Analysis of COS cell lysates after [³H]mevalonic acid labeling indicated that specific incorporation of radioactivity occurred only in the α(i1) subunits with the CVLS mutation. Immunoprecipitation of COS cell fractions after labeling with [³⁵S]methionine indicated that both WT and CVSL mutant proteins were localized predominantly in the particulate fraction, whereas GA and GA-CVLS mutant proteins were found primarily in the soluble fraction. These results directly demonstrate that the carboxyl-terminal sequence, CGLF, is incapable of leading to isoprenylation but that alteration of two residues (glycine to valine, phenylalanine to serine) is sufficient to promote isoprenylation.