DIFFERENTIAL ISOPRENYLATION OF CARBOXY-TERMINAL MUTANTS OF AN INHIBITORY G-PROTEIN ALPHA-SUBUNIT - NEITHER FARNESYLATION NOR GERANYLGERANYLATION IS SUFFICIENT FOR MEMBRANE ATTACHMENT

被引:18
作者
BUTRYNSKI, JE
JONES, TLZ
BACKLUND, PS
SPIEGEL, AM
机构
[1] NIDDKD,MOLEC PATHOPHYSIOL BRANCH,BLDG 10,ROOM 8C101,BETHESDA,MD 20892
[2] NIMH,GEN & COMPARAT BIOCHEM LAB,BETHESDA,MD 20892
关键词
D O I
10.1021/bi00149a037
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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(i)1 (WT) subunit, the soluble nonmyristoylated G-protein alpha(i)1 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(i)1 carboxy terminus. Only the GA-CVLS and GA-CALL proteins incorporated [H-3]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.
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页码:8030 / 8035
页数:6
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