Yamane H K, Farnsworth C C, Xie H Y, Evans T, Howald W N, Gelb M H, Glomset J A, Clarke S, Fung B K
Jules Stein Eye Institute, University of California, Los Angeles 90024.
Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):286-90. doi: 10.1073/pnas.88.1.286.
We showed previously that a 23-kDa guanine nucleotide-binding protein (G protein) purified from bovine brain membranes is carboxyl methylated and that this modification occurs at or near the membrane-binding domain. In the present study, we identified this small G protein as G25K (formerly termed Gp). We demonstrated that proteolytic digests of 3H-methylated G25K contained radiolabeled material that coeluted with synthetic S-(geranylgeranyl)cysteine methyl ester on reversed-phase HPLC. Further treatment by performic acid oxidation yielded radiolabeled material that coeluted with L-cysteic acid methyl ester, verifying that the isoprenoid moiety and carboxyl methyl ester are localized on a C-terminal cysteine residue. Analysis by gas chromatography-coupled mass spectrometry of material released from purified G25K by Raney nickel treatment positively identified the covalently bound lipid as an all-trans-geranylgeranyl (C20) isoprenoid moiety. These results suggest that geranylgeranyl modification and perhaps methyl esterification function in the membrane localization of this small G protein.
我们之前表明，从牛脑膜中纯化出的一种23 kDa鸟嘌呤核苷酸结合蛋白（G蛋白）被羧甲基化，且这种修饰发生在膜结合结构域或其附近。在本研究中，我们将这种小G蛋白鉴定为G25K（以前称为Gp）。我们证明，经3H甲基化的G25K的蛋白水解消化产物中含有放射性标记物质，该物质在反相高效液相色谱上与合成的S-（香叶基香叶基）半胱氨酸甲酯共洗脱。经过甲酸氧化的进一步处理产生了与L-半胱氨酸甲酯共洗脱的放射性标记物质，证实类异戊二烯部分和羧甲基酯位于C末端半胱氨酸残基上。通过气相色谱-质谱联用分析经雷尼镍处理从纯化的G25K释放出的物质，明确鉴定出共价结合的脂质为全反式香叶基香叶基（C20）类异戊二烯部分。这些结果表明，香叶基香叶基修饰以及可能的甲酯化作用于这种小G蛋白的膜定位。
