Yamane H K, Farnsworth C C, Xie H Y, Howald W, Fung B K, Clarke S, Gelb M H, Glomset J A
Jules Stein Eye Institute, Los Angeles, CA.
Proc Natl Acad Sci U S A. 1990 Aug;87(15):5868-72. doi: 10.1073/pnas.87.15.5868.
We have shown previously that guanine nucleotide-binding protein (G protein) beta gamma complexes purified from bovine brain membranes are methyl esterified on a C-terminal cysteine residue of the gamma polypeptide. In the present study, 3H-methylated G beta gamma complexes cleaved to their constituent amino acids by exhaustive proteolysis were shown to contain radiolabeled material that coeluted with geranylgeranylcysteine methyl ester on reversed-phase HPLC and two TLC systems. Further treatment by performic acid oxidation yielded radiolabeled material that coeluted with L-cysteic acid methyl ester, verifying that the prenyl modification occurs on a C-terminal cysteine residue. Analysis by gas chromatography-coupled mass spectrometry of material released from purified G beta gamma by treatment with Raney nickel positively identified the covalently bound lipid as an all-trans-geranylgeranyl (C20) isoprenoid moiety. To delineate the distribution of this modification among gamma subunits, purified G beta gamma complexes were separated into 5-kDa (gamma 5) and 6-kDa (gamma 6) forms of the gamma polypeptide by reversed-phase HPLC. Gas chromatography-coupled mass spectrometry analyses of Raney nickel-treated purified gamma 5 and gamma 6 subunits showed that both polypeptides were modified by geranylgeranylation. These results demonstrate that at least two forms of brain gamma subunit are posttranslationally modified by geranylgeranylation and carboxyl methylation. These modifications may be important for targeting G beta gamma complexes to membranes.
我们之前已经表明，从牛脑膜中纯化的鸟嘌呤核苷酸结合蛋白（G蛋白）βγ复合物在γ多肽的C末端半胱氨酸残基上发生甲基酯化。在本研究中，通过彻底的蛋白水解将3H甲基化的Gβγ复合物切割成其组成氨基酸，结果显示其中含有放射性标记物质，该物质在反相高效液相色谱和两种薄层层析系统上与香叶基香叶基半胱氨酸甲酯共洗脱。经过甲酸氧化进一步处理后，产生了与L-半胱氨酸甲酯共洗脱的放射性标记物质, 证实异戊二烯基修饰发生在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后从纯化的Gβγ中释放的物质，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。为了确定这种修饰在γ亚基中的分布情况，通过反相高效液相色谱将纯化的Gβγ复合物分离成5 kDa（γ5）和6 kDa（γ6）形式的γ多肽。对经雷尼镍处理的纯化γ5和γ6亚基进行气相色谱-质谱联用分析表明，这两种多肽都被香叶基香叶基化修饰。这些结果表明，至少两种形式的脑γ亚基在翻译后被香叶基香叶基化和羧基甲基化修饰。这些修饰对于将Gβγ复合物靶向到膜上可能很重要。
