Myristoylation of an inhibitory GTP-binding protein alpha subunit is essential for its membrane attachment.

We transfected COS cells with cDNAs for the alpha subunits of stimulatory and inhibitory GTP-binding proteins, alpha s and alpha i1, respectively, and immunoprecipitated the metabolically labeled products with specific peptide antibodies. Cells were separated into particulate and soluble fractions before immunoprecipitation; [35S]methionine-labeled alpha s and alpha i were both found primarily in the particulate fraction. [3H]Myristate was incorporated into endogenous and transfected alpha i but could not be detected in alpha s even when it was overexpressed. We converted the second residue, glycine, of alpha i1 into alanine by site-directed mutagenesis. Upon transfection of the mutant alpha i1 into COS cells, the [35S]methionine-labeled product was localized primarily to the soluble fraction, and, also unlike normal alpha i1, the mutant failed to incorporate [3H]myristate. The unmyristoylated mutant alpha i1 could still interact with the beta-gamma complex, since purified beta gamma subunits promoted pertussis toxin-catalyzed ADP-ribosylation of both the normal and mutant alpha i1 subunits. These results indicate that myristoylation is critical for membrane attachment of alpha i but not alpha s subunits. IMAGES:

我们分别将编码刺激性与抑制性GTP结合蛋白（GTP-binding protein）α亚基的互补脱氧核糖核酸（cDNA）αs和αi1转染至COS细胞中，采用特异性肽抗体对代谢标记的表达产物进行免疫沉淀实验。免疫沉淀前将细胞裂解物分为颗粒组分与可溶性组分；经[35S]甲硫氨酸标记的αs与αi均主要分布于颗粒组分中。[3H]肉豆蔻酸可掺入内源性及转染表达的αi蛋白，但即便αs过表达，也无法在αs中检测到该标记物。我们通过定点诱变技术将αi1的第二位氨基酸残基甘氨酸突变为丙氨酸。将该突变型αi1转染至COS细胞后，[35S]甲硫氨酸标记的突变产物主要定位于可溶性组分；且与正常αi1不同，该突变体无法掺入[3H]肉豆蔻酸。未发生肉豆蔻酰化的突变型αi1仍可与βγ复合物相互作用，因为纯化的βγ亚基可促进百日咳毒素催化的正常与突变型αi1亚基的ADP核糖基化反应。上述实验结果表明，肉豆蔻酰化对于αi亚基的膜附着至关重要，但对αs亚基并非必需。图像：