MYRISTOYLATION OF AN INHIBITORY GTP-BINDING PROTEIN ALPHA-SUBUNIT IS ESSENTIAL FOR ITS MEMBRANE ATTACHMENT
MYRISTOYLATION OF AN INHIBITORY GTP-BINDING PROTEIN ALPHA-SUBUNIT IS ESSENTIAL FOR ITS MEMBRANE ATTACHMENT
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DOI：
10.1073/pnas.87.2.568
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发表时间：
1990-01-01
影响因子：
11.1
通讯作者：
SPIEGEL, AM
中科院分区：
文献类型：
--
作者：
JONES, TLZ;SIMONDS, WF;SPIEGEL, AM
我们将α基因的cDNA导入COS细胞。刺激性和抑制性GTP结合蛋白的亚基分别为αs和αi1，并用特定的多肽抗体免疫沉淀代谢标记的产物。在免疫沉淀之前，细胞被分成颗粒组分和可溶性组分；[35S]蛋氨酸标记的αs和αi都主要存在于颗粒组分中。[~3H]肉豆蔻酸盐被掺入内源性和转染αi中，但即使在过量表达的情况下也不能在αi中检测到。我们通过定点突变将αI1的第二个残基甘氨酸转化为丙氨酸。当将突变的αi1导入COS细胞时，[35S]蛋氨酸标记的产物主要定位于可溶部分，而且，与正常的αi1不同的是，突变的αi1未能掺入[~3H]肉豆蔻酸。未肉豆蔻酸化的突变体αi1仍然可以与β-伽马相互作用。复合体，自提纯后。贝塔..伽马。亚基促进百日咳毒素催化的正常和突变αi1亚基的ADP核糖化。这些结果表明，肉豆蔻酰化对αi亚基的膜附着是关键的，但对αs亚基的附着不起关键作用。
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-direction 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.