Gutierrez L, Magee A I, Marshall C J, Hancock J F
National Institute for Medical Research, Mill Hill, London, UK.
EMBO J. 1989 Apr;8(4):1093-8. doi: 10.1002/j.1460-2075.1989.tb03478.x.
We have studied the post-translational processing of p21ras proteins. The primary translation product pro-p21 is cytosolic and is rapidly converted to a cytosolic form (c-p21) of higher mobility on SDS-PAGE. c-p21 is converted in turn to the membrane-bound mature palmitoylated form (m-p21) of slightly higher mobility. These processing steps are accompanied by increases in isoelectric point and in hydrophobicity as judged by Triton X-114 partitioning. Although the increases in electrophoretic mobility and hydrophobicity precede acylation we show that mutation of Cys186, which has been shown to block acylation, also abolishes the pro-p21 to c-p21 conversion. Thus the Cys186 residue is involved in the processing steps prior to acylation. We have identified two processing events which contribute to the pro-p21 conversion. Site-directed mutagenesis to insert tryptophan, which is not present in the wild type, followed by metabolic labelling with [3H]tryptophan has allowed us to map a proteolytic processing event which removes the three C-terminal residues. In addition, both the c-p21 and m-p21 forms are carboxyl-methylated. Approximately one methyl group is incorporated per molecule of p21 at steady state, which can partially account for the increase in isoelectric point. Unlike palmitate, methyl group turnover is not observed.
我们研究了p21ras蛋白的翻译后加工过程。初级翻译产物前体p21存在于胞质溶胶中，并迅速转化为在SDS-PAGE上迁移率更高的胞质溶胶形式（c-p21）。c-p21继而转化为迁移率略高的膜结合成熟棕榈酰化形式（m-p21）。根据Triton X-114分配判断，这些加工步骤伴随着等电点和疏水性的增加。尽管电泳迁移率和疏水性的增加先于酰化作用，但我们发现已证明能阻断酰化作用的Cys186突变也消除了前体p21到c-p21的转化。因此，Cys186残基参与了酰化作用之前的加工步骤。我们确定了两个有助于前体p21转化的加工事件。通过定点诱变插入野生型中不存在的色氨酸，然后用[3H]色氨酸进行代谢标记，使我们能够确定一个去除三个C末端残基的蛋白水解加工事件。此外，c-p21和m-p21形式都进行了羧甲基化。在稳态下，每分子p21大约掺入一个甲基，这可以部分解释等电点的增加。与棕榈酸不同，未观察到甲基的周转。
