Identification of a precursor in the biosynthesis of the p21 transforming protein of harvey murine sarcoma virus
Identification of a precursor in the biosynthesis of the p21 transforming protein of harvey murine sarcoma virus
复制标题
哈维鼠肉瘤病毒 p21 转化蛋白生物合成中前体的鉴定
作者：
T. Y. Shih;M. Weeks;P. Gruss;R. Dhar;S. Oroszlan;E. Scolnick
哈维鼠肉瘤病毒（Ha-MuSV）的v-ras基因编码的p21转化蛋白在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中以21，000和23，000道尔顿之间的双重带迁移。双联体的下带被指定为p21，上带被指定为pp 21，因为它与p21的磷酸化形式共迁移。通过用[35 S]甲硫氨酸脉冲标记，我们检测到p21前体pro-p21，其迁移好像比p21大大约1，000道尔顿。在每毫升100微克放线菌酮的存在下，通过脉冲追踪实验用[25 S]蛋氨酸建立了底物-产物关系，放线菌酮抑制所有从头蛋白质生物合成。在4小时内，pro-p21被完全追赶到p21中，并且在接下来的24小时内pp 21积累。因此，从p21形成pp 21不需要从头蛋白质合成。通过亚细胞分级进入胞质溶胶和膜组分，我们发现，pro-p21是在非膜结合状态下合成的，并且在其完全合成后不久，p21产物与膜组分相关联。通过用70%甲酸或金黄色葡萄球菌V8蛋白酶选择性切割p21的一个独特的天冬氨酸-脯氨酸残基，我们发现pro-p21加工的分子内位点位于pro-p21分子的C-末端部分。的可能性，前体参与组装的p21到质膜，或者，该处理是一个步骤中的激活p21的生化活性进行了讨论。
The p21 transforming protein coded for by the v-ras gene of Harvey murine sarcoma virus (Ha-MuSV) migrates as a doublet band between 21,000 and 23,000 daltons during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lower band of the doublet is designated p21, and the upper band is designated pp21 since it comigrates with the phosphorylated form of p21. By pulse-labeling with [35S] methionine, we detected a p21 precursor, pro-p21, which migrated as if it was approximately 1,000 daltons larger than p21. The precursor-product relationship was established by pulse-chase experiments with [25S] methionine in the presence of 100 micrograms of cycloheximide per ml, which inhibited all de novo protein biosynthesis. Within 4 h, pro-p21 was completely chased into p21, and during the next 24 h pp21 accumulated. Thus, formation of pp21 from p21 did not require de novo protein synthesis. By subcellular fractionation into cytosol amd membrane fractions, we found that pro-p21 was synthesized in a non-membrane-bound state and that shortly after its complete synthesis, the p21 product was associated with the membrane fraction. By selective cleavage of p21 at a unique aspartic acid-proline residue with 70% formic acid or with Staphylococcus aureus V8 protease, we found that the intramolecular site of pro-p21 processing was located in the C-terminal portion of the pro-p21 molecule. The possibilities that the precursor was involved in the assembly of p21 into the plasma membrane and, alternatively, that the processing was a step in the activation of p21 biochemical activities are discussed.