Eskridge E M, Shields D
J Biol Chem. 1983 Oct 10;258(19):11487-91.
The biosynthesis, segregation, and processing of preproinsulin (116 amino acids) was investigated to determine the mechanism(s) by which it is translocated across the endoplasmic reticulum membrane. Islet mRNA was translated in the wheat germ cell-free system, and at various times during preproinsulin synthesis, puromycin was added, followed by addition of microsomal membranes. Neither processing of preproinsulin nor translocation of proinsulin into microsomal membranes occurred in the presence of puromycin. Synchronization of preproinsulin translation by addition of 7-methylguanosine 5'-phosphate enabled the timing of preproinsulin synthesis and proinsulin (91 amino acids) segregation into microsomal membranes to be determined. Membrane binding occurs when about 60 amino acids have been polymerized, i.e. prior to the completion of the polypeptide chain. The binding of signal recognition particle to the nascent signal is demonstrated to be an absolute requirement for translocation and processing of preproinsulin. The results indicate that segregation and processing of preproinsulin are co-translational events; no evidence for a post-translational mechanism was found. Furthermore, this work, together with similar studies, suggests that presecretory polypeptides must be synthesized as part of a precursor with a minimum size of 60-80 amino acids in order to effect membrane binding and translocation of the polypeptide chain within the intracisternal space of the endoplasmic reticulum.
为确定前胰岛素原（116个氨基酸）跨内质网膜转运的机制，对其生物合成、分离及加工过程进行了研究。胰岛mRNA在麦胚无细胞系统中进行翻译，在前胰岛素原合成的不同时间加入嘌呤霉素，随后加入微粒体膜。在嘌呤霉素存在的情况下，前胰岛素原既未进行加工，胰岛素原也未转运至微粒体膜中。通过添加7-甲基鸟苷5'-磷酸使前胰岛素原翻译同步化，从而能够确定前胰岛素原合成及胰岛素原（91个氨基酸）分离至微粒体膜中的时间。当约60个氨基酸聚合时，即多肽链完成之前，会发生膜结合。已证明信号识别颗粒与新生信号的结合是前胰岛素原转运和加工的绝对必要条件。结果表明，前胰岛素原的分离和加工是共翻译事件；未发现翻译后机制的证据。此外，这项工作以及类似研究表明，分泌前多肽必须作为最小长度为60-80个氨基酸的前体的一部分进行合成才能在内质网池内实现膜结合和多肽链的转运。
