Cell-free processing and segregation of insulin precursors.
Cell-free processing and segregation of insulin precursors.
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DOI：
10.1016/s0021-9258(17)44253-0
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发表时间：
1983-10
期刊：
影响因子：
--
通讯作者：
E. Eskridge;D. Shields
中科院分区：
文献类型：
--
作者：
E. Eskridge;D. Shields
研究了胰岛素前原（116个氨基酸）的生物合成、分离和加工过程，以确定其在内质网膜上转运的机制。在小麦无生殖细胞系统中，胰岛mRNA被翻译，在胰岛素前原合成的不同时间，添加嘌呤霉素，然后添加微粒体膜。在嘌呤霉素存在的情况下，既不发生胰岛素前原的加工，也不发生胰岛素前原易位到微粒体膜上。通过添加7-甲基鸟苷5'-磷酸来同步胰岛素原体翻译，可以确定胰岛素原体合成和胰岛素原体（91个氨基酸）分离到微粒体膜的时间。当大约60个氨基酸被聚合时，即在多肽链完成之前，膜结合发生。信号识别粒子与新生信号的结合被证明是胰岛素前原易位和加工的绝对要求。结果表明，胰岛素前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，这项工作以及类似的研究表明，分泌前多肽必须作为前体的一部分合成，其大小至少为60-80个氨基酸，以影响内质网内质腔内多肽链的膜结合和易位。
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.