Cell-free processing and segregation of insulin precursors.
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.
D000818:Animals; D004285:Dogs; D007328:Insulin; D007425:Intracellular Membranes / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D010944:Plants / Q000378:metabolism; D011384:Proinsulin / Q000235:genetics*; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011691:Puromycin / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics*; D014908:Triticum / Q000378:metabolism
D007328:Insulin; D011498:Protein Precursors; D012333:RNA, Messenger; D011691:Puromycin; C013737:preproinsulin; D011384:Proinsulin