The role of leaders in intracellular transport and secretion of the insulin precursor in the yeast Saccharomyces cerevisiae

Pulse-chase analysis of folded and misfolded insulin precursor (IP) express ed in Saccharomyces cerevisiae was performed to establish the requirements for intracellular transport and the influence of the secretory pathway qual ity control mechanisms on secretion. Metabolic labelling of the IP expresse d in S. cerevisiae showed that the effect of a leader was to stabilise the IP in the endoplasmic reticulum (ER), and facilitate intracellular transpor t of the fusion protein and rapid secretion. The first metabolically labell ed IP appeared in the culture supernatant within 2-4 min of chase, and most of the secreted IP appeared within the first 15 min of chase. After enzyma tic removal of the leader in a late Golgi apparatus compartment, the IP fol lowed one of two routes: (1) to the plasma membrane and hence to the cultur e supernatant, or (2) to a Golgi or post-Golgi compartment from which secre tion was restricted. Combined secretion and intracellular retention of the IP reflected either saturation of a Golgi or post-Golgi compartment and sec retion as a consequence of overexpression, or competition between secretion and intracellular retention. IP which was misfolded, either due to amino a cid substitution or because disulphide bond formation had been prevented wi th dithiothreitol (DTT), was transported from the ER to the Golgi apparatus but then retained in a Golgi or post-Golgi compartment and not exported to the culture supernatant. (C) 1999 Elsevier Science B.V. All rights reserve d.