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) expressed in Saccharomyces cerevisiae was performed to establish the requirements for intracellular transport and the influence of the secretory pathway quality control mechanisms on secretion. Metabolic labelling of the IP expressed in S. cerevisiae showed that the effect of a leader was to stabilise the IP in the endoplasmic reticulum (ER), and facilitate intracellular transport of the fusion protein and rapid secretion. The first metabolically labelled 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 enzymatic removal of the leader in a late Golgi apparatus compartment, the IP followed one of two routes: (1) to the plasma membrane and hence to the culture supernatant, or (2) to a Golgi or post-Golgi compartment from which secretion was restricted. Combined secretion and intracellular retention of the IP reflected either saturation of a Golgi or post-Golgi compartment and secretion as a consequence of overexpression, or competition between secretion and intracellular retention. IP which was misfolded, either due to amino acid substitution or because disulphide bond formation had been prevented with 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.
Kjeldsen
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Insulin Research, Novo Nordisk A/S, Bagsvaerd, Denmark. thk@novo.dk
Pettersson
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AF
Hach
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D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D004229:Dithiothreitol / Q000494:pharmacology; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007700:Kinetics; D000072235:Mating Factor; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D007328:Insulin; D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000072235:Mating Factor; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases; C499266:YPS1 protein, S cerevisiae; D004229:Dithiothreitol