ALPHA-FACTOR PRO-PEPTIDE N-LINKED OLIGOSACCHARIDES FACILITATE SECRETION OF THE INSULIN PRECURSOR IN SACCHAROMYCES-CEREVISIAE

To evaluate the possible relationship between N-linked glycosylation o f the Saccharomyces cerevisiae alpha-factor pro-peptide and transport of the alpha-factor pro-peptide/insulin precursor fusion protein throu gh the Saccharomyces cerevisiae secretory pathway, we analysed secreti on of insulin precursor facilitated by alpha-factor pro-peptides with one or more of the three N-linked glycosylation sites removed. Mutatio n of the three alpha-factor pro-peptide N-linked glycosylation sites d rastically decreased insulin precursor secretion, The three alpha-fact or pro-peptide N-linked glycosylation sites differ in their ability to facilitate secretion of the insulin precursor, The two alpha-factor p ro-peptide N-linked glycosylation sites localized closest to the insul in precursor contributed significantly to secretion, whereas the most N-terminally linked glycosylation site did not appear to facilitate se cretion, Only correctly folded insulin precursor was found in the cult ure supernatant, regardless of the propeptide used for secretion, indi cating that alpha-factor pro-peptide N-linked oligosaccharide chains a re not necessary for correct folding of the insulin precursor. Thus, N -linked glycosylation facilitates intracellular transport of the alpha -factor propeptide/insulin precursor fusion protein through the Saccha romyces cerevisiae secretory pathway and secretion of the insulin prec ursor. N-linked glycosylation per se is not sufficient: to facilitate secretion of the insulin precursor; the position of the N-linked oligo saccharide chain on the alpha-factor pro-peptide is important for faci litating efficient secretion.