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 of the Saccharomyces cerevisiae alpha-factor pro-peptide and transport of the alpha-factor pro-peptide/insulin precursor fusion protein through the Saccharomyces cerevisiae secretory pathway, we analysed secretion of insulin precursor facilitated by alpha-factor pro-peptides with one or more of the three N-linked glycosylation sites removed. Mutation of the three alpha-factor pro-peptide N-linked glycosylation sites drastically decreased insulin precursor secretion. The three alpha-factor pro-peptide N-linked glycosylation sites differ in their ability to facilitate secretion of the insulin precursor. The two alpha-factor pro-peptide N-linked glycosylation sites localized closest to the insulin precursor contributed significantly to secretion, whereas the most N-terminally linked glycosylation site did not appear to facilitate secretion. Only correctly folded insulin precursor was found in the culture supernatant, regardless of the pro-peptide used for secretion, indicating that alpha-factor pro-peptide N-linked oligosaccharide chains are 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 Saccharomyces cerevisiae secretory pathway and secretion of the insulin precursor. N-linked glycosylation per se is not sufficient to facilitate secretion of the insulin precursor; the position of the N-linked oligosaccharide chain on the alpha-factor pro-peptide is important for facilitating efficient secretion.
Kjeldsen
T
T
Andersen
A S
AS
Hach
M
M
Diers
I
I
Nikolajsen
J
J
Markussen
J
J
D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D006031:Glycosylation; D007328:Insulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000078790:Insulin Secretion; D013058:Mass Spectrometry; D000072235:Mating Factor; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism*; D010455:Peptides / Q000235:genetics* / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D007328:Insulin; D009844:Oligosaccharides; D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D000072235:Mating Factor