Overexpression of GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae corrects defects in dolichol-linked saccharide formation and protein glycosylation.
Thermosensitive mutants of Saccharomyces cerevisiae, affected in the endoplasmic reticulum (ER) located glycosylation, i.e. in Dol-P-Man synthase (dpm1), in beta-1,4 mannosyl transferase (alg1) and in alpha-1,3 mannosyltransferase (alg2), were used to assess the role of GDP-Man availability for the synthesis of dolichol-linked saccharides. The mutants were transformed with the yeast gene MPG1 (PSA1/VIG9) encoding GDP-Man pyrophosphorylase catalyzing the final step of GDP-Man formation. We found that overexpression of MPG1 allows growth at non-permissive temperature and leads to an increase in the cellular content of GDP-Man. In the alg1 and alg2 mutants, complemented with MPG1 gene, N-glycosylation of invertase was in part restored, to a degree comparable to that of the wild-type control. In the dpm1 mutant, the glycosylation reactions that depend on the formation of Dol-P-Man, i.e. elongation of Man(5)GlcNAc(2)-PP-Dol, O-mannosylation of chitinase and synthesis of GPI anchor were normal when MPG1 was overexpressed. Our data indicate that an increased level of GDP-Man is able to correct defects in mannosylation reactions ascribed to the ER and to the Golgi.
Janik
Anna
A
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02 106, Warsaw, Poland.
Sosnowska
Monika
M
Kruszewska
Joanna
J
Krotkiewski
Hubert
H
Lehle
Ludwig
L
Palamarczyk
Grazyna
G
D015152:Blotting, Northern; D002688:Chitinases / Q000378:metabolism; D004286:Dolichols / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis; D006056:Golgi Apparatus / Q000378:metabolism; D008364:Mannosyltransferases / Q000378:metabolism; D009154:Mutation; D009713:Nucleotidyltransferases / Q000096:biosynthesis* / Q000235:genetics; D009844:Oligosaccharides / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D004286:Dolichols; D017261:Glycosylphosphatidylinositols; D009844:Oligosaccharides; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase; D009713:Nucleotidyltransferases; C026917:mannose 1-phosphate guanylyltransferase; D002688:Chitinases