Biosynthesis of glycoproteins in Candida albicans: activity of dolichol phosphate mannose synthase and protein mannosylation in a mixed membrane fraction.
A mixed membrane fraction (MMF) was isolated from yeast cells of Candida albicans with the ability to synthesize dolichol phosphate mannose (Dol-P-Man) from GDP-Man and dolichol phosphate (Dol-P) and transfer the sugar to proteins. Temperature of incubation (20-37 degrees C) did not affect the synthesis of Dol-P-Man but protein mannosylation occurred better at physiological temperatures (28 degrees C and 37 degrees C). Most of the sugar (87-93%) in the mannoproteins was O-linked as judged by its release by beta-elimination. Mannose was identified as the sole product after this treatment. Following incubation of MMF with the sugar donor, parallel levels of Dol-P-Man and mannosylated proteins were detected up to 30 min. Thereafter, Dol-P-Man levels reached a steady value whereas mannoproteins rapidly accumulated. Lipid-linked oligosaccharides were also detected in incubation mixtures, though in much lower amounts than those of Dol-P-Man or mannoproteins. Dol-P-Man synthase activity increased proportionally in response to increasing concentrations of either of the two enzyme substrates. A Km value of 0.36 microM for GDP-Man was calculated. MMF failed to use exogenous Dol-P-Man for protein glycosylation. Specific inhibition of Dol-P-Man synthesis with amphomycin was concomitant with a parallel decrease in protein mannosylation, indicating that most of the sugar is transferred to protein via the carrier lipid. Results are discussed in terms of the role of Dol-P-Man in protein glycosylation in C. albicans.
D002176:Candida albicans / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002474:Cell-Free System; D004288:Dolichol Phosphates / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D006031:Glycosylation; D008358:Mannose / Q000378:metabolism*; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D011499:Protein Processing, Post-Translational
D004288:Dolichol Phosphates; D005656:Fungal Proteins; D008562:Membrane Glycoproteins; C025363:mannoproteins; C028306:dolichol monophosphate; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase; D008358:Mannose