title: Uptake of radiolabeled GlcNAc into Saccharomyces cerevisiae via native hexose transporters and its in vivo incorporation into GPI precursors in cells expressing heterologous GlcNAc kinase.
abstract: Yeast glycan biosynthetic pathways are commonly studied through metabolic incorporation of an exogenous radiolabeled compound into a target glycan. In Saccharomyces cerevisiae glycosylphosphatidylinositol (GPI) biosynthesis, [(3) H]inositol has been widely used to identify intermediates that accumulate in conditional GPI synthesis mutants. However, this approach also labels non-GPI lipid species that overwhelm detection of early GPI intermediates during chromatography. In this study, we show that despite lacking the ability to metabolize N-acetylglucosamine (GlcNAc), S. cerevisiae is capable of importing low levels of extracellular GlcNAc via almost all members of the hexose transporter family. Furthermore, expression of a heterologous GlcNAc kinase gene permits efficient incorporation of exogenous [(14) C]GlcNAc into nascent GPI structures in vivo, dramatically lowering the background signal from non-GPI lipids. Utilizing this new method with several conditional GPI biosynthesis mutants, we observed and characterized novel accumulating lipids that were not previously visible using [(3) H]inositol labeling. Chemical and enzymatic treatments of these lipids indicated that each is a GPI intermediate likely having one to three mannoses and lacking ethanolamine phosphate (Etn-P) side-branches. Our data support a model of yeast GPI synthesis that bifurcates after the addition of the first mannose and that includes a novel branch that produces GPI species lacking Etn-P side-branches.
authors.affiliation: New England Biolabs, Ipswich, MA 0193-2723, USA.
authors.forename: John J
authors.initials: JJ
authors.lastname: Scarcelli
authors.forename: Paul A
authors.initials: PA
authors.lastname: Colussi
authors.forename: Anne-Lise
authors.initials: AL
authors.lastname: Fabre
authors.forename: Eckhard
authors.initials: E
authors.lastname: Boles
authors.forename: Peter
authors.initials: P
authors.lastname: Orlean
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Taron
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D004983:Ethanolamines / Q000737:chemistry / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D007294:Inositol / Q000378:metabolism; D008358:Mannose / Q000737:chemistry / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014316:Tritium / Q000378:metabolism*
chemical_list: D004983:Ethanolamines; D017261:Glycosylphosphatidylinositols; D009004:Monosaccharide Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D014316:Tritium; D007294:Inositol; C005448:phosphorylethanolamine; D017853:Phosphotransferases (Alcohol Group Acceptor); C018716:N-acetylglucosamine kinase; D008358:Mannose; D000117:Acetylglucosamine