Suppression of Rft1 expression does not impair the transbilayer movement of Man5GlcNAc2-P-P-dolichol in sealed microsomes from yeast.
To further evaluate the role of Rft1 in the transbilayer movement of Man(5)GlcNAc(2)-P-P-dolichol (M5-DLO), a series of experiments was conducted with intact cells and sealed microsomal vesicles. First, an unexpectedly large accumulation (37-fold) of M5-DLO was observed in Rft1-depleted cells (YG1137) relative to Glc(3)Man(9)GlcNAc(2)-P-P-Dol in wild type (SS328) cells when glycolipid levels were compared by fluorophore-assisted carbohydrate electrophoresis analysis. When sealed microsomes from wild type cells and cells depleted of Rft1 were incubated with GDP-[(3)H]mannose or UDP-[(3)H]GlcNAc in the presence of unlabeled GDP-Man, no difference was observed in the rate of synthesis of [(3)H]Man(9)GlcNAc(2)-P-P-dolichol or Man(9)[(3)H]GlcNAc(2)-P-P-dolichol, respectively. In addition, no difference was seen in the level of M5-DLO flippase activity in sealed wild type and Rft1-depleted microsomal vesicles when the activity was assessed by the transport of GlcNAc(2)-P-P-Dol(15), a water-soluble analogue. The entry of the analogue into the lumenal compartment was confirmed by demonstrating that [(3)H]chitobiosyl units were transferred to endogenous peptide acceptors via the yeast oligosaccharyltransferase when sealed vesicles were incubated with [(3)H]GlcNAc(2)-P-P-Dol(15) in the presence of an exogenously supplied acceptor peptide. In addition, several enzymes involved in Dol-P and lipid intermediate biosynthesis were found to be up-regulated in Rft1-depleted cells. All of these results indicate that although Rft1 may play a critical role in vivo, depletion of this protein does not impair the transbilayer movement of M5-DLO in sealed microsomal fractions prepared from disrupted cells.
Rush
Jeffrey S
JS
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536, USA.
Gao
Ningguo
N
Lehrman
Mark A
MA
Matveev
Sergey
S
Waechter
Charles J
CJ
D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D001692:Biological Transport; D004287:Dolichol Monophosphate Mannose / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005947:Glucose / Q000378:metabolism; D006602:Hexosyltransferases / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011103:Polyisoprenyl Phosphate Monosaccharides / Q000378:metabolism; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000032:analysis* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011103:Polyisoprenyl Phosphate Monosaccharides; D011104:Polyisoprenyl Phosphate Oligosaccharides; C486728:RFT1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C058641:mannosyl(5)-N-acetyl(2)-glucose diphosphate dolichol; D004287:Dolichol Monophosphate Mannose; C002968:dolichol-D-glucosylmonophosphate; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D019883:Alkyl and Aryl Transferases; C084921:cis-isoprenyltransferase; D017853:Phosphotransferases (Alcohol Group Acceptor); C020087:dolichol kinase; D005947:Glucose; D008358:Mannose