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 Man₅GlcNAc₂-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 Glc₃Man₉GlcNAc₂-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-[³H]mannose or UDP-[³H]GlcNAc in the presence of unlabeled GDP-Man, no difference was observed in the rate of synthesis of [³H]Man₉GlcNAc₂-P-P-dolichol or Man₉[₃H]GlcNAc₂-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 GlcNAc₂-P-P-Dol₁₅, a water-soluble analogue. The entry of the analogue into the lumenal compartment was confirmed by demonstrating that [³H]chitobiosyl units were transferred to endogenous peptide acceptors via the yeast oligosaccharyltransferase when sealed vesicles were incubated with [³H]GlcNAc₂-P-P-Dol₁₅ 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.