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 Man₉³H]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.The lipid-linked oligosaccharyl donor, Glc₃Man₉GlcNAc₂-P-P-dolichol (mature DLO²), in protein N-glycosylation is formed in two stages in the endoplasmic reticulum (ER) (1–4). In the first stage the lipid intermediates Man-P-dolichol (Man-P-Dol), Glc-P-dolichol (Glc-P-Dol), and Man₅GlcNAc₂-P-P-dolichol (M5-DLO) are formed on the cytoplasmic leaflet of the ER with GDP-Man, UDP-Glc, and UDP-GlcNAc, serving as the glycosyl donors. The biosynthesis of the mature DLO is completed with the addition of four more mannosyl units and the formation of the triglucosyl cap in the second stage after the transbilayer movement of Man-P-Dol, Glc-P-Dol, and M5-DLO to the lumenal monolayer. Although many details about the genetics, enzymology, and regulation of these 14 glycosylation reactions are known, there is virtually nothing known about the ER proteins that are presumably required to allow the lipid-bound hydrophilic glycosyl groups to traverse the hydrophobic core of the ER bilayer.The PER5/RFT1 gene was originally identified by Walter and coworkers (5) as a gene that was up-regulated by the unfolded protein response and required for efficient protein N-glycosylation in yeast. In a related study (6), the rft1 mutation was shown to be inscrutably suppressed by p53, a soluble protein that has not been found in yeast.Helenius et al. (7) have reported evidence from metabolic labeling experiments indicating that the RFT1 gene in Saccharomyces cerevisiae encodes a protein that is involved in the flipping of M5-DLO in vivo. More recently, a point mutation in the human orthologue of the RFT1 gene has been shown to result in the accumulation of M5-DLO in fibroblasts from a patient containing an R67C amino acid substitution (8). Although these results implicate Rft1 in the transverse diffusion of M5-DLO, the topological orientation of the accumulated intermediate in the mutant cells and the precise function of the protein in the transbilayer movement of the glycolipid intermediate remain to be defined.Two reports (9, 10) have demonstrated that Rft1 is not required for the “flipping” of M5-DLO in a reconstituted proteoliposomal system, raising questions about the precise relationship between Rft1 and the M5-DLO flippase. A more recent corroborative study further characterizing the reconstituted flippase activity indicates that the in vitro assay exhibits an impressive specificity for M5-DLO (11).The current study was conducted to further explore the possible role of Rft1 in the transbilayer movement of M5-DLO in the ER. Our results establish the accumulation of chemical amounts of M5-DLO in the Rft1-depleted cells by FACE analysis, supporting the results obtained by metabolic labeling in the yeast (7) and human (8) mutant cells. However, a series of experiments conducted with sealed microsomal vesicles indicate that, although Rft1 may be required to overcome a biophysical constraint for the flipping of M5-DLO in vivo, its depletion does not hinder the flipping of M5-DLO in sealed microsomal preparations in vitro. The resemblance of these results to the loss of the requirement for the Lec35 gene (12) in the transverse diffusion and/or utilization of Man-P-Dol and Glc-P-Dol for lipid intermediate biosynthesis during disruption of intact cells is discussed.