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. 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. The lipid-linked oligosaccharyl donor, Glc3Man9GlcNAc2-P-P-dolichol (mature DLO 2The abbreviations used are: DLOGlc3Man9GlcNAc2-P-P-DolERendoplasmic reticulumM5-DLOMan5GlcNAc2-P-P-Dolmature Man-P-DolmannosylphosphoryldolicholGlc-P-DolglucosylphosphoryldolicholCPYcarboxypeptidase YFACEfluorophore-assisted carbohydrate electrophoresisCHOChinese hamster ovaryCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidOTaseoligosaccharyltransferase. 2The abbreviations used are: DLOGlc3Man9GlcNAc2-P-P-DolERendoplasmic reticulumM5-DLOMan5GlcNAc2-P-P-Dolmature Man-P-DolmannosylphosphoryldolicholGlc-P-DolglucosylphosphoryldolicholCPYcarboxypeptidase YFACEfluorophore-assisted carbohydrate electrophoresisCHOChinese hamster ovaryCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidOTaseoligosaccharyltransferase.), in protein N-glycosylation is formed in two stages in the endoplasmic reticulum (ER) (1Snider M.D. Robbins P.W. J. Biol. Chem. 1982; 257: 6796-6801Abstract Full Text PDF PubMed Google Scholar, 2Herscovics A. Orlean P. FASEB J. 1993; 7: 540-550Crossref PubMed Scopus (437) Google Scholar, 3Schenk B. Fernandez F. Waechter C.J. Glycobiology. 2001; 11: 61R-70RCrossref PubMed Scopus (141) Google Scholar, 4Helenius A. Aebi M. Annu. Rev. Biochem. 2004; 73: 1019-1049Crossref PubMed Scopus (1593) Google Scholar). 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 (5Ng D.T. Spear E.D. Walter P. J. Cell Biol. 2000; 150: 77-88Crossref PubMed Scopus (273) Google Scholar) 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 (6Koerte A. Chong T. Li X. Wahane K. Cai M. J. Biol. Chem. 1995; 270: 22556-22564Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), the rft1 mutation was shown to be inscrutably suppressed by p53, a soluble protein that has not been found in yeast.Helenius et al. (7Helenius J. Ng D.T. Marolda C.L. Walter P. Valvano M.A. Aebi M. Nature. 2002; 415: 447-450Crossref PubMed Scopus (217) Google Scholar) 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 (8Haeuptle M.A. Pujol F.M. Neupert C. Winchester B. Kastaniotis A.J. Aebi M. Hennet T. Am. J. Hum. Genet. 2008; 82: 600-606Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). 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 (9Frank C.G. Sanyal S. Rush J.S. Waechter C.J. Menon A.K. Nature. 2008; 454: E3-5Crossref PubMed Scopus (57) Google Scholar, 10Sanyal S. Frank C.G. Menon A.K. Biochemistry. 2008; 47: 7937-7946Crossref PubMed Scopus (71) Google Scholar) 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 (11Sanyal S. Menon A.K. Proc. PubMed Scopus Google study was conducted to further the role of Rft1 in the transbilayer movement of M5-DLO in the results the accumulation of of M5-DLO in the Rft1-depleted cells by the results by metabolic labeling in the yeast (7Helenius J. Ng D.T. Marolda C.L. Walter P. Valvano M.A. Aebi M. Nature. 2002; 415: 447-450Crossref PubMed Scopus (217) Google Scholar) and human (8Haeuptle M.A. Pujol F.M. Neupert C. Winchester B. Kastaniotis A.J. Aebi M. Hennet T. Am. J. Hum. Genet. 2008; 82: 600-606Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) mutant cells. a series of experiments conducted with sealed microsomal vesicles indicate although Rft1 may be required to a for the flipping of M5-DLO in vivo, depletion does not the flipping of M5-DLO in sealed microsomal in The of these results to the of the for the gene M. Rush J.S. S. M.A. J. J. Waechter C.J. M.A. Biol. 2001; PubMed Scopus Google Scholar) in the transverse diffusion of and for lipid intermediate biosynthesis of intact cells is has been for many that play in in and the of glycolipid intermediates in and biosynthesis A.K. Cell Biol. 1995; Full Text PDF PubMed Scopus Google Scholar, T. Menon A.K. PubMed Scopus Google Scholar, T. A. J. Cell 2004; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the many and of the has been in and ER proteins involved in the of Man-P-Dol, Glc-P-Dol, and the evidence (7Helenius J. Ng D.T. Marolda C.L. Walter P. Valvano M.A. Aebi M. Nature. 2002; 415: 447-450Crossref PubMed Scopus (217) Google Scholar) that Rft1 in the transbilayer movement of M5-DLO in this The of the in vitro reported was to more evidence to a role of Rft1 in the transbilayer movement of M5-DLO sealed microsomal vesicles from yeast as a evidence that large of M5-DLO accumulated in Rft1-depleted yeast several of experiments indicate that Rft1 is not required for the transverse diffusion of M5-DLO from the cytoplasmic leaflet to the lumenal in sealed microsomal the precise of depletion of Rft1 in the vesicles used as a is on a of endogenous Rft1 into the cells after the from to as the cells of the of Rft1 after to after is on the that Rft1 synthesis the to as and that Rft1 is not the formation of was by sealed microsomes with these in vitro sealed vesicles from wild type and Rft1-depleted cells formed and no accumulation of was The of into and was in sealed vesicles from Rft1-depleted cells compared with wild type that was up-regulated in response to of Rft1 be that the enzymes that Dol-P as acceptor and an in the of exogenously that microsomal fractions from Rft1-depleted cells levels of endogenous Dol-P for lipid intermediate biosynthesis relative to wild type cells. is that this of to the of M5-DLO after Rft1 depletion that M5-DLO was in sealed the was by endogenous by the microsomal with UDP-[3H]GlcNAc by the addition of unlabeled The that was by and Rft1-depleted microsomes evidence that formed M5-DLO was in sealed microsomes from wild type and Rft1-depleted cells the is that the of formed by Rft1-depleted microsomes was the is that the large of M5-DLO by there was no evidence that the M5-DLO, was by the large endogenous is that a of the M5-DLO in a compartment as observed in M.A. Glycobiology. PubMed Scopus Google evidence for M5-DLO flippase was the transport of GlcNAc2-P-P-Dol15, a water-soluble analogue of that has been shown to be presumably by the flippase the transbilayer movement of M5-DLO, in microsomes from a of and yeast F. C. A. P. PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Waechter C.J. Biochem. PubMed Scopus Google Scholar, C. PubMed Scopus Google Scholar). The results from this that the analogue the sealed microsomal vesicles from wild type and Rft1-depleted cells the The that transport of the water-soluble analogue to a in Rft1-depleted vesicles that the Rft1-depleted microsomal vesicles are more in ER or may have a as a of the unfolded protein of the analogue to the lumenal compartment was by that the was transferred to a peptide results a are in with recent related (9Frank C.G. Sanyal S. Rush J.S. Waechter C.J. Menon A.K. Nature. 2008; 454: E3-5Crossref PubMed Scopus (57) Google Scholar, 10Sanyal S. Frank C.G. Menon A.K. Biochemistry. 2008; 47: 7937-7946Crossref PubMed Scopus (71) Google Scholar, S. Menon A.K. Proc. PubMed Scopus Google Scholar) have demonstrated that the flipping of M5-DLO in reconstituted assessed by a in the presence or of The reconstituted flippase activity be from Rft1 and a by S. Frank C.G. Menon A.K. Biochemistry. 2008; 47: 7937-7946Crossref PubMed Scopus (71) Google Scholar), and the flippase activity between the intermediate that is M5-DLO, and (11Sanyal S. Menon A.K. Proc. PubMed Scopus Google study the of the mutant cells by that several enzymes involved in Dol-P and lipid intermediate biosynthesis are as a result of the of Rft1 In addition, the rate of transport of and was in vesicles from Rft1-depleted cells relative to wild type cells not that and as as the enzymes involved in Dol-P and lipid intermediate synthesis in be up-regulated as a response to the transbilayer movement or accumulation of M5-DLO in vivo. In related were identified on the of for an intact unfolded protein response for (5Ng D.T. Spear E.D. Walter P. J. Cell Biol. 2000; 150: 77-88Crossref PubMed Scopus (273) Google Scholar), and et al. J.S. Walter P. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) have reported that enzymes in the and are up-regulated the unfolded protein response in S. cerevisiae on the results in this not the that Rft1 a role in the flipping of M5-DLO in intact cells. that or that Rft1 for flipping in no after cells are is of the function of is for the of and in intact cells not in ER vesicles M. Rush J.S. S. M.A. J. J. Waechter C.J. M.A. Biol. 2001; PubMed Scopus Google Scholar). is that of the of the ER are when cells are disrupted the of microsomal In more and be required to the precise function of Rft1 in and to ER proteins that are involved in the transbilayer movement of M5-DLO as as and The lipid-linked oligosaccharyl donor, Glc3Man9GlcNAc2-P-P-dolichol (mature DLO 2The abbreviations used are: DLOGlc3Man9GlcNAc2-P-P-DolERendoplasmic reticulumM5-DLOMan5GlcNAc2-P-P-Dolmature Man-P-DolmannosylphosphoryldolicholGlc-P-DolglucosylphosphoryldolicholCPYcarboxypeptidase YFACEfluorophore-assisted carbohydrate electrophoresisCHOChinese hamster ovaryCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidOTaseoligosaccharyltransferase. 2The abbreviations used are: DLOGlc3Man9GlcNAc2-P-P-DolERendoplasmic reticulumM5-DLOMan5GlcNAc2-P-P-Dolmature Man-P-DolmannosylphosphoryldolicholGlc-P-DolglucosylphosphoryldolicholCPYcarboxypeptidase YFACEfluorophore-assisted carbohydrate electrophoresisCHOChinese hamster ovaryCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidOTaseoligosaccharyltransferase.), in protein N-glycosylation is formed in two stages in the endoplasmic reticulum (ER) (1Snider M.D. Robbins P.W. J. Biol. Chem. 1982; 257: 6796-6801Abstract Full Text PDF PubMed Google Scholar, 2Herscovics A. Orlean P. FASEB J. 1993; 7: 540-550Crossref PubMed Scopus (437) Google Scholar, 3Schenk B. Fernandez F. Waechter C.J. Glycobiology. 2001; 11: 61R-70RCrossref PubMed Scopus (141) Google Scholar, 4Helenius A. Aebi M. Annu. Rev. Biochem. 2004; 73: 1019-1049Crossref PubMed Scopus (1593) Google Scholar). 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 Glc3Man9GlcNAc2-P-P-Dol endoplasmic reticulum fluorophore-assisted carbohydrate electrophoresis hamster acid Glc3Man9GlcNAc2-P-P-Dol endoplasmic reticulum fluorophore-assisted carbohydrate electrophoresis hamster acid The PER5/RFT1 gene was originally identified by Walter and coworkers (5Ng D.T. Spear E.D. Walter P. J. Cell Biol. 2000; 150: 77-88Crossref PubMed Scopus (273) Google Scholar) 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 (6Koerte A. Chong T. Li X. Wahane K. Cai M. J. Biol. Chem. 1995; 270: 22556-22564Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), the rft1 mutation was shown to be inscrutably suppressed by p53, a soluble protein that has not been found in yeast. et al. (7Helenius J. Ng D.T. Marolda C.L. Walter P. Valvano M.A. Aebi M. Nature. 2002; 415: 447-450Crossref PubMed Scopus (217) Google Scholar) 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 (8Haeuptle M.A. Pujol F.M. Neupert C. Winchester B. Kastaniotis A.J. Aebi M. Hennet T. Am. J. Hum. Genet. 2008; 82: 600-606Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). 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 reports (9Frank C.G. Sanyal S. Rush J.S. Waechter C.J. Menon A.K. Nature. 2008; 454: E3-5Crossref PubMed Scopus (57) Google Scholar, 10Sanyal S. Frank C.G. Menon A.K. Biochemistry. 2008; 47: 7937-7946Crossref PubMed Scopus (71) Google Scholar) 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 (11Sanyal S. Menon A.K. Proc. PubMed Scopus Google Scholar). The study was conducted to further the role of Rft1 in the transbilayer movement of M5-DLO in the results the accumulation of of M5-DLO in the Rft1-depleted cells by the results by metabolic labeling in the yeast (7Helenius J. Ng D.T. Marolda C.L. Walter P. Valvano M.A. Aebi M. Nature. 2002; 415: 447-450Crossref PubMed Scopus (217) Google Scholar) and human (8Haeuptle M.A. Pujol F.M. Neupert C. Winchester B. Kastaniotis A.J. Aebi M. Hennet T. Am. J. Hum. Genet. 2008; 82: 600-606Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) mutant cells. a series of experiments conducted with sealed microsomal vesicles indicate although Rft1 may be required to a for the flipping of M5-DLO in vivo, depletion does not the flipping of M5-DLO in sealed microsomal in The of these results to the of the for the gene M. Rush J.S. S. M.A. J. J. Waechter C.J. M.A. Biol. 2001; PubMed Scopus Google Scholar) in the transverse diffusion of and for lipid intermediate biosynthesis of intact cells is has been for many that play in in and the of glycolipid intermediates in and biosynthesis A.K. Cell Biol. 1995; Full Text PDF PubMed Scopus Google Scholar, T. Menon A.K. PubMed Scopus Google Scholar, T. A. J. Cell 2004; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the many and of the has been in and ER proteins involved in the of Man-P-Dol, Glc-P-Dol, and the evidence (7Helenius J. Ng D.T. Marolda C.L. Walter P. Valvano M.A. Aebi M. Nature. 2002; 415: 447-450Crossref PubMed Scopus (217) Google Scholar) that Rft1 in the transbilayer movement of M5-DLO in this The of the in vitro reported was to more evidence to a role of Rft1 in the transbilayer movement of M5-DLO sealed microsomal vesicles from yeast as a evidence that large of M5-DLO accumulated in Rft1-depleted yeast several of experiments indicate that Rft1 is not required for the transverse diffusion of M5-DLO from the cytoplasmic leaflet to the lumenal in sealed microsomal the precise of depletion of Rft1 in the vesicles used as a is on a of endogenous Rft1 into the cells after the from to as the cells of the of Rft1 after to after is on the that Rft1 synthesis the to as and that Rft1 is not the formation of was by sealed microsomes with these in vitro sealed vesicles from wild type and Rft1-depleted cells formed and no accumulation of was The of into and was in sealed vesicles from Rft1-depleted cells compared with wild type that was up-regulated in response to of Rft1 be that the enzymes that Dol-P as acceptor and an in the of exogenously that microsomal fractions from Rft1-depleted cells levels of endogenous Dol-P for lipid intermediate biosynthesis relative to wild type cells. is that this of to the of M5-DLO after Rft1 depletion that M5-DLO was in sealed the was by endogenous by the microsomal with UDP-[3H]GlcNAc by the addition of unlabeled The that was by and Rft1-depleted microsomes evidence that formed M5-DLO was in sealed microsomes from wild type and Rft1-depleted cells the is that the of formed by Rft1-depleted microsomes was the is that the large of M5-DLO by there was no evidence that the M5-DLO, was by the large endogenous is that a of the M5-DLO in a compartment as observed in M.A. Glycobiology. PubMed Scopus Google evidence for M5-DLO flippase was the transport of GlcNAc2-P-P-Dol15, a water-soluble analogue of that has been shown to be presumably by the flippase the transbilayer movement of M5-DLO, in microsomes from a of and yeast F. C. A. P. PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Waechter C.J. Biochem. PubMed Scopus Google Scholar, C. PubMed Scopus Google Scholar). The results from this that the analogue the sealed microsomal vesicles from wild type and Rft1-depleted cells the The that transport of the water-soluble analogue to a in Rft1-depleted vesicles that the Rft1-depleted microsomal vesicles are more in ER or may have a as a of the unfolded protein of the analogue to the lumenal compartment was by that the was transferred to a peptide results a are in with recent related (9Frank C.G. Sanyal S. Rush J.S. Waechter C.J. Menon A.K. Nature. 2008; 454: E3-5Crossref PubMed Scopus (57) Google Scholar, 10Sanyal S. Frank C.G. Menon A.K. Biochemistry. 2008; 47: 7937-7946Crossref PubMed Scopus (71) Google Scholar, S. Menon A.K. Proc. PubMed Scopus Google Scholar) have demonstrated that the flipping of M5-DLO in reconstituted assessed by a in the presence or of The reconstituted flippase activity be from Rft1 and a by S. Frank C.G. Menon A.K. Biochemistry. 2008; 47: 7937-7946Crossref PubMed Scopus (71) Google Scholar), and the flippase activity between the intermediate that is M5-DLO, and (11Sanyal S. Menon A.K. Proc. PubMed Scopus Google study the of the mutant cells by that several enzymes involved in Dol-P and lipid intermediate biosynthesis are as a result of the of Rft1 In addition, the rate of transport of and was in vesicles from Rft1-depleted cells relative to wild type cells not that and as as the enzymes involved in Dol-P and lipid intermediate synthesis in be up-regulated as a response to the transbilayer movement or accumulation of M5-DLO in vivo. In related were identified on the of for an intact unfolded protein response for (5Ng D.T. Spear E.D. Walter P. J. Cell Biol. 2000; 150: 77-88Crossref PubMed Scopus (273) Google Scholar), and et al. J.S. Walter P. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) have reported that enzymes in the and are up-regulated the unfolded protein response in S. cerevisiae on the results in this not the that Rft1 a role in the flipping of M5-DLO in intact cells. that or that Rft1 for flipping in no after cells are is of the function of is for the of and in intact cells not in ER vesicles M. Rush J.S. S. M.A. J. J. Waechter C.J. M.A. Biol. 2001; PubMed Scopus Google Scholar). is that of the of the ER are when cells are disrupted the of microsomal In more and be required to the precise function of Rft1 in and to ER proteins that are involved in the transbilayer movement of M5-DLO as as and has been for many that play in in and the of glycolipid intermediates in and biosynthesis A.K. Cell Biol. 1995; Full Text PDF PubMed Scopus Google Scholar, T. Menon A.K. PubMed Scopus Google Scholar, T. A. J. Cell 2004; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the many and of the has been in and ER proteins involved in the of Man-P-Dol, Glc-P-Dol, and the evidence (7Helenius J. Ng D.T. Marolda C.L. Walter P. Valvano M.A. Aebi M. Nature. 2002; 415: 447-450Crossref PubMed Scopus (217) Google Scholar) that Rft1 in the transbilayer movement of M5-DLO in this The of the in vitro reported was to more evidence to a role of Rft1 in the transbilayer movement of M5-DLO sealed microsomal vesicles from yeast as a Although evidence that large of M5-DLO accumulated in Rft1-depleted yeast several of experiments indicate that Rft1 is not required for the transverse diffusion of M5-DLO from the cytoplasmic leaflet to the lumenal in sealed microsomal Although the precise of depletion of Rft1 in the vesicles used as a is on a of endogenous Rft1 into the cells after the from to as the cells of the of Rft1 after to after is on the that Rft1 synthesis the to as and that Rft1 is not In the formation of was by sealed microsomes with these in vitro sealed vesicles from wild type and Rft1-depleted cells formed and no accumulation of was The of into and was in sealed vesicles from Rft1-depleted cells compared with wild type that was up-regulated in response to of Rft1 be that the enzymes that Dol-P as acceptor and an in the of exogenously that microsomal fractions from Rft1-depleted cells levels of endogenous Dol-P for lipid intermediate biosynthesis relative to wild type cells. is that this of to the of M5-DLO after Rft1 depletion To that M5-DLO was in sealed the was by endogenous by the microsomal with UDP-[3H]GlcNAc by the addition of unlabeled The that was by and Rft1-depleted microsomes evidence that formed M5-DLO was in sealed microsomes from wild type and Rft1-depleted cells the is that the of formed by Rft1-depleted microsomes was the is that the large of M5-DLO by there was no evidence that the M5-DLO, was by the large endogenous is that a of the M5-DLO in a compartment as observed in M.A. Glycobiology. PubMed Scopus Google Scholar). To evidence for M5-DLO flippase was the transport of GlcNAc2-P-P-Dol15, a water-soluble analogue of that has been shown to be presumably by the flippase the transbilayer movement of M5-DLO, in microsomes from a of and yeast F. C. A. P. PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Waechter C.J. Biochem. PubMed Scopus Google Scholar, C. PubMed Scopus Google Scholar). The results from this that the analogue the sealed microsomal vesicles from wild type and Rft1-depleted cells the The that transport of the water-soluble analogue to a in Rft1-depleted vesicles that the Rft1-depleted microsomal vesicles are more in ER or may have a as a of the unfolded protein of the analogue to the lumenal compartment was by that the was transferred to a peptide The results a are in with recent related (9Frank C.G. Sanyal S. Rush J.S. 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