Suppression of Rft1 expression does not impair the transbilayer movement of Man5GlcNAc2-P-P-dolichol in sealed microsomes from yeast

Jeffrey S. Rush, Ningguo Gao, Mark A. Lehrman, Sergey Matveev, Charles J. Waechter

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)19835-19842
Number of pages8
JournalJournal of Biological Chemistry
Volume284
Issue number30
DOIs
StatePublished - Jul 24 2009

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Microsomes
Yeast
Yeasts
Dolichol
Peptides
Uridine Diphosphate
Fluorophores
Glycolipids
Biosynthesis
Mannose
Electrophoresis
Guanosine Diphosphate Mannose
Carbohydrates
Lipids
mannosyl(5)-N-acetyl(2)-glucose diphosphate dolichol
Water
Enzymes
Proteins
Experiments

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Suppression of Rft1 expression does not impair the transbilayer movement of Man5GlcNAc2-P-P-dolichol in sealed microsomes from yeast. / Rush, Jeffrey S.; Gao, Ningguo; Lehrman, Mark A.; Matveev, Sergey; Waechter, Charles J.

In: Journal of Biological Chemistry, Vol. 284, No. 30, 24.07.2009, p. 19835-19842.

Research output: Contribution to journalArticle

Rush, Jeffrey S. ; Gao, Ningguo ; Lehrman, Mark A. ; Matveev, Sergey ; Waechter, Charles J. / Suppression of Rft1 expression does not impair the transbilayer movement of Man5GlcNAc2-P-P-dolichol in sealed microsomes from yeast. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 30. pp. 19835-19842.
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abstract = "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.",
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