Abstract
During protein N-glycosylation, dolichyl pyrophosphate (Dol-P-P) is discharged in the lumenal monolayer of the endoplasmic reticulum (ER). Dol-P-P is then cleaved to Dol-P by Dol-P-P phosphatase (DPPase). Studies with the yeast mutant cwh8Delta, lacking DPPase activity, indicate that recycling of Dol-P produced by DPPase contributes significantly to the pool of Dol-P utilized for lipid intermediate biosynthesis on the cytoplasmic leaflet. Whether Dol-P formed in the lumen diffuses directly back to the cytoplasmic leaflet or is first dephosphorylated to dolichol has not been determined. Incubation of sealed ER vesicles from calf brain with acetyl-Asn-Tyr-Thr-NH(2), an N-glycosylatable peptide, to generate Dol-P-P in the lumenal monolayer produced corresponding increases in the rates of Man-P-Dol, Glc-P-Dol, and GlcNAc-P-P-Dol synthesis in the absence of CTP. No changes in dolichol kinase activity were observed. When streptolysin-O permeabilized CHO cells were incubated with an acceptor peptide, N-glycopeptide synthesis, requiring multiple cycles of the dolichol pathway, occurred in the absence of CTP. The results obtained with sealed microsomes and CHO cells indicate that Dol-P, formed from Dol-P-P, returns to the cytoplasmic leaflet where it can be reutilized for lipid intermediate biosynthesis, and dolichol kinase is not required for recycling. It is possible that the flip-flopping of the carrier lipid is mediated by a flippase, which would provide a mechanism for the recycling of Dol-P derived from Man-P-Dol-mediated reactions in N-, O-, and C-mannosylation of proteins, GPI anchor assembly, and the three Glc-P-Dol-mediated reactions in Glc(3)Man(9)GlcNAc(2)-P-P-Dol (DLO) biosynthesis.
Highlights
The observation that lipid intermediate biosynthesis and protein N-glycosylation are impaired in the yeast mutant, cwh8⌬, lacking the lumenally oriented dolichyl pyrophosphate (Dol-P-P) phosphatase [4, 6], suggests that recycling of Dol-P-P from the lumenal leaflet contributes significantly to the pool of Dol-P utilized for lipid intermediate biosynthesis
Because no enzymatic degradation of dolichol has been reported, it is likely that the glycosyl carrier lipid participates in multiple rounds of lipid intermediate biosynthesis
The possibility that Dol-P-P diffused transversely to the cytoplasmic leaflet, where it could be converted to Dol-P was not explored since the yeast [4] and mammalian [5] Dol-P-P phosphatases have been shown to have lumenally oriented active sites and Dol-P-P accumulates in the cwh8⌬ yeast strain [4]
Summary
Materials—[3H]Mannose (60 Ci/mmol), UDP-[3H]glucose (20 Ci/mmol), UDP-N-[14C]acetylglucosamine (55 mCi/ mmol), and [␥-32P]ATP (ϳ500 Ci/mmol) were purchased from American Radiolabeled Chemicals Assays for the In Vitro Synthesis of Man-P-Dol, GlcNAc1–2-PP-Dol, Glc-P-Dol, and Glc3Man9GlcNAc2-P-P-Dol—Reaction mixtures contained 50 mM Tris-HCl, pH 8.0, 0.25 M sucrose, 5 mM CaCl2, 5 mM MgCl2, 4 mM AMP, calf brain microsomal fraction (0.8 mg membrane protein) and either 5 M GDP[3H]Man (4,200 dpm/pmol), 5 M UDP-[3H]Glc (2,000 dpm/ pmol), or 5 M UDP-[14C]GlcNAc (349 dpm/pmol) in a total volume of 0.1 ml. None of these assay mixtures contained CTP. Analytical Procedures—Protein concentrations were determined using the BCA protein assay (Pierce) following precipitation of membrane proteins with deoxycholate and trichloroacetic acid according to the Pierce Biotechnology Bulletin, “Eliminate Interfering Substances from Samples for BCA Protein Assay.” Samples were analyzed for radioactivity by scintillation spectrometry in a Packard Tri-Carb 2100TR liquid scintillation spectrometer following the addition of 0.5 ml of 1% SDS and 4 ml of Econosafe Economical Biodegradable Counting Mixture (Research Products International, Corp., Mount Prospect, IL)
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