title: Expression of functional bacterial undecaprenyl pyrophosphate synthase in the yeast rer2{Delta} mutant and CHO cells.
abstract: During evolution the average chain length of polyisoprenoid glycosyl carrier lipids increased from C55 (prokaryotes) to C75 (yeast) to C95 (mammalian cells). In this study, the ability of the E. coli enzyme, undecaprenyl pyrophosphate synthase (UPPS), to complement the loss of the yeast cis-isoprenyltransferase in the rer2Δ mutant was tested to determine if (55)dolichyl phosphate (Dol-P) could functionally substitute in the protein N-glycosylation pathway for (75)Dol-P, the normal isoprenologue synthesized in S. cerevisiae. First, expression of UPPS in the yeast mutant was found to complement the growth and the hypoglycosylation of carboxypeptidase Y defects suggesting that the (55)polyprenyl-P-P intermediate was converted to (55)Dol-P and that (55)Dol-P could effectively substitute for (75)Dol-P in the biosynthesis and function of Man-P-Dol, Glc-P-Dol and Glc(3)Man(9)GlcNAc(2)-P-P-Dol (mature DLO) in the protein N-glycosylation pathway and glycosylphosphatidylinositol anchor assembly. In support of this conclusion, mutant cells expressing UPPS (1) synthesized (55)Dol-P based on MS analysis, (2) utilized (55)Dol-P to form Man-P-(55)Dol in vitro and in vivo, and (3) synthesized N-linked glycoproteins at virtually normal rates as assessed by metabolic labeling with [(3)H]mannose. In addition, an N-terminal GFP-tagged construct of UPPS was shown to localize to the endoplasmic reticulum of Chinese hamster ovary cells. Consistent with the synthesis of (55)Dol-P by the transfected cells, microsomes from the transfected cells synthesized the [(14)C](55)polyprenyl-P-P intermediate when incubated with [(14)C]isopentenyl pyrophosphate and [(3)H]Man-P-(55)Dol when incubated with GDP-[(3)H]Man. These results indicate that (C55)polyisoprenoid chains, significantly shorter than the natural glycosyl carrier lipid, can function in the transbilayer movement of DLOs in the endoplasmic reticulum of yeast and mammalian cells, and that conserved sequences in the cis-isoprenyltransferases are recognized by, yet to be identified, binding partners in the endoplasmic reticulum of mammalian cells.
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, BBSRB, 741 S Limestone St, Lexington, KY 40536, USA.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Rush
authors.forename: Sergey
authors.initials: S
authors.lastname: Matveev
authors.forename: Ziqiang
authors.initials: Z
authors.lastname: Guan
authors.forename: Christian R H
authors.initials: CR
authors.lastname: Raetz
authors.forename: C J
authors.initials: CJ
authors.lastname: Waechter
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004122:Dimethylallyltranstransferase; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D005816:Genetic Complementation Test; D009154:Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D019883:Alkyl and Aryl Transferases; D004122:Dimethylallyltranstransferase; C485360:RER2 protein, S cerevisiae; C023266:undecaprenyl pyrophosphate synthetase