Format

Send to

Choose Destination
Glycobiology. 2010 Dec;20(12):1585-93. doi: 10.1093/glycob/cwq107. Epub 2010 Aug 4.

Expression of functional bacterial undecaprenyl pyrophosphate synthase in the yeast rer2{Delta} mutant and CHO cells.

Author information

1
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, BBSRB, 741 S Limestone St, Lexington, KY 40536, USA.

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.

PMID:
20685834
PMCID:
PMC3003547
DOI:
10.1093/glycob/cwq107
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Silverchair Information Systems Icon for PubMed Central
Loading ...
Support Center