Expression of functional bacterial undecaprenyl pyrophosphate synthase in the yeast rer2Δ mutant and CHO cells

Jeffrey S. Rush, Sergey Matveev, Ziqiang Guan, Christian R.H. Raetz, C. J. Waechter

Research output: Contribution to journalArticlepeer-review

10 Scopus citations


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 Glc3Man9GlcNAc2-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 [3H]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 [14C](55)polyprenyl-P-P intermediate when incubated with [ 14C]isopentenyl pyrophosphate and [3H]Man-P-(55)Dol when incubated with GDP-[3H]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.

Original languageEnglish
Pages (from-to)1585-1593
Number of pages9
Issue number12
StatePublished - Dec 2010

Bibliographical note

Funding Information:
This work was supported by National Institutes of Health (NIH) grant GM36035 (CJW), and the mass spectrometry facility in the Department of Biochemistry of the Duke University Medical Center and Dr. Ziqiang Guan are supported by the LIPID MAPS Large Scale Collaborative grant number GM-069338 from NIH.


  • CHO cells
  • cis-isoprenyltransferase
  • dolichyl phosphate
  • undecaprenyl pyrophosphate synthase
  • yeast mutant

ASJC Scopus subject areas

  • Biochemistry


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