Document Detail


Expression of functional bacterial undecaprenyl pyrophosphate synthase in the yeast rer2{Delta} mutant and CHO cells.
MedLine Citation:
PMID:  20685834     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Jeffrey S Rush; Sergey Matveev; Ziqiang Guan; Christian R H Raetz; C J Waechter
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2010-08-04
Journal Detail:
Title:  Glycobiology     Volume:  20     ISSN:  1460-2423     ISO Abbreviation:  Glycobiology     Publication Date:  2010 Dec 
Date Detail:
Created Date:  2010-11-05     Completed Date:  2011-02-17     Revised Date:  2013-05-29    
Medline Journal Info:
Nlm Unique ID:  9104124     Medline TA:  Glycobiology     Country:  England    
Other Details:
Languages:  eng     Pagination:  1585-93     Citation Subset:  IM    
Affiliation:
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, BBSRB, 741 S Limestone St, Lexington, KY 40536, USA.
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MeSH Terms
Descriptor/Qualifier:
Alkyl and Aryl Transferases / biosynthesis*,  genetics
Animals
CHO Cells
Cricetinae
Cricetulus
Dimethylallyltranstransferase*
Endoplasmic Reticulum / enzymology,  genetics
Escherichia coli / enzymology*,  genetics
Escherichia coli Proteins / biosynthesis*,  genetics
Gene Expression*
Genetic Complementation Test
Mutation
Recombinant Proteins / biosynthesis*,  genetics
Saccharomyces cerevisiae / enzymology*,  genetics
Saccharomyces cerevisiae Proteins*
Grant Support
ID/Acronym/Agency:
GM-069338/GM/NIGMS NIH HHS; GM36035/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Escherichia coli Proteins; 0/Recombinant Proteins; 0/Saccharomyces cerevisiae Proteins; EC 2.5.-/Alkyl and Aryl Transferases; EC 2.5.1.1/Dimethylallyltranstransferase; EC 2.5.1.1/RER2 protein, S cerevisiae; EC 2.5.1.31/undecaprenyl pyrophosphate synthetase
Comments/Corrections

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