Identification and characterization of a cDNA encoding a long-chain cis-isoprenyltranferase involved in dolichyl monophosphate biosynthesis in the ER of brain cells

Preetha Shridas, Jeffrey S. Rush, Charles J. Waechter

Research output: Contribution to journalArticlepeer-review

34 Scopus citations

Abstract

A long-chain cis-isoprenyltransferase (cis-IPTase) located in the endoplasmic reticulum (ER) catalyzes the chain elongation stage in the pathway for the de novo biosynthesis of dolichyl monophosphate (Dol-P) in eukaryotic cells. In Saccharomyces cerevisiae, the ER-associated cis-IPTase is encoded by the RER2 gene. Mutations in the RER2 gene result in defects in growth and protein N-glycosylation. In this study a cDNA isolated from human brain (Accession No. AK023164.1), which has substantial homology to cis-IPTases from bacteria, Arabidopsis, and S. cerevisiae, has been shown to: (1) complement the growth defect; (2) restore cis-IPTase activity; dolichol and Dol-P synthesis; and (3) restore normal N-glycosylation of carboxypeptidase Y (CPY) in the yeast rer2Δ mutant. Consistent with a role in Dol-P biosynthesis, overexpression of the human cis-isoprenyltransferase (hCIT) cDNA also suppresses the temperature-sensitive growth and CPY hypoglycosylation phenotypes in sec59-1 cells which are defective in Dol-P biosynthesis due to a temperature-sensitive mutation in dolichol kinase. Overexpression of hCIT in Chinese hamster ovary (CHO) cells results in a modest increase in cis-IPTase activity associated with microsomal fractions and the appearance of a new 38kDa polypeptide that co-localizes with calnexin in the ER, the site of Dol-P biosynthesis, even though no transmembrane domains are predicted by a hydropathy plot.

Original languageEnglish
Pages (from-to)1349-1356
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume312
Issue number4
DOIs
StatePublished - Dec 26 2003

Bibliographical note

Funding Information:
The authors thank Dr. Myung-Hee Kim for many helpful suggestions with the immunofluorescence and other aspects of this work, and Leroy Pakkiri for carefully editing the manuscript. The authors also acknowledge the technical assistance of Dr. Susan Wang. This research was supported by National Institutes of Health Grant GM36035 awarded to C.J.W.

Keywords

  • Allylic polyprenyl pyrophosphate
  • Dolichyl monophosphate
  • Endoplasmic reticulum
  • cis-Isoprenyltransferase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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