Identification and characterization of a cDNA encoding a long-chain cis-isoprenyltranferase involved in dolichyl monophosphate biosynthesis in the ER of brain cells.
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 rer2Delta 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.
Shridas
Preetha
P
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, USA.
Rush
Jeffrey S
JS
Waechter
Charles J
CJ
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D001923:Brain Chemistry; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D004288:Dolichol Phosphates / Q000096:biosynthesis*; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology*; D005656:Fungal Proteins / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006019:Glycolysis / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D018076:DNA, Complementary; D004288:Dolichol Phosphates; D005656:Fungal Proteins; C028306:dolichol monophosphate; D019883:Alkyl and Aryl Transferases; C084921:cis-isoprenyltransferase