Expression and characterization of a human cDNA that complements the temperature-sensitive defect in dolichol kinase activity in the yeast sec59-1 mutant: the enzymatic phosphorylation of dolichol and diacylglycerol are catalyzed by separate CTP-mediated kinase activities in Saccharomyces cerevisiae

Expression and characterization of a human cDNA that complements the temperature-sensitive defect... Dolichol kinase (DK) catalyzes the CTP-mediated phosphorylation of dolichol in eukaryotic cells, the terminal step in dolichyl monophosphate (Dol-P) biosynthesis de novo . In S. cerevisiae , the SEC59 gene encodes a protein essential for the expression of DK, an enzyme activity that is required for cell viability and normal rates of lipid intermediate synthesis and protein N-glycosylation. This study identifies a cDNA clone from human brain that encodes the mammalian homolog of DK (hDK1p). hDK1 is capable of complementing the growth defect, elevating DK activity, and consequently increasing Dol-P levels in vivo and restoring normal N-glycosylation of carboxypeptidase Y at the restrictive temperature in the temperature-sensitive mutant sec59-1 . The CTP-mediated phosphorylation of diacylglycerol (DAG) is unaffected by either the temperature-sensitive mutation in the sec59-1 strain, overexpression of the SEC59 gene, or the mammalian homolog hDK1 under conditions that produced a loss or elevation in the level of DK activity. Additionally, overexpression of hDK1p in Sf-9 cells resulted in a 15-fold increase in DK activity but not DAG kinase activity in crude microsomal fractions. The cloned cDNA contains an open reading frame that would encode a protein with 538 amino acids and a molecular weight of 59,268 kDa. Consistent with this prediction, new polypeptides were detected with an apparent molecular weight of 59–60 kDa when His 6 -tagged constructs of hDK1 or the SEC59 gene were expressed in Sf-9 cells or the temperature-sensitive sec59-1 mutant cells, respectively. These results identify the first cDNA clone encoding a protein required for the expression of DK activity, possibly the catalytic subunit, in a mammalian cell, and establish that the phosphorylation of dolichol and DAG are catalyzed by separate kinase activities in yeast. Key words http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Glycobiology Oxford University Press

Expression and characterization of a human cDNA that complements the temperature-sensitive defect in dolichol kinase activity in the yeast sec59-1 mutant: the enzymatic phosphorylation of dolichol and diacylglycerol are catalyzed by separate CTP-mediated kinase activities in Saccharomyces cerevisiae

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Publisher
Oxford University Press
Copyright
Copyright © 2015 Oxford University Press
ISSN
0959-6658
eISSN
1460-2423
DOI
10.1093/glycob/cwf068
Publisher site
See Article on Publisher Site

Abstract

Dolichol kinase (DK) catalyzes the CTP-mediated phosphorylation of dolichol in eukaryotic cells, the terminal step in dolichyl monophosphate (Dol-P) biosynthesis de novo . In S. cerevisiae , the SEC59 gene encodes a protein essential for the expression of DK, an enzyme activity that is required for cell viability and normal rates of lipid intermediate synthesis and protein N-glycosylation. This study identifies a cDNA clone from human brain that encodes the mammalian homolog of DK (hDK1p). hDK1 is capable of complementing the growth defect, elevating DK activity, and consequently increasing Dol-P levels in vivo and restoring normal N-glycosylation of carboxypeptidase Y at the restrictive temperature in the temperature-sensitive mutant sec59-1 . The CTP-mediated phosphorylation of diacylglycerol (DAG) is unaffected by either the temperature-sensitive mutation in the sec59-1 strain, overexpression of the SEC59 gene, or the mammalian homolog hDK1 under conditions that produced a loss or elevation in the level of DK activity. Additionally, overexpression of hDK1p in Sf-9 cells resulted in a 15-fold increase in DK activity but not DAG kinase activity in crude microsomal fractions. The cloned cDNA contains an open reading frame that would encode a protein with 538 amino acids and a molecular weight of 59,268 kDa. Consistent with this prediction, new polypeptides were detected with an apparent molecular weight of 59–60 kDa when His 6 -tagged constructs of hDK1 or the SEC59 gene were expressed in Sf-9 cells or the temperature-sensitive sec59-1 mutant cells, respectively. These results identify the first cDNA clone encoding a protein required for the expression of DK activity, possibly the catalytic subunit, in a mammalian cell, and establish that the phosphorylation of dolichol and DAG are catalyzed by separate kinase activities in yeast. Key words

Journal

GlycobiologyOxford University Press

Published: Sep 1, 2002

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