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.
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.
Fernandez
Fabiana
F
Institut fur Mikrobiologie, ETH Zentrum, CH-8092 Zurich, Switzerland.
Shridas
Preetha
P
Jiang
Songmin
S
Aebi
Markus
M
Waechter
Charles J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003570:Cytidine Triphosphate / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary; D004075:Diglycerides / Q000378:metabolism*; D004286:Dolichols / Q000378:metabolism*; D005800:Genes, Fungal; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera
D017931:DNA Primers; D018076:DNA, Complementary; D004075:Diglycerides; D004286:Dolichols; D003570:Cytidine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020087:dolichol kinase