Isolation of a chinese hamster ovary (CHO) cDNA encoding phosphatidylglycerophosphate (PGP) synthase, expression of which corrects the mitochondrial abnormalities of a PGP synthase-defective mutant of CHO-K1 cells.
Phosphatidylglycerophosphate (PGP) synthase catalyzes the first step in the cardiolipin (CL) branch of phospholipid biosynthesis in mammalian cells. In this study, we isolated a Chinese hamster ovary (CHO) cDNA encoding a putative protein similar in sequence to the yeast PGS1 gene product, PGP synthase. The gene for the isolated CHO cDNA was named PGS1. Expression of the CHO PGS1 cDNA in CHO-K1 cells and production of a recombinant CHO PGS1 protein with a N-terminal extension in Escherichia coli resulted in 15-fold and 90-fold increases of PGP synthase specific activity, respectively, establishing that CHO PGS1 encodes PGP synthase. A PGP synthase-defective CHO mutant, PGS-S, isolated previously (Ohtsuka, T., Nishijima, M., and Akamatsu, Y. (1993) J. Biol. Chem. 268, 22908-22913) exhibits striking reductions in biosynthetic rate and cellular content of phosphatidylglycerol (PG) and CL and shows mitochondrial morphological and functional abnormalities. The CHO PGS-S mutant transfected with the CHO PGS1 cDNA exhibited 620-fold and 7-fold higher PGP synthase activity than mutant PGS-S and wild type CHO-K1 cells, respectively, and had a normal cellular content and rate of biosynthesis of PG and CL. In contrast to mutant PGS-S, the transfectant had morphologically normal mitochondria. When the transfectant and mutant PGS-S cells were cultivated in a glucose-depleted medium, in which cellular energy production mainly depends on mitochondrial function, the transformant but not mutant PGS-S was capable of growth. These results demonstrated that the morphological and functional defects displayed by the PGS-S mutant are due directly to the reduced ability to make normal levels of PG and/or CL.
Kawasaki
K
K
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan.
Kuge
O
O
Chang
S C
SC
Heacock
P N
PN
Rho
M
M
Suzuki
K
K
Nishijima
M
M
Dowhan
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002308:Cardiolipins / Q000096:biosynthesis; D006224:Cricetinae; D018076:DNA, Complementary / Q000302:isolation & purification; D006801:Humans; D008854:Microscopy, Electron; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009154:Mutation; D010085:Oxidative Phosphorylation; D010715:Phosphatidylglycerols / Q000096:biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D017855:Transferases (Other Substituted Phosphate Groups) / Q000096:biosynthesis / Q000235:genetics*
D002308:Cardiolipins; D018076:DNA, Complementary; D010715:Phosphatidylglycerols; D012333:RNA, Messenger; D017855:Transferases (Other Substituted Phosphate Groups); C020676:CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase