Transfection of the ketogenic mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase cDNA into Mev-1 cells corrects their auxotrophy for mevalonate.
A somatic cell mutant of the Chinese hamster ovary (CHO)-K1 (called Mev-1), auxotrophic for mevalonate by virtue of a complete lack of detectable cytosolic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase activity, was transfected with a plasmid containing the cDNA for ketogenic mitochondrial HMG-CoA synthase under the control of SV40 early promoter. The resulting stable cell line (Mev-SM) was able to grow in the absence of mevalonate. Analysis of Western blot showed that the new cell line strongly expressed mitochondrial HMG-CoA synthase protein. Immunocytochemical studies using specific antibodies against mitochondrial HMG-CoA synthase showed that the protein was located exclusively inside the mitochondria. The prototroph cell line Mev-SM can incorporate labeled acetate into cholesterol in the absence of mevalonate. These results show that the new cell line may circumvent the lack of cytosolic HMG-CoA synthase activity by producing cholesterol-convertible HMG-CoA inside the mitochondria.
Unit of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Spain.
J A
JA
Ortiz
G
G
Gil-Gómez
R P
RP
Casaroli-Marano
S
S
Vilaró
F G
FG
Hegardt
D
D
Haro
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002784:Cholesterol / Q000096:biosynthesis; D006224:Cricetinae; D018076:DNA, Complementary; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000235:genetics* / Q000378:metabolism; D007659:Ketones / Q000378:metabolism*; D015636:Magnesium Chloride / Q000494:pharmacology; D008798:Mevalonic Acid / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008928:Mitochondria / Q000201:enzymology* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009154:Mutation; D014162:Transfection
D018076:DNA, Complementary; D007659:Ketones; D015636:Magnesium Chloride; D002784:Cholesterol; D006904:Hydroxymethylglutaryl-CoA Synthase; D008798:Mevalonic Acid