cDNA cloning of MEV, a mutant protein that facilitates cellular uptake of mevalonate, and identification of the point mutation responsible for its gain of function.
We report the expression cloning of pMev, a cDNA that facilitates cellular uptake of mevalonate. pMev was isolated from the met-18b-2 clone of Chinese hamster ovary (CHO) cells, which were selected for growth in low concentrations of mevalonate when synthesis is blocked by compactin (Faust, J. R., and Krieger, M. (1987) J. Biol. Chem. 262, 1996-2004). pMev encodes a 494-residue protein, Mev, that is predicted to have 12 membrane-spanning regions, consistent with a membrane transporter. Surprisingly, levels of Mev mRNA and protein are similar in CHO and met-18b-2 cells. The Mev gene differs from the wild-type gene by a single base change that substitutes a cysteine for phenylalanine in the 10th membrane-spanning region. met-18b-2 cells are heterozygous for this dominant gain-of-function mutation. Transfection of a cDNA encoding pMev, but not the wild-type cDNA, elicited a marked increase in [3H]mevalonate uptake and incorporation into cellular lipids in stably and transiently transfected cells. The availability of pMev will facilitate studies of [3H]mevalonate incorporation into trace products, including p21ras and other prenylated proteins.
Kim
C M
CM
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.
Goldstein
J L
JL
Brown
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D016466:CHO Cells; D002352:Carrier Proteins; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D015723:Gene Library; D005799:Genes, Dominant; D006801:Humans; D008148:Lovastatin / Q000031:analogs & derivatives / Q000494:pharmacology; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008647:Mesocricetus; D008798:Mevalonic Acid / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014162:Transfection
D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D012333:RNA, Messenger; C077949:Mev protein, Cricetulus griseus; C012258:mevastatin; D008148:Lovastatin; D008798:Mevalonic Acid