Rat mitochondrial and cytosolic 3-hydroxy-3-methylglutaryl-CoA synthases are encoded by two different genes.

Abstract

We report the isolation and characterization of a 1994-base-pair cDNA that encompasses the entire transcription unit of the mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (EC 4.1.3.5.) gene from rat. Analysis of the nucleotide sequence reveals that the cDNA encodes a polypeptide of 508 residues and 56,918-Da molecular mass. Identity of the cDNA clone isolated as mitochondrial HMG-CoA synthase was confirmed by the following criteria: (i) Amino acid residues are 65% homologous with hamster cytosolic HMG-CoA synthase. (i) A 19-amino acid sequence probably corresponding to the catalytic site is highly homologous (90%) to that reported for chicken liver mitochondrial HMG-CoA synthase. (iii) The expression product of the cDNA in Escherichia coli has HMG-CoA synthase activity. (iv) The protein includes a sequence of 37 amino acid residues at the N terminus that is not present in the cytosolic enzyme. The predominantly basic, hydrophobic, and hydroxylated nature of the residues of this sequence suggests that it is a leader peptide to target HMG-CoA synthase inside mitochondria. These data plus the hybridization pattern in genomic Southern blot analysis, the different transcript size (2.0 kilobases versus 3.4 kilobases for the cytosolic enzyme), and the different expression pattern shown in RNA blot experiments suggest the presence of two HMG-CoA synthase genes, one for the cytosolic and another for the mitochondrial enzyme.3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (EC 4.1.3.5.) catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form HMG-CoA and CoA. In liver this reaction occurs in two essentially different pathways: (i) the production of mevalonate from HMG-CoA prior to the synthesis of sterols and isoprenoids of various functions; and (ii) the conversion of HMG-CoA into acetoacetate and then into other related compounds by means of the ketogenetic pathway that occurs in mitochondria.Mitochondrial HMG-CoA synthase has been assumed to be the rate-limiting enzyme of ketogenesis in rat liver (1, 2). Although carnitine palmitoyl transferase I is also considered an important control of the ketogenic flux in liver, under certain conditions control shifts away from this enzyme to an intramitochondrial control site (3, 4). Many groups suggest that this control lies within the pathway from acetyl-CoA to ketonic bodies, the HMG-CoA pathway (5, 6).Gil et al. (7)(8)(9) speculated that both cytosolic and mitochondrial HMG-CoA synthase enzymes were products of the same gene, transcribed through an alternative splicing mechanism. This hypothesis was generated from the observation that the catalytic domain of both synthases was nearly homologous (7, 10) and that an alternative splicing pattern of exon 2 in cytosolic HMG-CoA synthase occurs among various tissues through development. This also holds true between human and hamster genes (8, 9).However, because mitochondrial and cytoplasmic HMGCoA synthases differ in charge and immunological properties (11) and no clear splicing...

Analysis of the polyadenylation consensus sequence context in the genes of nuclear encoded mitochondrial proteins

“…(H CK); rat fumarase [76], (R mtFumra); human branched chain ~-keto acid dehydrogenase complex subunit E1 ~ [77], (H E1 BCKD); rat brain and liver cytochrome oxidase subunit IV [78], (R coxlV); bovine uncoupling protein [79], (B Ucp); rat cytochrome c oxidase subunh Via (Vb) [80], (R coxVb); human cytochrome c oxidase subunit Via [81], (H coxVia); human NAD-dependent methylene tetrahydrofolate dehydrogenase-cyc!ohydrolase [82], (H MTHFdeh); rat short chain acyl-CoA dehydrogenase and isovaleryl-CoA dehydrogenase [83], (R SCCode, R IVD); mouse cytochrome oxidase subunit Ya [84], (M coxVa); human cytochrome bcl complex core protein 2 [85]; human cytOchrome c oxidase subunit VIIa [8 ! ], (H coxVIla); human ADP/ATP translocase [87], (H AD/TPtl, H AD/TPt2); bovine ADP/ATP translocase [88], (B AD/TPtl, B AD/TPt2); rat cytochrome c oxidase subunit Va [89], (R coxVa); bovine ATP synthase subunit 7' [90], (B ATPsynt); rat 3-hydroxy-3-methyl-glutarylCoA synthase [91], (R HMGCoAs); human sarcomerespecific creatine kinase [92], (H sarc.CK); humar, cytochrome c oxidase subunit VIIc [93], (H coxVIIc); rat monoamine oxidase A [94] %G 16 17 19 17 14 Z1 17 25 19 24 25 21 22 23 %A 35 22 26 29 24 23 28 26 30 30 31 33 28 28 %T 34 34 36 33 2d 36 35 28 33 28 31 29 30 32 tC 16 28 i~ 21 28 20 19 20 18 IB 13 17 20 17 poaxuxon -13 -12 -ii -10 IG 11 21 20 15 15 12 17 21 17 11 25 19 10 0 %~ 42 26 23 26 26 37 29 35 27 24 23 24 31 9 7 %T 39 35 42 42 28 31 32 30 34 47 42 41 25 2 %C 8 18 15 16 31 20 22 15 22 18 I0 16 Table t for every gene. This bank contains the presently known 110 prem~gNA ends and is most likely repre~enta~cive for all genes of nuclear encoded mitochondrial proteins.…”

Prevention of diabetic alterations in transgenic mice overexpressing Myc in the liver.

“…CPT I and CPT II), two membrane-bound enzymes responsible for the entrance of acyl-CoA into mitochondria, and the mitochondrial HMG-CoA synthase, a regulatory enzyme of the ketone-body pathway, have been considered to control liver ketogenesis (35)(36)(37)(38)(39). These enzyme activities increase during diabetes.…”

Isolation of pig mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene promoter: characterization of a peroxisome proliferator-responsive element

“…Such changes of gene expression have been correlated with nutritional and hormonal The transcription start site, indicated by arrows, was determined by 5-RACE for the human [44] and pig (the present study) genes, and by primer extension and S1 experiments for the rat [52] gene. The functionality of PPRE was shown experimentally for the rat [13] and pig gene (the present study).…”