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. 1990 May;87(10):3874–3878. doi: 10.1073/pnas.87.10.3874

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

J Ayté 1, G Gil-Gómez 1, D Haro 1, P F Marrero 1, F G Hegardt 1
PMCID: PMC54006  PMID: 1971108

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. Identify 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. (ii) 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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boon M. R., Zammit V. A. Use of a selectively permeabilized isolated rat hepatocyte preparation to study changes in the properties of overt carnitine palmitoyltransferase activity in situ. Biochem J. 1988 Feb 1;249(3):645–652. doi: 10.1042/bj2490645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Clinkenbeard K. D., Reed W. D., Mooney R. A., Lane M. D. Intracellular localization of the 3-hydroxy-3-methylglutaryl coenzme A cycle enzymes in liver. Separate cytoplasmic and mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A generating systems for cholesterogenesis and ketogenesis. J Biol Chem. 1975 Apr 25;250(8):3108–3116. [PubMed] [Google Scholar]
  6. Dashti N., Ontko J. A. Rate-limiting function of 3-hydroxy-3-methylglutaryl-coenzyme A synthase in ketogenesis. Biochem Med. 1979 Dec;22(3):365–374. doi: 10.1016/0006-2944(79)90024-3. [DOI] [PubMed] [Google Scholar]
  7. Decaux J. F., Robin D., Robin P., Ferré P., Girard J. Intramitochondrial factors controlling hepatic fatty acid oxidation at weaning in the rat. FEBS Lett. 1988 May 9;232(1):156–158. doi: 10.1016/0014-5793(88)80407-1. [DOI] [PubMed] [Google Scholar]
  8. Gil G., Brown M. S., Goldstein J. L. Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase from the hamster. II. Isolation of the gene and characterization of the 5' flanking region. J Biol Chem. 1986 Mar 15;261(8):3717–3724. [PubMed] [Google Scholar]
  9. Gil G., Goldstein J. L., Slaughter C. A., Brown M. S. Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase from the hamster. I. Isolation and sequencing of a full-length cDNA. J Biol Chem. 1986 Mar 15;261(8):3710–3716. [PubMed] [Google Scholar]
  10. Gil G., Smith J. R., Goldstein J. L., Brown M. S. Optional exon in the 5'-untranslated region of 3-hydroxy-3-methylglutaryl coenzyme A synthase gene: conserved sequence and splicing pattern in humans and hamsters. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1863–1866. doi: 10.1073/pnas.84.7.1863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grant P. M., Tellam J., May V. L., Strauss A. W. Isolation and nucleotide sequence of a cDNA clone encoding rat mitochondrial malate dehydrogenase. Nucleic Acids Res. 1986 Aug 11;14(15):6053–6066. doi: 10.1093/nar/14.15.6053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Holness M. J., French T. J., Schofield P. S., Sugden M. C. The relationship between fat synthesis and oxidation in the liver after re-feeding and its regulation by thyroid hormone. Biochem J. 1987 Nov 1;247(3):621–626. doi: 10.1042/bj2470621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Khandjian E. W. UV crosslinking of RNA to nylon membrane enhances hybridization signals. Mol Biol Rep. 1986;11(2):107–115. doi: 10.1007/BF00364822. [DOI] [PubMed] [Google Scholar]
  14. Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 1984 Jan 25;12(2):857–872. doi: 10.1093/nar/12.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Long B. H., Huang C. Y., Pogo A. O. Isolation and characterization of the nuclear matrix in Friend erythroleukemia cells: chromatin and hnRNA interactions with the nuclear matrix. Cell. 1979 Dec;18(4):1079–1090. doi: 10.1016/0092-8674(79)90221-6. [DOI] [PubMed] [Google Scholar]
  17. Lowe D. M., Tubbs P. K. Succinylation and inactivation of 3-hydroxy-3-methylglutaryl-CoA synthase by succinyl-CoA and its possible relevance to the control of ketogenesis. Biochem J. 1985 Nov 15;232(1):37–42. doi: 10.1042/bj2320037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mehrabian M., Callaway K. A., Clarke C. F., Tanaka R. D., Greenspan M., Lusis A. J., Sparkes R. S., Mohandas T., Edmond J., Fogelman A. M. Regulation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A synthase and the chromosomal localization of the human gene. J Biol Chem. 1986 Dec 5;261(34):16249–16255. [PubMed] [Google Scholar]
  19. Miziorko H. M., Behnke C. E. Amino acid sequence of an active site peptide of avian liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase. J Biol Chem. 1985 Nov 5;260(25):13513–13516. [PubMed] [Google Scholar]
  20. Obaru K., Nomiyama H., Shimada K., Nagashima F., Morino Y. Cloning and sequence analysis of mRNA for mouse aspartate aminotransferase isoenzymes. J Biol Chem. 1986 Dec 25;261(36):16976–16983. [PubMed] [Google Scholar]
  21. Reed W. D., Clinkenbeard D., Lane M. D. Molecular and catalytic properties of mitochondrial (ketogenic) 3-hydroxy-3-methylglutaryl coenzyme A synthase of liver. J Biol Chem. 1975 Apr 25;250(8):3117–3123. [PubMed] [Google Scholar]
  22. Rogers S., Wells R., Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science. 1986 Oct 17;234(4774):364–368. doi: 10.1126/science.2876518. [DOI] [PubMed] [Google Scholar]
  23. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Suzuki T., Sato M., Yoshida T., Tuboi S. Rat liver mitochondrial and cytosolic fumarases with identical amino acid sequences are encoded from a single gene. J Biol Chem. 1989 Feb 15;264(5):2581–2586. [PubMed] [Google Scholar]
  26. Vollmer S. H., Mende-Mueller L. M., Miziorko H. M. Identification of the site of acetyl-S-enzyme formation on avian liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase. Biochemistry. 1988 Jun 14;27(12):4288–4292. doi: 10.1021/bi00412a014. [DOI] [PubMed] [Google Scholar]
  27. Volpe J. J., Obert K. A. Coordinate regulation of cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A synthase but not 3-hydroxy-3-methylglutaryl coenzyme A reductase in C-6 glia. Arch Biochem Biophys. 1981 Nov;212(1):88–97. doi: 10.1016/0003-9861(81)90346-5. [DOI] [PubMed] [Google Scholar]
  28. Williamson D. H., Bates M. W., Krebs H. A. Activity and intracellular distribution of enzymes of ketone-body metabolism in rat liver. Biochem J. 1968 Jul;108(3):353–361. doi: 10.1042/bj1080353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. von Heijne G. Mitochondrial targeting sequences may form amphiphilic helices. EMBO J. 1986 Jun;5(6):1335–1342. doi: 10.1002/j.1460-2075.1986.tb04364.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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