Skip to main content
PMC home page
Genetics logoLink to Genetics
. 2002 Jul;161(3):1043–1052. doi: 10.1093/genetics/161.3.1043

Mutational bisection of the mitochondrial DNA stability and amino acid biosynthetic functions of ilv5p of budding yeast.

Joseph M Bateman 1, Philip S Perlman 1, Ronald A Butow 1
PMCID: PMC1462179  PMID: 12136009

Abstract

Ilv5p is a bifunctional yeast mitochondrial enzyme required for branched chain amino acid biosynthesis and for the stability of mitochondrial DNA (mtDNA) and its parsing into nucleoids. The latter occurs when the general amino acid control (GAC) pathway is activated. We have isolated ilv5 mutants that lack either the enzymatic (a(-)D(+)) or the mtDNA stability function (a(+)D(-)) of the protein. The affected residues in these two mutant classes cluster differently when mapped to the 3-D structure of the spinach ortholog of Ilv5p. a(-)D(+) mutations map to conserved internal domains known to be important for substrate and cofactor binding, whereas the a(+)D(-) mutations map to a C-terminal region on the surface of the protein. The a(+)D(-) mutants also have a temperature-sensitive phenotype when grown on a glycerol medium, which correlates with their degree of mtDNA instability. Analysis of an a(+)D(-) mutant with a strong mtDNA instability phenotype shows that it is also unable to parse mtDNA into nucleoids when activated by the GAC pathway. Finally, the wild-type Escherichia coli ortholog of Ilv5p behaves like a(+)D(-) mutants when expressed and targeted to mitochondria in ilv5Delta yeast cells, suggesting that yeast Ilv5p acquired its mtDNA function after the endosymbiotic event.

Full Text

The Full Text of this article is available as a PDF (581.3 KB).

Selected References

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

  1. Biou V., Dumas R., Cohen-Addad C., Douce R., Job D., Pebay-Peyroula E. The crystal structure of plant acetohydroxy acid isomeroreductase complexed with NADPH, two magnesium ions and a herbicidal transition state analog determined at 1.65 A resolution. EMBO J. 1997 Jun 16;16(12):3405–3415. doi: 10.1093/emboj/16.12.3405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Diffley J. F., Stillman B. A close relative of the nuclear, chromosomal high-mobility group protein HMG1 in yeast mitochondria. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7864–7868. doi: 10.1073/pnas.88.17.7864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dumas R., Butikofer M. C., Job D., Douce R. Evidence for two catalytically different magnesium-binding sites in acetohydroxy acid isomeroreductase by site-directed mutagenesis. Biochemistry. 1995 May 9;34(18):6026–6036. doi: 10.1021/bi00018a004. [DOI] [PubMed] [Google Scholar]
  4. Guex N., Peitsch M. C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis. 1997 Dec;18(15):2714–2723. doi: 10.1002/elps.1150181505. [DOI] [PubMed] [Google Scholar]
  5. Kaufman B. A., Newman S. M., Hallberg R. L., Slaughter C. A., Perlman P. S., Butow R. A. In organello formaldehyde crosslinking of proteins to mtDNA: identification of bifunctional proteins. Proc Natl Acad Sci U S A. 2000 Jul 5;97(14):7772–7777. doi: 10.1073/pnas.140063197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. MacAlpine D. M., Perlman P. S., Butow R. A. The numbers of individual mitochondrial DNA molecules and mitochondrial DNA nucleoids in yeast are co-regulated by the general amino acid control pathway. EMBO J. 2000 Feb 15;19(4):767–775. doi: 10.1093/emboj/19.4.767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Megraw T. L., Chae C. B. Functional complementarity between the HMG1-like yeast mitochondrial histone HM and the bacterial histone-like protein HU. J Biol Chem. 1993 Jun 15;268(17):12758–12763. [PubMed] [Google Scholar]
  8. Megraw T. L., Kao L. R., Chae C. B. The mitochondrial histone HM: an evolutionary link between bacterial HU and nuclear HMG1 proteins. Biochimie. 1994;76(10-11):909–916. doi: 10.1016/0300-9084(94)90015-9. [DOI] [PubMed] [Google Scholar]
  9. Miyakawa I., Okazaki-Higashi C., Higashi T., Furutani Y., Sando N. Isolation and characterization of mitochondrial nucleoids from the yeast Pichia jadinii. Plant Cell Physiol. 1996 Sep;37(6):816–824. doi: 10.1093/oxfordjournals.pcp.a029017. [DOI] [PubMed] [Google Scholar]
  10. Miyakawa I., Sando N., Kawano S., Nakamura S., Kuroiwa T. Isolation of morphologically intact mitochondrial nucleoids from the yeast, Saccharomyces cerevisiae. J Cell Sci. 1987 Nov;88(Pt 4):431–439. doi: 10.1242/jcs.88.4.431. [DOI] [PubMed] [Google Scholar]
  11. Mueller P. P., Hinnebusch A. G. Multiple upstream AUG codons mediate translational control of GCN4. Cell. 1986 Apr 25;45(2):201–207. doi: 10.1016/0092-8674(86)90384-3. [DOI] [PubMed] [Google Scholar]
  12. Newman S. M., Zelenaya-Troitskaya O., Perlman P. S., Butow R. A. Analysis of mitochondrial DNA nucleoids in wild-type and a mutant strain of Saccharomyces cerevisiae that lacks the mitochondrial HMG box protein Abf2p. Nucleic Acids Res. 1996 Jan 15;24(2):386–393. doi: 10.1093/nar/24.2.386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. OGUR M., ST. JOHN R., NAGAI S. Tetrazolium overlay technique for population studies of respiration deficiency in yeast. Science. 1957 May 10;125(3254):928–929. doi: 10.1126/science.125.3254.928. [DOI] [PubMed] [Google Scholar]
  14. Petersen J. G., Holmberg S. The ILV5 gene of Saccharomyces cerevisiae is highly expressed. Nucleic Acids Res. 1986 Dec 22;14(24):9631–9651. doi: 10.1093/nar/14.24.9631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Suzuki T., Kawano S., Kuroiwa T. Structure of three-dimensionally rod-shaped mitochondrial nucleoids isolated from the slime mould Physarum polycephalum. J Cell Sci. 1982 Dec;58:241–261. doi: 10.1242/jcs.58.1.241. [DOI] [PubMed] [Google Scholar]
  17. Thompson J. D., Gibson T. J., Plewniak F., Jeanmougin F., Higgins D. G. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997 Dec 15;25(24):4876–4882. doi: 10.1093/nar/25.24.4876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Van Dyck E., Foury F., Stillman B., Brill S. J. A single-stranded DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB. EMBO J. 1992 Sep;11(9):3421–3430. doi: 10.1002/j.1460-2075.1992.tb05421.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Van Tuyle G. C., McPherson M. L. A compact form of rat liver mitochondrial DNA stabilized by bound proteins. J Biol Chem. 1979 Jul 10;254(13):6044–6053. [PubMed] [Google Scholar]
  20. Wessel P. M., Biou V., Douce R., Dumas R. A loop deletion in the plant acetohydroxy acid isomeroreductase homodimer generates an active monomer with reduced stability and altered magnesium affinity. Biochemistry. 1998 Sep 15;37(37):12753–12760. doi: 10.1021/bi980411g. [DOI] [PubMed] [Google Scholar]
  21. Williamson D. H., Fennell D. J. Visualization of yeast mitochondrial DNA with the fluorescent stain "DAPI". Methods Enzymol. 1979;56:728–733. doi: 10.1016/0076-6879(79)56065-0. [DOI] [PubMed] [Google Scholar]
  22. Zelenaya-Troitskaya O., Newman S. M., Okamoto K., Perlman P. S., Butow R. A. Functions of the high mobility group protein, Abf2p, in mitochondrial DNA segregation, recombination and copy number in Saccharomyces cerevisiae. Genetics. 1998 Apr;148(4):1763–1776. doi: 10.1093/genetics/148.4.1763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Zelenaya-Troitskaya O., Perlman P. S., Butow R. A. An enzyme in yeast mitochondria that catalyzes a step in branched-chain amino acid biosynthesis also functions in mitochondrial DNA stability. EMBO J. 1995 Jul 3;14(13):3268–3276. doi: 10.1002/j.1460-2075.1995.tb07330.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES