Genetic and biochemical interactions involving tricarboxylic acid cycle (TCA) function using a collection of mutants defective in all TCA cycle genes.

The eight enzymes of the tricarboxylic acid (TCA) cycle are encoded by at least 15 different nuclear genes in Saccharomyces cerevisiae. We have constructed a set of yeast strains defective in these genes as part of a comprehensive analysis of the interactions among the TCA cycle proteins. The 15 major TCA cycle genes can be sorted into five phenotypic categories on the basis of their growth on nonfermentable carbon sources. We have previously reported a novel phenotype associated with mutants defective in the IDH2 gene encoding the Idh2p subunit of the NAD+-dependent isocitrate dehydrogenase (NAD-IDH). Null and nonsense idh2 mutants grow poorly on glycerol, but growth can be enhanced by extragenic mutations, termed glycerol suppressors, in the CIT1 gene encoding the TCA cycle citrate synthase and in other genes of oxidative metabolism. The TCA cycle mutant collection was utilized to search for other genes that can suppress idh2 mutants and to identify TCA cycle genes that display a similar suppressible growth phenotype on glycerol. Mutations in 7 TCA cycle genes were capable of functioning as suppressors for growth of idh2 mutants on glycerol. The only other TCA cycle gene to display the glycerol-suppressor-accumulation phenotype was IDH1, which encodes the companion Idh1p subunit of NAD-IDH. These results provide genetic evidence that NAD-IDH plays a unique role in TCA cycle function.

[1]  L. McAlister-Henn,et al.  Molecular genetics of yeast TCA cycle isozymes. , 1997, Progress in nucleic acid research and molecular biology.

[2]  T. Boller,et al.  Cloning and disruption of a gene required for growth on acetate but not on ethanol: The acetyl‐coenzyme a synthetase gene of Saccharmoyces cerevisiae , 1992, Yeast.

[3]  R. Rothstein.  Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. , 1991, Methods in enzymology.

[4]  A. Tzagoloff,et al.  Suppression of sdh1 mutations by the SDH1b gene of Saccharomyces cerevisiae , 1998, Yeast.

[5]  A. Tzagoloff,et al.  Structure and regulation of KGD2, the structural gene for yeast dihydrolipoyl transsuccinylase , 1990, Molecular and cellular biology.

[6]  P. Srere,et al.  Complexes of sequential metabolic enzymes. , 1987, Annual review of biochemistry.

[7]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[8]  B. Sumegi,et al.  Cloning and sequencing of a cDNA encoding Saccharomyces cerevisiae carnitine acetyltransferase. Use of the cDNA in gene disruption studies. , 1993, The Journal of biological chemistry.

[9]  M. McCammon,et al.  Mutations in the IDH2 gene encoding the catalytic subunit of the yeast NAD+-dependent isocitrate dehydrogenase can be suppressed by mutations in the CIT1 gene encoding citrate synthase and other genes of oxidative metabolism. , 1997, Archives of biochemistry and biophysics.

[10]  J. Boeke,et al.  SDH1, the gene encoding the succinate dehydrogenase flavoprotein subunit from Saccharomyces cerevisiae. , 1992, Gene.

[11]  R. Brodeur,et al.  Metabolic studies on Saccharomyces cerevisiae containing fused citrate synthase/malate dehydrogenase. , 1994, Biochemistry.

[12]  M. Veenhuis,et al.  Association of glyoxylate and beta-oxidation enzymes with peroxisomes of Saccharomyces cerevisiae , 1990, Journal of bacteriology.

[13]  P. Brown,et al.  Exploring the metabolic and genetic control of gene expression on a genomic scale. , 1997, Science.

[14]  Wen-Ning Zhao,et al.  Assembly and Function of a Cytosolic Form of NADH-specific Isocitrate Dehydrogenase in Yeast (*) , 1996, The Journal of Biological Chemistry.

[15]  L. McAlister-Henn,et al.  Isolation, nucleotide sequence analysis, and disruption of the MDH2 gene from Saccharomyces cerevisiae: evidence for three isozymes of yeast malate dehydrogenase , 1991, Molecular and cellular biology.

[16]  J. Pronk,et al.  Pyruvate Metabolism in Saccharomyces cerevisiae , 1996, Yeast.

[17]  A. Tzagoloff,et al.  Structure and regulation of KGD1, the structural gene for yeast alpha-ketoglutarate dehydrogenase , 1989, Molecular and cellular biology.

[18]  B. Sumegi,et al.  Metabolism of [3-13C]pyruvate in TCA cycle mutants of yeast. , 1992, Biochemistry.

[19]  K. Mosbach,et al.  Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate dehydrogenase. , 1994, Biochemistry.

[20]  C. Herbert,et al.  The CIT3 gene of Saccharomyces cerevisiae encodes a second mitochondrial isoform of citrate synthase , 1997, Molecular microbiology.

[21]  G. Fink,et al.  Methods in enzymology vol 194 guide to yeast genetics and molecular biology , 1991 .

[22]  R. S. Muir,et al.  Gene disruption with PCR products in Saccharomyces cerevisiae. , 1995, Gene.

[23]  L. Guarente,et al.  Metabolic changes in Saccharomyces cerevisiae strains lacking citrate synthases. , 1988, The Journal of biological chemistry.

[24]  B. Sumegi,et al.  Organizational aspects of the citric acid cycle. , 1987, Biochemical Society symposium.

[25]  Wen-Ning Zhao,et al.  Affinity Purification and Kinetic Analysis of Mutant Forms of Yeast NAD+-specific Isocitrate Dehydrogenase* , 1997, The Journal of Biological Chemistry.

[26]  D. Marguet,et al.  Molecular cloning of the yeast mitochondrial aconitase gene (ACO1) and evidence of a synergistic regulation of expression by glucose plus glutamate , 1990, Molecular and cellular biology.

[27]  L. McAlister-Henn,et al.  Kinetic analysis of NAD(+)-isocitrate dehydrogenase with altered isocitrate binding sites: contribution of IDH1 and IDH2 subunits to regulation and catalysis. , 1993, Biochemistry.

[28]  J. E. Lawson,et al.  Characterization of PDH beta 1, the structural gene for the pyruvate dehydrogenase beta subunit from Saccharomyces cerevisiae. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[29]  H. Tabak,et al.  Peroxisomal β‐oxidation of polyunsaturated fatty acids in Saccharomyces cerevisiae: isocitrate dehydrogenase provides NADPH for reduction of double bonds at even positions , 1998, The EMBO journal.

[30]  C. Dieckmann,et al.  PET genes of Saccharomyces cerevisiae. , 1990, Microbiological reviews.

[31]  M. McCammon,et al.  Yeast mutants of glucose metabolism with defects in the coordinate regulation of carbon assimilation. , 1999, Archives of biochemistry and biophysics.

[32]  J. Dickinson,et al.  Functional analysis in Saccharomyces cerevisiae of naturally occurring amino acid substitutions in human dihydrolipoamide dehydrogenase. , 1996, Human molecular genetics.

[33]  I. Scheffler,et al.  Cloning and characterization of the iron-sulfur subunit gene of succinate dehydrogenase from Saccharomyces cerevisiae. , 1990, The Journal of biological chemistry.

[34]  L. Thompson,et al.  Isolation and expression of the gene encoding yeast mitochondrial malate dehydrogenase , 1987, Journal of bacteriology.

[35]  M. Bolotin-Fukuhara,et al.  Structure and regulation of SDH3, the yeast gene encoding the cytochrome b560 subunit of respiratory complex II. , 1994, The Journal of biological chemistry.

[36]  B. Lemire,et al.  Isolation and characterization of the Saccharomyces cerevisiae SDH4 gene encoding a membrane anchor subunit of succinate dehydrogenase. , 1994, The Journal of biological chemistry.

[37]  M. Berg,et al.  Transient mRNA responses in chemostat cultures as a method of defining putative regulatory elements: application to genes involved in Saccharomyces cerevisiae acetyl-coenzyme A metabolism. , 1998 .

[38]  Gerald R. Fink,et al.  Guide to yeast genetics and molecular biology , 1993 .

[39]  M. Teige,et al.  Model of a quinary structure between Krebs TCA cycle enzymes: a model for the metabolon. , 1997, Biochemistry.

[40]  M. Veenhuis,et al.  Proliferation of microbodies in Saccharomyces cerevisiae , 1987, Yeast.

[41]  G. Schatz,et al.  Isolation of the nuclear yeast genes for citrate synthase and fifteen other mitochondrial proteins by a new screening method. , 1984, The EMBO journal.

[42]  L. Grivell,et al.  Yeast mitochondrial NAD(+)-dependent isocitrate dehydrogenase is an RNA-binding protein. , 1993, Nucleic acids research.

[43]  M. McCammon,et al.  An unassembled subunit of NAD(+)-dependent isocitrate dehydrogenase is insoluble and covalently modified. , 1998, Archives of biochemistry and biophysics.

[44]  R. Haselbeck,et al.  Function and expression of yeast mitochondrial NAD- and NADP-specific isocitrate dehydrogenases. , 1993, The Journal of biological chemistry.

[45]  H. Küntzel,et al.  Mitochondrial and Cytoplasmic Polysomes from Neurospora crassa , 1967, Nature.

[46]  B. Lemire,et al.  Covalent Attachment of FAD to the Yeast Succinate Dehydrogenase Flavoprotein Requires Import into Mitochondria, Presequence Removal, and Folding (*) , 1996, The Journal of Biological Chemistry.

[47]  L. McAlister-Henn,et al.  NAD(+)-dependent isocitrate dehydrogenase. Cloning, nucleotide sequence, and disruption of the IDH2 gene from Saccharomyces cerevisiae. , 1991, Journal of Biological Chemistry.

[48]  L. McAlister-Henn,et al.  Metabolic effects of altering redundant targeting signals for yeast mitochondrial malate dehydrogenase. , 1997, Archives of biochemistry and biophysics.

[49]  I. Dawes,et al.  The nucleotide sequence of the LPD1 gene encoding lipoamide dehydrogenase in Saccharomyces cerevisiae: comparison between eukaryotic and prokaryotic sequences for related enzymes and identification of potential upstream control sites. , 1988, Journal of general microbiology.

[50]  L. Guarente,et al.  Saccharomyces cerevisiae contains two functional citrate synthase genes , 1986, Molecular and cellular biology.

[51]  M. Wu,et al.  Mitochondrial and cytoplasmic fumarases in Saccharomyces cerevisiae are encoded by a single nuclear gene FUM1. , 1987, The Journal of biological chemistry.

[52]  G. Mannaerts,et al.  [Peroxisomal beta-oxidation]. , 1993, Verhandelingen - Koninklijke Academie voor Geneeskunde van Belgie.

[53]  L. McAlister-Henn,et al.  Cloning and characterization of the gene encoding the IDH1 subunit of NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae. , 1992, The Journal of biological chemistry.

[54]  M. McCammon,et al.  Genes of succinyl-CoA ligase from Saccharomyces cerevisiae. , 1998, European journal of biochemistry.

[55]  M. McCammon,et al.  Mutants of Saccharomyces cerevisiae with defects in acetate metabolism: isolation and characterization of Acn- mutants. , 1996, Genetics.