Metabolic effects of altering redundant targeting signals for yeast mitochondrial malate dehydrogenase.

Eukaryotic cells contain highly homologous isozymes of malate dehydrogenase which catalyze the same reaction in different cellular compartments. To examine whether the metabolic functions of these isozymes are interchangeable, we have altered the cellular localization of mitochondrial malate dehydrogenase (MDH1) in yeast. Since a previous study showed that removal of the targeting presequence from MDH1 does not prevent mitochondrial import in vivo, we tested the role of a putative cryptic targeting sequence near the amino terminus of the mature polypeptide. Three residues in this region were changed to residues present in analogous positions in the other two yeast MDH isozymes. Alone, these replacements did not affect activity or localization of MDH1 but, in combination with deletion of the presequence, prevented mitochondrial import in vivo. Measurable levels of the resulting cytosolic form of MDH1 were low with expression from a centromere-based plasmid but were comparable to normal cellular levels with expression from a multicopy plasmid. The cytosolic form of MDH1 restored the ability of a deltaMDH1 disruption strain to grow on ethanol or acetate, suggesting that mitochondrial localization of MDH1 is not essential for its function in the TCA cycle. This TCA cycle function observed for the cytosolic form of MDH1 is unique to that isozyme since overexpression of MDH2 and of a cytosolic form of MDH3 in a deltaMDH1 strain failed to restore growth. Finally, only partial restoration of growth of a deltaMDH2 disruption mutant was attained with the cytosolic form of MDH1, suggesting that MDH2 may also have unique metabolic functions.

[1]  D. Mecke,et al.  Evidence for catabolite degradation in the glucose-dependent inactivation of yeast cytoplasmic malate dehydrogenase. , 1978, European journal of biochemistry.

[2]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[3]  L. McAlister-Henn,et al.  Subunit structure, expression, and function of NAD(H)-specific isocitrate dehydrogenase in Saccharomyces cerevisiae , 1990, Journal of bacteriology.

[4]  G. Daum,et al.  Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. , 1982, The Journal of biological chemistry.

[5]  P. Lazarow,et al.  Peroxisomal biogenesis: multiple pathways of protein import. , 1994, The Journal of biological chemistry.

[6]  S. Subramani,et al.  Protein import into peroxisomes and biogenesis of the organelle. , 1993, Annual review of cell biology.

[7]  L. Banaszak,et al.  Refined crystal structure of cytoplasmic malate dehydrogenase at 2.5-A resolution. , 1989, Biochemistry.

[8]  M. Blaber,et al.  Complete nucleotide sequence of the Escherichia coli gene encoding malate dehydrogenase. , 1987, Nucleic acids research.

[9]  R. Roggenkamp.  Targeting signals for protein import into peroxisomes , 1992, Cell biochemistry and function.

[10]  W. N. Burnette,et al.  "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. , 1981, Analytical biochemistry.

[11]  L. Banaszak,et al.  Refined crystal structure of mitochondrial malate dehydrogenase from porcine heart and the consensus structure for dicarboxylic acid oxidoreductases. , 1994, Biochemistry.

[12]  J. Goodman,et al.  An oligomeric protein is imported into peroxisomes in vivo , 1994, The Journal of cell biology.

[13]  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.

[14]  C. Richardson,et al.  DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[15]  D. Andrews,et al.  Saccharomyces cerevisiae peroxisomal thiolase is imported as a dimer. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[16]  C. Gietl,et al.  Glyoxysomal malate dehydrogenase from watermelon is synthesized with an amino-terminal transit peptide. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[17]  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.

[18]  J. Steffan,et al.  Isolation and characterization of the yeast gene encoding the MDH3 isozyme of malate dehydrogenase. , 1992, The Journal of biological chemistry.

[19]  L. McAlister-Henn,et al.  Glucose-induced degradation of the MDH2 isozyme of malate dehydrogenase in yeast. , 1992, The Journal of biological chemistry.

[20]  Y. Peleg,et al.  The single translation product of the FUM1 gene (fumarase) is processed in mitochondria before being distributed between the cytosol and mitochondria in Saccharomyces cerevisiae , 1994, Molecular and cellular biology.

[21]  L. Grivell,et al.  Isolation and inactivation of the nuclear gene encoding the rotenone-insensitive internal NADH: ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae. , 1991, European journal of biochemistry.

[22]  L. Thompson,et al.  Gene sequence and primary structure of mitochondrial malate dehydrogenase from Saccharomyces cerevisiae. , 1988, Biochemistry.

[23]  T. Tsuzuki,et al.  Cloning and sequence analysis of cDNAs encoding mammalian mitochondrial malate dehydrogenase. , 1987, Biochemistry.

[24]  J. Steffan,et al.  Expression and Function of a Mislocalized Form of Peroxisomal Malate Dehydrogenase (MDH3) in Yeast (*) , 1995, The Journal of Biological Chemistry.

[25]  K. Faber,et al.  Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Hansenula polymorpha. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[26]  N. Wolins,et al.  Specific binding of the peroxisomal protein targeting sequence to glyoxysomal membranes. , 1994, The Journal of biological chemistry.

[27]  D. Kinney,et al.  Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions. , 1986, Gene.

[28]  S Subramani,et al.  Identification of Peroxisomal Targeting Signals Located at the Carboxy Terminus of Four Peroxisomal Proteins Materials and Methods Reagents , 1988 .

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

[30]  M. Hall,et al.  Crystal structure of a ternary complex of Escherichia coli malate dehydrogenase citrate and NAD at 1.9 A resolution. , 1993, Journal of molecular biology.

[31]  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.

[32]  B. Minshew,et al.  Use of an immunoperoxidase method for identification of Bacteroides fragilis , 1979, Journal of clinical microbiology.

[33]  D. Botstein,et al.  Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. , 1979, Gene.

[34]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations , 1983 .

[35]  T. Suzuki,et al.  Evidence that rat liver mitochondrial and cytosolic fumarases are synthesized from one species of mRNA by alternative translational initiation at two in-phase AUG codons. , 1992, European journal of biochemistry.

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

[37]  L. Thompson,et al.  Dispensable presequence for cellular localization and function of mitochondrial malate dehydrogenase from Saccharomyces cerevisiae. , 1989, The Journal of biological chemistry.

[38]  H. Tabak,et al.  Peroxisomal and mitochondrial carnitine acetyltransferases of Saccharomyces cerevisiae are encoded by a single gene. , 1995, The EMBO journal.