Assembly and function of a cytosolic form of NADH-specific isocitrate dehydrogenase in yeast.
Mitochondrial NAD-dependent isocitrate dehydrogenase catalyzes a rate-limiting step in the tricarboxylic acid cycle. Yeast isocitrate dehydrogenase is an octomer composed of two subunits (IDH1 and IDH2) encoded by different genes and possessing independent mitochondrial targeting presequences. Oligonucleotide-directed mutagenesis was used to remove the presequences from each gene and from both genes carried on centromere-based expression plasmids. Effects on cellular localization were examined in a yeast strain containing chromosomal disruptions of IDH1 and IDH2 loci. Each subunit was found to be dependent upon its presequence for mitochondrial localization, and the subunits are independently imported into mitochondria under most growth conditions. Furthermore, an active holoenzyme can be assembled in the cytosol and this ''cytosolic'' form of isocitrate dehydrogenase can reverse the acetate- growth phenotype characteristic of the DeltaIDH1/ DeltaIDH2 disruption strain, indicating functional replacement of the mitochondrial enzyme. However, transformants containing plasmids lacking either the IDH1 or IDH2 presequence coding regions were unexpectedly found to be capable of growth on acetate medium. Further investigation demonstrated that cellular localization of the IDH1 subunit can be biased by this stringent growth pressure.
Zhao
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WN
Department of Biochemistry, University of Texas Health Science Center, San Antonio, 78284-7760, USA.
McAlister-Henn
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002451:Cell Compartmentation; D002952:Citric Acid Cycle; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers / Q000737:chemistry; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D010101:Oxygen Consumption; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development
D017931:DNA Primers; D007527:Isoenzymes; D009243:NAD; D007521:Isocitrate Dehydrogenase