title: Submitochondrial localization, cell-free synthesis, and mitochondrial import of 2-isopropylmalate synthase of yeast.
abstract: 2-Isopropylmalate synthase (EC 4.1.3.12) of yeast is a mitochondrial enzyme. We now provide evidence showing that a large part of the 2-isopropylmalate synthase activity that is associated with the mitochondria is located in the mitochondrial matrix. In vitro translation of total yeast RNA followed by immunoprecipitation with anti-2-isopropylmalate synthase antibody yields two polypeptides. The larger of these has an apparent molecular weight identical to that of purified 2-isopropylmalate synthase subunit (ca. 65,000). It is incorporated into isolated yeast mitochondria with no detectable change in molecular weight. The import requires energy. The smaller polypeptide migrates to a position corresponding to a molecular weight of 63,000-64,000. It is not taken up by mitochondria. Both polypeptides, which also can be obtained by immunoprecipitation of crude extracts, become labeled when in vitro translation is performed in the presence of N-formyl[35S]methionyl-tRNAf. Mutants with no detectable 2-isopropylmalate synthase activity are deficient in either one or both synthase-related polypeptides. These results are discussed in the light of recent evidence for two 2-isopropylmalate synthase-encoding genes in yeast.
mesh_terms: D015079:2-Isopropylmalate Synthase / Q000096:biosynthesis*; D002474:Cell-Free System; D008928:Mitochondria / Q000378:metabolism; D008970:Molecular Weight; D009154:Mutation; D007652:Oxo-Acid-Lyases / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013367:Submitochondrial Particles / Q000201:enzymology
chemical_list: D015079:2-Isopropylmalate Synthase; D007652:Oxo-Acid-Lyases