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Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation
14:44

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Published on: March 15, 2014

Submitochondrial localization, cell-free synthesis, and mitochondrial import of 2-isopropylmalate synthase of yeast

D M Hampsey, A S Lewin, G B Kohlhaw

    Proceedings of the National Academy of Sciences of the United States of America
    |March 1, 1983
    PubMed

    Insights

    Yeast 2-isopropylmalate synthase, a mitochondrial enzyme, has activity primarily in the mitochondrial matrix. Evidence shows a larger polypeptide is imported into mitochondria, while a smaller one is not.

    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Yeast Genetics

    Background:

    • 2-Isopropylmalate synthase (EC 4.1.3.12) is a key enzyme in leucine biosynthesis.
    • Its localization within yeast mitochondria has been a subject of investigation.

    Purpose of the Study:

    • To determine the precise mitochondrial localization of 2-isopropylmalate synthase activity in yeast.
    • To investigate the import and processing of 2-isopropylmalate synthase subunits into yeast mitochondria.

    Main Methods:

    • In vitro translation of yeast RNA followed by immunoprecipitation.
    • Import studies using isolated yeast mitochondria.
    • Analysis of yeast mutants deficient in 2-isopropylmalate synthase activity.

    Main Results:

    • Yeast 2-isopropylmalate synthase activity is largely localized to the mitochondrial matrix.
    • Two distinct polypeptides related to the enzyme were identified via in vitro translation and immunoprecipitation.
    • A larger polypeptide (approx. 65 kDa) is imported into mitochondria in an energy-dependent manner without processing, while a smaller polypeptide (63-64 kDa) is not imported.
    • Yeast mutants lacking enzyme activity are deficient in one or both polypeptides.

    Conclusions:

    • The study provides strong evidence for the mitochondrial matrix localization of 2-isopropylmalate synthase.
    • The import of the larger polypeptide into mitochondria suggests a specific targeting mechanism.
    • The existence of two distinct polypeptides supports recent findings of two yeast genes encoding 2-isopropylmalate synthase.

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