A regulatory factor, Fil1p, involved in derepression of the isocitrate lyase gene in Saccharomyces cerevisiae

A possible mitochondrial protein necessary for protein synthesis in mitochondria

Authors

  • Tamotsu Kanai,

    1. Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan
    Search for more papers by this author
  • Shigeru Takeshita,

    1. Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan
    Search for more papers by this author
  • Haruyuki Atomi,

    1. Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan
    Search for more papers by this author
  • Ken Umemura,

    1. Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan
    Search for more papers by this author
  • Mitsuyoshi Ueda,

    1. Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan
    Search for more papers by this author
  • Atsuo Tanaka

    1. Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan
    Search for more papers by this author

  • Correspondence to A. Tanaka, Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan

  • Fax: +81 75 753 5534.

  • Phone: +81 75 753 5524.

  • E-mail:atsuo@sbchem.kyoto-u.ac.jp

  • Abbreviations. CSRE, carbon-source-responsive element; UAS, upstream activating sequence; UPR-ICL, 5′ upstream region of Candida tropicalis isocitrate lyase gene; ORF, open reading frame; RRF, ribosome recycling factor.

Abstract

A mutant was isolated that failed to derepress the 5′ upstream region of Candida tropicalis isocitrate lyase gene (UPR-ICL)-mediated gene expression in acetate medium, and the gene (FIL1) that complemented this mutation was isolated. The fil1 null mutant in which FIL1 is disrupted (Δfil1 strain) could not grow on acetate or ethanol, and the derepression of the isocitrate lyase encoded by ICL1 in Saccharomyces cerevisiae was also defected. The amino acid sequence of Fil1p (230 amino acids) showed similarity to ribosome recycling factors (RRFs) of prokaryotes. Compared to prokaryotic RRFs, Fil1p had an N-terminal 46-amino-acid extension which was shown to be able to function as a mitochondrial-targeting sequence. The subcellular fractionation of the Δfil1 strain showed that protein constituents of the mitochondrial fraction of the Δfil1 strain differed from those of the wild-type strain, but resembled those of chloramphenicol-treated cells or ρ+ cells. The specific activity of cytochrome c oxidase, was severely decreased in Δfil1, ρ+ and chloramphenicol-treated cells compared with wild-type cells, while enzymatic levels of mitochondrial NAD+-linked isocitrate dehydrogenase, which is encoded by nuclear DNA, were not affected. These results suggest that Fil1p is necessary for protein synthesis in mitochondria of S. cerevisiae. Furthermore, cells treated with antimycin A, along with chloramphenicol-treated, ρ+, and Δfil1 cells, showed deficiency in derepression of isocitrate lyase. Northern-blot analysis showed that this can be ascribed to no increase in transcription of ICL1 and FBP1 encoding fructose 1,6-bisphosphatase. The results indicate the presence of a communication pathway between mitochondria and the nucleus which represses expression of genes encoding the key enzymes of the glyoxylate cycle and gluconeogenic pathway when there is a deficiency in the mitochondrial respiratory chain.

Ancillary