Sequences of the iron-sulfur protein precursor necessary for its import and two-step processing in yeast mitochondria.
Gene fusion techniques were used to examine whether the presequence of the iron-sulfur protein contains sufficient information for the import of attached mouse cytosolic enzyme dihydrofolate reductase into isolated yeast mitochondria and its subsequent two-step processing. Genes encoding amino-terminal segments of the iron-sulfur precursor protein including the proposed first presequence (residues 1-21), the complete presequence (residues 1-30), and various lengths of the precursor protein from 31 to 160 residues were fused, in frame, to dihydrofolate reductase. All of the fusion proteins, after synthesis in an in vitro transcription-translation system, were imported into a protease-resistant compartment of mitochondria where a single proteolytic cleavage was observed at the first processing site. The second cleavage, however, was not observed after import of any of the chimeric proteins, suggesting that the second cleavage may be strongly influenced by the presence of the passenger protein. A deletion mutant of the iron-sulfur protein precursor lacking residues 161-180 underwent two proteolytic cleavages similar to those observed for the wild-type iron-sulfur protein after import into mitochondria. These results suggest that the complete sequence of the mature form of the iron-sulfur protein including the amino acid residues involved in binding the iron sulfur clusters is not necessary for the second cleavage to occur. In addition, the hydrophobic sequence present in residues 55-66 of the precursor protein which has been suggested to anchor the iron-sulfur protein to the inner membrane, was not necessary for the import and two-step processing of the protein, since a deletion mutant lacking residues 55-66 was processed correctly.
Department of Biochemistry, West Virginia University, School of Medicine, Morgantown 26506-9142.
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D001692:Biological Transport; D002451:Cell Compartmentation; D004252:DNA Mutational Analysis; D014450:Electron Transport Complex III; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
D007506:Iron-Sulfur Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C037989:Rieske iron-sulfur protein; D013762:Tetrahydrofolate Dehydrogenase; D014450:Electron Transport Complex III