SEQUENCES OF THE IRON-SULFUR PROTEIN-PRECURSOR NECESSARY FOR ITS IMPORT AND 2-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 impor t of attached mouse cytosolic enzyme dihydrofolate reductase into isol ated 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 pro tein from 31 to 160 residues were fused, in frame, to dihydrofolate re ductase. All of the fusion proteins, after synthesis in an in vitro tr anscription-translation system, were imported into a protease-resistan t compartment of mitochondria where a single proteolytic cleavage was observed at the first processing site. The second cleavage, however, w as not observed after import of any of the chimeric proteins, suggesti ng that the second cleavage may be strongly influenced by the presence of the passenger protein. A deletion mutant of the iron-sulfur protei n precursor lacking residues 161-180 underwent two proteolytic cleavag es similar to those observed for the wild-type iron-sulfur protein aft er import into mitochondria. These results suggest that the complete s equence of the mature form of the iron-sulfur protein including the am ino acid residues involved in binding the iron sulfur clusters is not necessary for the second cleavage to occur. In addition, the hydrophob ic sequence present in residues 55-66 of the precursor protein which h as been suggested to anchor the iron-sulfur protein to the inner membr ane, was not necessary for the import and two-step processing of the p rotein, since a deletion mutant lacking residues 55-66 was processed c orrectly. (C) 1994 Academie Press, Inc.