Mutations in a 19-Amino-Acid Hydrophobic Region of the Yeast Cytochrome cl Presequence Prevent Sorting to the

Most mitochondrial proteins destined for the intermembrane space (IMS) carry in their presequence information for localization to the IMS in addition to information for their import. By selecting for mutants in the yeast Saccharomyces cerevisiae that mislocalize an IMS-targeted fusion protein, we identified mutations in the IMS sorting signal of the cytochrome cl protein. Amino acid substitutions or deletions in a stretch of 19 hydrophobic amino acids of the cytochrome cl presequence resulted in accumulation of the intermediate form of the cytochrome cl protein in the matrix. In some cases, the accumulated intermediate appeared to be slowly exported from the matrix, across the inner membrane to the IMS. Our results support the hypothesis that the cytochrome cl precursor is normally imported completely into the matrix and then exported to the IMS. Most mitochondrial proteins are encoded by nuclear genes, synthesized in the cytoplasm, and imported into the mitochondria (1, 13). Proteins imported into mitochondria are specifically targeted to one of the four mitochondrial compartments: matrix, inner membrane, intermembrane space (IMS), and outer membrane. Most proteins destined for the three interior compartments of the mitochondria ar