Import of proteins into mitochondria. Import and maturation of the mitochondrial intermembrane space enzymes cytochrome b2 and cytochrome c peroxidase in intact yeast cells.

The import of cytochrome b2 and cytochrome c peroxidase into mitochondria was investigated by pulse-chase experiments with intact yeast cells combined with subcellular fractionation. Import and processing of the precursors of these intermembrane space proteins is blocked by uncouplers of oxidative phosphorylation, indicating that an "energized" inner membrane is required. Cytochrome b2 is processed in two steps. The first step involves energy-dependent transport across both mitochondrial membranes and cleavage by a matrix-located protease to yield an intermediate which is smaller than the precursor, but larger than the mature protein. The second step involves conversion of the intermediate to the mature form. Whereas the precursor and the mature form are soluble, the intermediate is membrane-bound and exposed to the intermembrane space. The maturation of cytochrome c peroxidase is much slower than that of cytochrome b2. Proteolytic processing rather than import is rate-limiting since cytochrome c peroxidase precursor labeled during a 3-min pulse is already found attached to the outer face of the mitochondrial inner membrane. Import of cytochrome b2 and probably also of cytochrome c peroxidase thus involves energy-dependent transport to the matrix and cleavage by a matrix-localized protease. Maturation of cytochrome b2 proceeds in the sequence: soluble precursor leads to membrane-bound intermediate form leads to soluble mature form.