SORTING OF CYTOCHROME B(2) TO THE INTERMEMBRANE SPACE OF MITOCHONDRIA- KINETIC-ANALYSIS OF INTERMEDIATES DEMONSTRATES PASSAGE THROUGH THE MATRIX

Precytochrome b(2) is targeted to the mitochondrial intermembrane spac e by a dual targeting sequence comprising 80 amino acids. A kinetic an alysis of intramitochondrial sorting was performed. The intermediate-s ize form accumulated transiently in the matrix. When import was perfor med in the presence of metal chelators to prevent the first processing by the matrix processing peptidase, >40% of the imported precursor wa s localized in the matrix. A deletion of 13 amino acids in the interme mbrane space sorting sequence caused partial inhibition of the first p rocessing, and a transient accumulation of the precursor form in the m atrix was also observed. The decrease in this matrix-localized precurs or form paralleled an increase in the mature-size form in the intermem brane space. A point mutation in the mitochondrial targeting sequence (N-terminal to the sorting sequence) resulted in missorting to the mat rix space. Furthermore, a chimeric protein consisting of the initial 8 5 residues of cytochrome b(2) fused to dihydrofolate reductase was par tially targeted to the matrix at 15 degrees C, but not at 25 degrees C . Together, the results presented here indicate that cytochrome b(2) p asses through the matrix on its sorting pathway to the intermembrane s pace.