TARGETING OF CYTOCHROME-B2 INTO THE MITOCHONDRIAL INTERMEMBRANE SPACE- SPECIFIC RECOGNITION OF THE SORTING SIGNAL

Cytochrome b2 contains 2-fold targeting information: an amino-terminal signal for targeting to the mitochondrial matrix, followed by a secon d cleavable sorting signal that functions in directing the precursor i nto the mitochondrial intermembrane space. The role of the second sort ing sequence was analyzed by replacing one, two or all of the three po sitively charged amino acid residues which are present at the amino-te rminal side of the hydrophobic core by uncharged residues or an acidic residue. With a number of these mutant precursor proteins, processing to the mature form was reduced or completely abolished and at the sam e time targeting to the matrix space occurred. The accumulation in the matrix depended on a high level of intramitochondrial ATP. At low lev els of matrix ATP, the mutant proteins were sorted into the intermembr ane space like the wild-type precursors. The results: (i) suggest the existence of one or more matrix components that specifically recognize the second sorting signal and thereby trigger the translocation into the intermembrane space; (ii) indicate that the mutant signals have re duced ability to interact with the recognition component(s) and then e mbark on the default pathway into the matrix by interacting with mitoc hondrial hsp70 in conjunction with matrix ATP; (iii) strongly argue ag ainst a mechanism by which the hydrophobic segment of the sorting sequ ence stops translocation in the hydrophobic phase of the inner membran e.