A NOVEL INTERMEDIATE ON THE IMPORT PATHWAY OF CYTOCHROME B(2) INTO MITOCHONDRIA - EVIDENCE FOR CONSERVATIVE SORTING

Cytochrome b(2) is sorted into the intermembrane spaceia by a bipartit e N-terminal targeting and sorting presequence. In an attempt to defin e the sorting pathway we have identified an as yet unknown import inte rmediate. Cytochrome b(2)-dihydrofolate reductase (DHFR) fusion protei ns were arrested in the presence of methotrexate (MTX) so that the DHF R domain was at the surface of the outer membrane while the N-terminus reached into the intermembrane space where the sorting signal was rem oved. This membrane-spanning, mature-sized species was efficiently cha sed into the mitochondria upon removal of MTX. Thus, an intermediate w as generated which was exposed to the intermembrane space but was stil l associated with the inner membrane. This intermediate was also found upon direct import of cytochrome b(2) and derived fusion proteins. Th ese membrane-bound mature-sized cytochrome bz species loop through the matrix and could be recovered in a complex with mt-Hsp70 and the inne r membrane MIM44/ISP45, a component of the inner membrane import appar atus. This novel sorting intermediate can only be explained by a pathw ay in which cytochrome b(2) passes through the matrix. The existence o f such an intermediate is inconsistent with a pathway by which entranc e of the mature part of cytochrome b(2) into the matrix is stopped by the sorting sequence; however, its presence is fully consistent with t he conservative sorting pathway.