The sorting route of cytochrome b2 branches from the general mitochondrial import pathway at the preprotein translocase of the inner membrane.

Cytochrome b2 is synthesized in the cytosol with a bipartite presequence. The first part of the presequence targets the protein to mitochondria and mediates translocation into the mitochondrial matrix compartment; the second part contains the sorting signal that is required for delivery of the protein to the intermembrane space. The localization of the structures that recognize the sorting signal is unclear. Here we show that upon import in vivo, the sorting signal of cytochrome b2 causes an early divergence from the general matrix import pathway and thereby prevents translocation of a folded C-terminal domain into mitochondria. By co-immunoprecipitations we find that translocation intermediates of cytochrome b2 are associated with Tim23, a component of the inner membrane protein import machinery. Cytochrome b2 constructs with an alteration in the sorting signal are mistargeted to the matrix of wild-type mitochondria. In mitochondria containing a mutant form of Tim23, however, the translocation of the altered sorting signal across the inner membrane is inhibited, and cytochrome b2 is correctly sorted to the intermembrane space. We suggest that the sorting signal of cytochrome b2 is recognized within the inner membrane in close vicinity to Tim23.