Functional cooperation and stoichiometry of protein translocases of the outer and inner membranes of mitochondria.

The qualitative relationship between preprotein translocases in the mitochondrial outer and inner membranes was determined by both a functional analysis and a determination of characteristic components of the translocases. Translocation contact sites of isolated mitochondria were saturated with intermediates of a matrix-targeted precursor of the beta-subunit of the F1-ATPase (pF1beta), and import of preproteins into the different mitochondrial subcompartments was monitored. A strong inhibition (75-95%) was observed for preproteins with an N-terminal matrix targeting signal, indicating that a significant portion of the contact sites was blocked by accumulated F1beta. Insertion of preproteins into the outer membrane and import into the intermembrane space of preproteins without matrix targeting signals was inhibited by about 45%, indicating that functional outer membrane translocases were available despite saturation of contact sites. Similarly, import of members of the mitochondrial carrier family into the inner membrane was only partly inhibited (40-50%), demonstrating that functional Tim22 translocases were available to cooperate with the Tom machinery in the import of carrier proteins. The stoichiometry of Tom40, Tim23, and Tim22 in mitochondria was determined to be 5:1:0.22. We conclude that translocases of the outer membrane are present in excess over translocases of the inner membrane.