TRANSLOCATION AND INSERTION OF PRECURSOR PROTEINS INTO ISOLATED OUTERMEMBRANES OF MITOCHONDRIA

Nuclear-encoded proteins destined for mitochondria must cross the oute r or both outer and inner membranes to reach their final sub-mitochond rial locations. While the inner membrane can translocate preproteins b y itself, it is not known whether the outer membrane also contains an endogenous protein translocation activity which can function independe ntly of the inner membrane. To selectively study the protein transport into and across the outer membrane of Neurospora crassa mitochondria, outer membrane vesicles were isolated which were sealed, in a right-s ide-out orientation, and virtually free of inner membranes. The vesicl es were functional in the insertion and assembly of various outer memb rane proteins such as porin, MOM19, and MOM22. Like with intact mitoch ondria, import into isolated outer membranes was dependent on protease -sensitive surface receptors and led to correct folding and membrane i ntegration. The vesicles were also capable of importing a peripheral c omponent of the inner membrane, cytochrome c heme lyase (CCHL), in a r eceptor-dependent fashion. Thus, the protein translocation machinery o f the outer mitochondrial membrane can function as an independent enti ty which recognizes, inserts, and translocates mitochondrial preprotei ns of the outer membrane and the intermembrane space. In contrast, pro teins which have to be translocated into or across the inner membrane were only specifically bound to the vesicles, but not imported. This s uggests that transport of such proteins involves the participation of components of the intermembrane space and/or the inner membrane, and t hat in these cases the outer membrane translocation machinery has to a ct in concert with that of the inner membrane.