Cytoplasmic chaperones determine the targeting pathway of precursor proteins to mitochondria.

Two ATP-dependent cytosolic chaperones, mitochondrial import stimulation factor (MSF) and hsp70, are known to be involved in the import of precursor proteins into mitochondria. Hsp70 generally recognizes unfolded proteins, while MSF specifically recognizes mitochondrial precursor proteins and targets them to mitochondria in a NEM-sensitive manner. Here we analyzed the relative contribution of these chaperones in the import process and confirmed that the precursor proteins are targeted to mitochondria via two distinct pathways: one requiring MSF and the other requiring hsp70. Both pathways depend on distinct proteinaceous components of the outer mitochondrial membrane. The MSF-dependent pathway is NEM-sensitive and requires the hydrolysis of extra-mitochondrial ATP for the release of MSF from the mitochondrial import receptor, whereas the hsp70-dependent pathway is NEM-sensitive and does not require extra-mitochondrial ATP. The NEM-insensitive, hsp70-dependent import became NEM-sensitive depending on the amount of MSF added. The relative importance of the two pathways appears to be determined by the affinities of MSF and hsp70 for the precursor proteins.