Unfolding of preproteins upon import into mitochondria

Unfolding of preproteins and translocation across the mitochondrial membran es requires their interaction with mt-Hsp70 and Tim44 at the inner face of the inner membrane and ATP as an energy source. We measured the temperature dependence of the rates of unfolding and import into the matrix of two fol ded passenger domains, the tightly folded heme-binding domain (HBD) of cyto chrome b(2) and the loosely folded mouse dihydrofolate reductase (DHFR), De spite the stability of the HBD, its rates of thermal breathing were fast an d the preprotein was imported rapidly at all temperatures, In contrast, rat es of unfolding and import of DHFR were strongly temperature dependent and import was significantly slower than unfolding. In addition, import rates o f DHFR were strongly dependent on the length of the presequence, We propose that the mitochondrial import motor does not exert a constant pulling forc e. Rather, mt-Hsp70 appears to release a translocating polypeptide chain su ch that the precursor can then slide back and refold on the surface of the mitochondria. Refolding competes with translocation, and passengers may und ergo several rounds of unfolding and refolding prior to their import.