The mitochondrial Hsp70‐dependent import system actively unfolds preproteins and shortens the lag phase of translocation

Unfolding is an essential process during translocation of preproteins into mitochondria; however, controversy exists as to whether mitochondria play an active role in unfolding. We have established an in vitro system with a kinetic saturation of the mitochondrial import machinery, yielding translocation rates comparable to in vivo import rates. Preproteins with short N‐terminal segments in front of a folded domain show a characteristic delay of the onset of translocation (lag phase) although the maximal import rate is similar to that of longer preproteins. The lag phase is shortened by extending the N‐terminal segment to improve the accessibility to matrix heat shock protein 70 and abolished by unfolding of the preprotein. A mutant mtHsp70 defective in binding to the inner membrane prolongs the lag phase and reduces the translocation activity. A direct comparison of the rate of spontaneous unfolding in solution with that during translocation demonstrates that unfolding by mitochondria is significantly faster, proving an active unfolding process. We conclude that access of mtHsp70 to N‐terminal preprotein segments is critical for active unfolding and initiation of translocation.