Separation of structural and dynamic functions of the mitochondrial translocase:: Tim44 is crucial for the inner membrane import sites in translocation of tightly folded domains, but not of loosely folded preproteins

The essential gene TIM44 encodes a subunit of the inner mitochondrial membrane preprotein translocase that forms a complex with the matrix heat-shock protein Hsp70, The specific role of Tim44 in protein import has not yet been defined because of the lack of means to block its function. Here we report on a Saccharomyces cerevisiae mutant allele of TIM44 that allows selective and efficient inactivation of Tim44 in organello, Surprisingly, the mutant mitochondria are still able to import preproteins. The import rate is only reduced by similar to 30% compared with wild-type as long as the preproteins do not carry stably folded domains. Moreover,. the number of import sites is not reduced. However, the mutant mitochondria are strongly impaired in pulling folded domains of preproteins close to the outer membrane and in promoting their unfolding. Our results demonstrate that Tim44 is not an essential structural component of the import channel, but is crucial for import of folded domains. We suggest that the concerted action of Tim44 and mtHsp70 drives unfolding of preproteins and accelerates translocation of loosely folded preproteins. While mtHsp70 is essential for import of both tightly and loosly folded preproteins, Tim44 plays a more specialized role in translocation of tightly folded domains.