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 h eat-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 fun ction. Here we report on a Saccharomyces cerevisiae mutant allele of T IM44 that allows selective and efficient inactivation of Tim44 in orga nello, Surprisingly, the mutant mitochondria are still able to import preproteins. The import rate is only reduced by similar to 30% compare d with wild-type as long as the preproteins do not carry stably folded domains. Moreover,. the number of import sites is not reduced. Howeve r, the mutant mitochondria are strongly impaired in pulling folded dom ains of preproteins close to the outer membrane and in promoting their unfolding. Our results demonstrate that Tim44 is not an essential str uctural component of the import channel, but is crucial for import of folded domains. We suggest that the concerted action of Tim44 and mtHs p70 drives unfolding of preproteins and accelerates translocation of l oosely folded preproteins. While mtHsp70 is essential for import of bo th tightly and loosly folded preproteins, Tim44 plays a more specializ ed role in translocation of tightly folded domains.