Biogenesis of the yeast frataxin homolog Yfh1p - Tim44-dependent transfer to mtHsp70 facilitates folding of newly imported proteins in mitochondria

Tim44 is an essential component of the mitochondrial inner membrane protein import machinery. In this study we asked if Tim44 is of relevance in intra mitochondrial protein folding. We investigated the role of Tim44 in the bio genesis of the authentic mitochondrial protein Yfh1p, the yeast homolog of mammalian frataxin, which was recently implicated in Friedreich ataxia. Aft er inactivation of Tim44, binding of mitochondrial heat shock protein (mtHs p)70 to translocating Yfh1p and subsequent folding to the native state was nearly completely blocked. Residual amounts of imported Yfh1p showed an inc reased tendency to aggregate. To further characterize the functions of Tim4 4 in the matrix, we imported dihydrofolate reductase (DHFR) as a model prot ein. Depletion of Tim44 allowed import of DHFR, although folding of the new ly imported DHFR was delayed. Moreover, the depletion of Tim44 caused a str ongly reduced binding of mtHsp70 and Mge1 to the translocating polypeptide. Subsequent dissociation of mtHsp70 from imported DHFR was delayed, indicat ing that mtHsp70-substrate complexes formed independently of Tim44 differ f rom the complexes that form under the control of Tim44. We conclude that Ti m44 not only plays a role in protein translocation but also in the pathways of mitochondrial protein folding.