Identification of cytosolic factors interacting with mitochondrial preproteins

Abstract

5. Summary During the evolutionary evolvement of mitochondria, the mitochondrial genome got reduced and has almost completely been transferred to the host genome. Therefore most mitochondrial proteins are synthesized in the cytosol and delivered to the organelle by the help of molecular chaperones in an unfolded state. Cytosolic chaperones like Hsp90 and Hsp70 in mammalian cells act in a large cytosolic complex that helps in the delivery of some mitochondrial proteins to the organelle by docking on the import receptor Tom70. These chaperones are abundant ones, which have many other cellular functions, suggesting that the specificity for the targeting of mitochondrial proteins probably requires addition of specific factors within the targeting complex. Most mitochondrial precursor proteins comprise a cleavable N-terminal targeting sequence, also known as presequence, that targets them to the mitochondrial import receptor Tom20. Many of these proteins associate during or after synthesis with cytosolic chaperones to keep them in an import competent state. So far, only little is known about the identity and the potential function of specific cytosolic chaperones assisting in import of mitochondrial precursor proteins containing N-terminal targeting sequence. The aim of this study was to identify cytosolic factors that interact with presequence-containing mitochondrial precursor proteins, to characterize their involvement in stabilization and targeting of precursor proteins and to study their physiological role in mitochondrial biogenesis. To this end, in vivo site-directed photo-crosslinking method in Saccharomyces cerevisiae was performed. I identified the (co)-chaperones Sti1, Ssa1, Ydj1, and Hsp82 as cytosolic factors, which interact specifically with mitochondrial precursor proteins. Sti1 as a co-chaperone of Hsp90/Hsp70 complex was further investigated. I could demonstrate that sti1Δ cells displayed a decreased growth rate in comparison to wild type. Additionally mitochondrial morphology was affected in cells lacking Sti1. I also found genetic interaction of Sti1 with the import components Tom20 and Mim1. Furthermore pull-down experiments with recombinant GST-tagged versions of Tom70 and Tom20 revealed association of Sti1 with Tom70 whereas Sis1 and Ydj1 interacted with Tom20. Based on the results from this study, I can conclude that Sti1, Sis1, Ydj1, Ssa1 and Hsp82 are involved in the import of presequence-containing proteins to mitochondria.