Energy Coupling Mechanisms And Lipid-Mediated Subunit Interactions Of The Mitochondrial Protein Transport Machinery

The TIM23 complex of the mitochondrial inner membrane mediates the translocation and integration of nuclear-encoded polypeptides that are targeted to the mitochondrion. Using a host of in organello and model membrane systems, we have analyzed the conformational alterations of key TIM23 subunits and site-specific interactions that occur between subunits during different steps of the transport process. First, using a fluorescence mapping strategy, we have analyzed the structural dynamics of the central voltage-gated channel-forming subunit, Tim23. We show dramatic alterations in the conformation of Tim23 transmembrane segments of the channel region that occur in a manner coupled to the magnitude of the proton-motive force across the inner membrane. Second, using a series of model membrane systems and reductionist approaches, we have analyzed site-specific interactions that occur between the Tim23 channel and the central receptor Tim50. Using Tim23 reconstituted into nanodiscs of different lipid composition, we identify the Tim50-interactive regions within the soluble and channel domains of Tim23. We find a critical role for cardiolipin, the signature anionic phospholipid of the mitochondrion, in promoting Tim23-Tim50 interactions. Using a combination of biochemical, mass spectrometry, and molecular dynamics approaches, we identify sites of the Tim50 receptor that bind to cardiolipin-containing lipid bilayers. The headgroup and acyl chain regions of cardiolipin that are critical in mediating Tim50 binding have been addressed using mitochondria from yeast strains defective in different steps of cardiolipin remodeling as well as model membranes of different lipid composition. Finally, we use small angle x-ray scattering (SAXS) to generate ab initio models of the Tim50 receptor domain and its lipid-interactive surface. These results illuminate the early receptor-mediated stages of protein translocation by the TIM23 complex and on the role of cardiolipin in promoting subunit interactions within the translocon.