The Mitochondrial Tim23 Protein Transport Complex Undergoes Conformational Dynamics Coupled to the Energized State of the Inner Membrane

Abstract

The TIM23 Complex of the mitochondrial inner membrane (IM) is a multi-component assembly that mediates the translocation of matrix-targeted precursor proteins as well as the integration of membrane proteins. This complex is energetically coupled to both an ATPase motor and the electrochemical proton potential across the IM. The central subunit of this complex, Tim23, forms a voltage-gated channel and contains a large soluble domain in the intermembrane space that functions as a substrate receptor. To analyze the structural dynamics that are attendant with changes in the energized state of the membrane, we employed a high-resolution fluorescence mapping approach in which small extrinsic probes were cotranslationally incorporated into specific sites of Tim23 from Saccharomyces cerevisiae and analyzed in active mitochondria by steady-state and time-resolved measurements under different physiologically relevant states. Analysis of the channel-facing transmembrane segment (TMS2) of Tim23 revealed that changes in the energized status of the inner membrane caused dramatic structural alterations in the channel region. In an energized membrane, TMS2 formed a continuous α-helix that was inaccessible to the aqueous intermembrane space. Upon depolarization, the helical periodicity of TMS2 was disrupted and the channel became exposed to the IMS. Real time kinetic measurements confirmed that changes in TMS2 conformation coincided with depolarization. This analysis is extended to the soluble receptor domain of Tim23, where we show proton-motive force-coupled structural changes and key protein interactions that are mediated by specific lipids within the inner membrane. These results reveal how the energized state of the membrane drives functionally relevant structural dynamics in membrane proteins that are coupled to processes such as channel gating.