Structural changes in the mitochondrial Tim23 channel are coupled to the proton-motive force

Abstract

Tim23, the central subunit of the TIM23 protein-translocation complex, forms a voltage-gated channel in the mitochondrial inner membrane (MIM), an energy-conserving membrane that generates a proton-motive force to drive vital processes. Using high-resolution fluorescence mapping of a channel-facing transmembrane segment (TMS2) of Tim23 from Saccharomyces cerevisiae, we demonstrate that changes in the energized state of the MIM cause marked structural alterations in the channel region. In an energized membrane, TMS2 forms a continuous α-helix that is inaccessible to the aqueous intermembrane space (IMS). Upon depolarization, the helical periodicity of TMS2 is disrupted, and the channel becomes exposed to the IMS. Kinetic measurements confirm that changes in TMS2 conformation coincide with depolarization. These results reveal how the energized state of the membrane drives functionally relevant structural dynamics in membrane proteins coupled to processes such as channel gating.

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis

“…The mitochondrial nucleoid encodes only for 13 structural proteins, while the rest of the mitochondrial proteome is nuclear-encoded and transported to the mitochondria through a sophisticated protein import machinery. [19][20][21][22][23][24][25][26][27][28] The translocase of the outer mitochondrial membrane (TOM), the entry gate to the mitochondria, 20 passes on the cargos to the sorting and assembly machinery (SAM) complex, to the mitochondrial intermembrane space assembly (MIA) complex and to the translocases of the inner mitochondrial membrane TIM22 or TIM23 complexes for delivery to the outer membrane, the intermembrane space, the inner membrane or the matrix, respectively. [19][20][21][22]25,26,28 Unexpectedly, we found that GB mitochondrial entry is independent of the TOM-TIM23 complexes, but requires instead the Sam50 and Tim22 channels and mitochondrial heat-shock protein 70 (mtHsp70).…”

Role of Tim17 Transmembrane Regions in Regulating the Architecture of Presequence Translocase and Mitochondrial DNA Stability

“…Recent reports highlight that the TM2 helix of Tim23 forms a core of the protein-conducting channel with one side facing the nonpolar environment and the opposite side facing the aqueous channel lumen. At the same time, the TM2 region of Tim23 undergoes structural changes in the absence of Δ m , leading to the channel opening toward the more polar environment (54,55). Intriguingly, our previous study showed that the TM2 region of Tim23 plays an inevitable role in interacting with Tim17, which in turn helps in recruiting the PAM machinery.…”

“…A preliminary analysis using reconstituted proteoliposomes suggests that Tim23 forms a gated twin pore and that depletion of Tim17 or its truncation at the N terminus disrupts the architecture of the core channel (21,23,53). Intriguingly, impaired interaction of Tim17 with the TM2 region of Tim23 has been largely attributed to loss of Δ leading to defective import (22,54,55). These observations collectively indicate that Tim17 plays an important role in modulating the voltage-dependent structural dynamics of the TIM23 channel.…”

Dynamic organization of the mitochondrial protein import machinery

“…Two energy sources drive protein translocation. The membrane potential (Δψ) across the inner membrane activates the channel protein Tim23 and moves the positively charged presequences in an electrophoretic manner (Martin et al, 1991;Truscott et al, 2001;Shariff et al, 2004;Krayl et al, 2007;van der Laan et al, 2007;Malhotra et al, 2013). PAM with the matrix heat shock protein of ~70 kDa (mtHsp70) is driven by ATP (Kang et al, 1990;Wickner and Schekman, 2005).…”

“…The lateral opening involves Tim17, a partner protein of Tim23, and the lateral gatekeeper protein Mgr2 van der Laan et al, 2007van der Laan et al, , 2013Malhotra et al, 2013;Ieva et al, 2014;Steffen and Koehler, 2014). Tim21, which is associated with Mgr2, binds to the respiratory chain complexes and positions them in close vicinity of the TIM23 translocation channel (Figure 3) (van der Laan et al, 2006;Wiedemann et al, 2007;Gebert et al, 2012).…”