Mitochondrial life cycle and protein import are intricate cellular processes, which require precise coordination between the transport machineries of outer and inner mitochondrial membranes. Presequence translocase performs the indispensable function of translocating preproteins having N-terminal targeting sequences across the inner membrane. Tim23 forms the core of the voltage-gated import channel, while Tim17 is presumed to maintain the stoichiometry of the translocase. However, mechanistic insights into how Tim17 coordinates these regulatory events within the complex remained elusive. We demonstrate that Tim17 harbors conserved G/AXXXG/A motifs within its transmembrane regions and plays an imperative role in the translocase assembly through interaction with Tim23. Tandem motifs are highly essential, as most of the amino acid substitutions lead to nonviability due to the complete destabilization of the TIM23 channel. Importantly, Tim17 transmembrane regions regulate the dynamic assembly of translocase to form either the TIM23 (PAM)-complex or TIM23 (SORT)-complex by recruiting the presequence translocase-associated motor (PAM) machinery or Tim21, respectively. To a greater significance, tim17 mutants displayed mitochondrial DNA (mtDNA) instability, membrane potential loss, and defective import, resulting in organellar dysfunction. We conclude that the integrity of Tim17 transmembrane regions is critical for mitochondrial function and protein turnover.KEYWORDS mitochondria, presequence translocase, mtDNA stability, preprotein import, membrane potential T he vast majority of mitochondrial proteins are encoded by nuclear genes and synthesized on cytosolic ribosomes with an amino-terminal positively charged cleavable signal sequence. The efficient import of mitochondrial proteins requires precise coordination between the translocases of the outer membrane (TOM complex) and the inner membrane (TIM23 complex) (1-9). After initial recognition and transport through TOM complex, these preproteins are actively sorted based on their destination. The preproteins directed toward the matrix compartment possess an N-terminal positively charged presequence and are imported into the matrix compartment with the aid of presequence translocase (TIM23 complex) (1-10). The core of presequence translocase consists of a membrane-bound channel component formed by . Tim23 and Tim17 are phylogenetically related integral membrane proteins and have similar transmembrane topology. Tim23 contributes to the formation of voltage-gated protein-conducting pores, while the evidence suggests that Tim17 is majorly involved in regulating the channel activity (16)(17)(18)(19)(20)(21)(22)(23). Other components such as Tim50 and Tim21 act as receptors and coordinate with the intermembrane space (IMS) domain of Tim23 to facilitate preprotein recognition and transfer from the TOM complex to presequence translocase (16, 17, 22, 24-32). Mgr2 has been recently

Mgr2 regulates the gating behavior of the TIM23 complex and mgr2∆ and causes aberrations in mitochondrial dynamics. Here, we show that Mgr2 directly associates with channel-forming Tim23. Additionally, the Mgr2 transmembrane region plays a crucial role in coupling the TIM23 complex with OXPHOS machinery and thus regulates mitochondrial protein import and dynamics.

Mitochondrial biogenesis requires efficient sorting of various proteins into different mitochondrial sub-compartments, mediated by dedicated protein machinery present in the outer and inner membrane. Among them, the TIM22 complex enables the integration of complex membrane proteins with internal targeting signals into the inner membrane. Although the Tim22 protein forms the core of the complex, the dynamic recruitment of subunits to the channel is still enigmatic. In this study, we highlight that the intermembrane space (IMS) and transmembrane 4 (TM4) regions of Tim22 are critically required for interactions with the membrane-embedded subunits, including Tim54, Tim18, and Sdh3, and thereby maintain the functional architecture of the TIM22 translocase. Furthermore, we find that the TM1 and TM2 regions of Tim22 are important for association with Tim18, whereas TM3 is exclusively required for the interaction with Sdh3. Moreover, impairment of TIM22 complex assembly influences its translocase activity, the mitochondrial network, and the viability of cells lacking mitochondrial DNA. Overall, our findings provide compelling evidence highlighting the significance of conserved regions of Tim22 that are important for the maintenance of the TIM22 complex and mitochondrial integrity.

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