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

Abstract

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

Protein Translocation into the Intermembrane Space and Matrix of Mitochondria: Mechanisms and Driving Forces

“…Tim23 and Tim17 are two structurally related multi-spanning inner membrane proteins that form the protein-conducting channel of the inner membrane (Truscott et al, 2001 ; Meier et al, 2005 ; Zarsky and Dolezal, 2016 ). Tim17, which shows the highest degree of sequence conservation of all TOM and TIM subunits, coordinates the membrane potential-dependent opening of the channel with the binding of the import motor on the matrix site via the membrane-associated subunit Tim44 (Meier et al, 2005 ; Martinez-Caballero et al, 2007 ; Ramesh et al, 2016 ; Demishtein-Zohary et al, 2017 ; Matta et al, 2017 ; Ting et al, 2017 ). Tim44 serves as binding site for the mitochondrial Hsp70 (mtHsp70) of the matrix which binds tightly to incoming polypeptides upon hydrolysis of its bound ATP to ADP in a reaction regulated by Pam18/Tim14 and Pam16/Tim16, a J protein and a J-like protein, respectively, that are associated with the TIM23 complex.…”

“…From this perspective, the power stroke model can be seen as an extension rather than an alternative to the Brownian Ratchet model. The tight contact of mtHsp70 to the protein-conducting channel of the TIM23 translocase, which is critical for both mechanisms, is mediated by Tim44, Pam18/Tim14, and Pam16/Tim16 (Demishtein-Zohary et al, 2017 ; Matta et al, 2017 ; Ting et al, 2017 ).…”

Mutations in TIMM50 cause severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology

“…In fact, the mitochondrial import machinery has been reported to be linked to the mitochondrial inner membrane organizing system, which is necessary for maintaining mitochondrial cristae structure (Becker, Böttinger, & Pfanner, 2012). In addition, alterations in TIM17, another component of TIM23 complex, have been recently found to be associated with abnormal mitochondrial morphology (Matta, Pareek, Bankapalli, Oblesha, & D'Silva, 2017).…”

Mgr2 regulates mitochondrial preprotein import by associating with channel-forming Tim23 subunit

“…Visualization of mitochondrial morphology was performed as per previously published protocols ( Matta et al. , 2017 ).…”

“…The antisera used for immunodecoration against yeast-specific proteins such as Tim23, Tim17, Tim50, Tim44, Tim9, GST, Pam18, and Pam16 was raised in rabbits, as reported earlier ( Pareek et al. , 2013 ; Matta et al. , 2017 ).…”