Molecular Insights Revealing Interaction of Tim23 and Channel Subunits of Presequence Translocase

Abstract

Tim23 is an essential channel-forming subunit of the presequence translocase recruiting multiple components for assembly of the core complex, thereby regulating the protein translocation process. However, understanding of the precise interaction of subunits associating with Tim23 remains largely elusive. Our findings highlight that transmembrane helix 1 (TM1) is required for homodimerization of Tim23, while, together with TM2, it is involved in preprotein binding within the channel. Based on our evidence, we predict that the TM1 and TM2 from each dimer are involved in the formation of the central translocation pore, aided by Tim17. Furthermore, TM2 is also involved in the recruitment of Tim21 and the presequence-associated motor (PAM) subcomplex to the Tim23 channel, while the matrix-exposed loop L1 generates specificity in their association with the core complex. Strikingly, our findings indicate that the C-terminal sequence of Tim23 is dispensable for growth and functions as an inhibitor for binding of Tim21. Our model conceptually explains the cooperative function between Tam41 and Pam17 subunits, while the antagonistic activity of Tim21 predominantly determines the bound and free forms of the PAM subcomplex during import. Mitochondria are indispensable organelles required for a wide spectrum of cellular processes, including energy metabolism. The mitochondrial genome encodes only a few proteins across the kingdoms, ranging from 7 in Saccharomyces cerevisiae to 13 in humans (1, 2). Almost all the several hundreds of proteins residing in various mitochondrial compartments are encoded by the nuclear genome and synthesized on cytosolic ribosomes. Therefore, active mitochondrial function and biogenesis require efficient protein transport destined for the different subcompartments (1-9). Greater than 60% of mitochondrial proteins are targeted to the matrix compartment and are translocated through the presequence translocase (Tim23 complex), an inner membranebound multisubunit machinery whose components are highly conserved. The presequence translocase consists of central channel-forming components mainly constituted by the Tim23 protein together with Tim17, which maintains the integrity of the translocation pore (10-15), while other subunits, such as Tim50 and Tim21, which contains a single transmembrane helix with a large intermembrane space (IMS)-exposed domain, assist with presorting and guided channeling of preproteins via interaction with the translocase of outer membrane (TOM) complex (16)(17)(18)(19)(20). Besides channel-forming components, the Tim23 complex recruits a peripherally associated import motor whose functions are essential for the translocation of proteins destined for the matrix compartment (21-23). Mitochondrial heat shock protein Hsp70 (Ssc1 in yeast) forms the core of the import motor recruited for preprotein binding in the ATP-bound state to the channel via a membrane tether, Tim44 (21-24). Tim44 provides the platform and recruits two additional terminal regulatory components, J protein Pam...

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

“…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.…”

“…Translocation via presequence translocase is energetically driven by active membrane potential (Δ m ) across the inner membrane. Reduced Δ m has been implicated in diminished rates of protein import across the inner membrane (22,36,53). To assess whether the mutations in G/AXXXG/A motifs of Tim17 impart loss in Δ m , we stained the cells with the polarity-sensitive JC-1 dye (69).…”

“…To check whether the loss of Δ m leads to decrease in the organellar functionality, wild-type and mutant cells were stained with MitoTracker Deep Red-A dye, which is a membrane potential-dependent indicator of active mitochondrial mass (70)(71)(72). After inducing the phenotype by heat shock, the wild type and tim17 mutants were subjected to functional mass estimation using MitoTracker Deep Red-A fluorescence through flow-cytometric analysis (22). In agreement with our hypothesis, compared to the wild type, all the tim17 mutants exhibited a significant decrease in fluorescence, indicating that mutations in G/AXXXG/A motifs of Tim17 caused the reduction in functional mitochondria (Fig.…”

“…At the amino acid sequence level, both Tim17 and Tim23 contain a large number of G/AXXXG/A motifs within the transmembrane segments. Recent reports highlight that conditional mutants isolated within the G/AXXXG/A motifs of Tim23 transmembrane regions were found to impair Tim23 homodimerization as well as interactions with other components such as Tim17 (22,65).…”

“…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).…”