Tim23 Channel Research Articles

Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexes.

Nearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM complex), followed by a procession of alternative molecular machines, conducting transport to their final destination. The pre-sequence translocase of the inner membrane (TIM23-complex) imports proteins with cleavable pre-sequences. Progress in understanding these transport mechanisms has been hampered by the poor sensitivity and time resolution of import assays. However, with the development of an assay based on split NanoLuc luciferase, we can now explore this process in greater detail. Here, we apply this new methodology to understand how ∆ψ and ATP hydrolysis, the two main driving forces for import into the matrix, contribute to the transport of pre-sequence-containing precursors (PCPs) with varying properties. Notably, we found that two major rate-limiting steps define PCP import time: passage of PCP across the outer membrane and initiation of inner membrane transport by the pre-sequence - the rates of which are influenced by PCP size and net charge. The apparent distinction between transport through the two membranes (passage through TOM is substantially complete before PCP-TIM engagement) is in contrast with the current view that import occurs through TOM and TIM in a single continuous step. Our results also indicate that PCPs spend very little time in the TIM23 channel - presumably rapid success or failure of import is critical for maintenance of mitochondrial fitness.

Conformational ensemble of an intrinsically flexible loop in mitochondrial import protein Tim21 studied by modeling and molecular dynamics simulations

BackgroundTim21, a subunit of a highly dynamic translocase of the inner mitochondrial membrane (TIM23) complex, translocates proteins by interacting with subunits in the translocase of the outer membrane (TOM) complex and Tim23 channel in the TIM23 complex. A loop segment in Tim21, which is in close proximity of the binding site of Tim23, has different conformations in X-ray, NMR and new crystal contact-free space (CCFS) structures. MD simulations can provide information on the structure and dynamics of the loop in solution. MethodsThe conformational ensemble of the loop was characterized using loop modeling and molecular dynamics (MD) simulations. ResultsMD simulations confirmed mobility of the loop. Multidimensional scaling and clustering were used to characterize the dynamic conformational ensemble of the loop. Free energy landscape showed that the CCFS crystal structure occupied a low energy region as compared to the conventional X-ray crystal structure. Analysis of crystal packing indicates that the CCFS provides larger conformational space for the motions of the loop. ConclusionsOur work reported the conformational ensemble of the loop in solution, which is in agreement with the structure obtained from CCFS approach. The combination of the experimental techniques and computational methods is beneficial for studying highly flexible regions of proteins. General significanceComputational methods, such as loop modeling and MD simulations, have proved to be useful for studying conformational flexibility of proteins. These methods in integration with experimental techniques such as CCFS has the potential to transform the studies on flexible regions of proteins.

Loss of Mgr2P Destabilizes the TIM23 Channel and Reduces Mitochondrial Emission of Reactive Oxygen Species

The TIM23 complex is a hub for translocation of preproteins into or across the mitochondrial inner membrane. This dual sorting mechanism is currently being investigated, and in yeast it seems to be regulated by a recently discovered subunit, the Mgr2 protein. Deletion of Mgr2p has been found to delay protein translocation into the matrix and accumulation in the inner membrane. This result and other findings suggested that Mgr2p controls the lateral release of inner membrane proteins harboring a stop-transfer signal that follows an N-terminal acid presequence. However, the mechanism of lateral release is unknown. Also unknown is whether the presence or absence of yeast Mgr2p might modulate reactive oxygen species (ROS), like its mammalian counterpart, Romo1. Here, we used patch clamp electrophysiology and spectrofluorimetry to investigate the effect of Mgr2p deletion upon the channel activity of TIM23 and upon emission of ROS from mitochondria. Our results suggest that Mgr2p deletion both destabilizes the TIM23 channel and decreases ROS emission. However, these two effects seem independent from each other, because ROS emission was not affected in the presence of presequence peptides.

Deletion of Mgr2p Affects the Gating Behavior of the TIM23 Complex.

The TIM23 complex is a hub for translocation of preproteins into or across the mitochondrial inner membrane. This dual sorting mechanism is currently being investigated, and in yeast appears to be regulated by a recently discovered subunit, the Mgr2 protein. Deletion of Mgr2p has been found to delay protein translocation into the matrix and accumulation in the inner membrane. This result and other findings suggested that Mgr2p controls the lateral release of inner membrane proteins harboring a stop-transfer signal that follows an N-terminal amino acid signal. However, the mechanism of lateral release is unknown. Here, we used patch clamp electrophysiology to investigate the role of Mgr2p on the channel activity of TIM23. Deletion of Mgr2p decreased normal channel frequency and increased occurrence of a residual TIM23 activity. The residual channel lacked gating transitions but remained sensitive to synthetic import signal peptides. Similarly, a G145L mutation in Tim23p displaced Mgr2p from the import complex leading to gating impairment. These results suggest that Mgr2p regulates the gating behavior of the TIM23 channel.

Motor recruitment to the TIM23 channel\u2019s lateral gate restricts polypeptide release into the inner membrane

The presequence translocase of the mitochondrial inner membrane (TIM23 complex) facilitates anterograde precursor transport into the matrix and lateral release of precursors with stop-transfer signal into the membrane (sorting). Sorting requires precursor exit from the translocation channel into the lipid phase through the lateral gate of the TIM23 complex. How the two transport modes are regulated and balanced against each other is unknown. Here we show that the import motor J-protein Pam18, which is essential for matrix import, controls lateral protein release into the lipid bilayer. Constitutively translocase-associated Pam18 obstructs lateral precursor transport. Concomitantly, Mgr2, implicated in precursor quality control, is displaced from the translocase. We conclude that during motor-dependent matrix protein transport, the transmembrane segment of Pam18 closes the lateral gate to promote anterograde polypeptide movement. This finding explains why a motor-free form of the translocase facilitates the lateral movement of precursors with a stop-transfer signal.

Cation selectivity of the presequence translocase channel Tim23 is crucial for efficient protein import.

Virtually all mitochondrial matrix proteins and a considerable number of inner membrane proteins carry a positively charged, N-terminal presequence and are imported by the TIM23 complex (presequence translocase) located in the inner mitochondrial membrane. The voltage-regulated Tim23 channel constitutes the actual protein-import pore wide enough to allow the passage of polypeptides with a secondary structure. In this study, we identify amino acids important for the cation selectivity of Tim23. Structure based mutants show that selectivity is provided by highly conserved, pore-lining amino acids. Mutations of these amino acid residues lead to reduced selectivity properties, reduced protein import capacity and they render the Tim23 channel insensitive to substrates. We thus show that the cation selectivity of the Tim23 channel is a key feature for substrate recognition and efficient protein import.

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

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.

Lipid-Dependence of the Membrane Interactions of the Tim23 Channel Subunit of the Mitochondrial Protein Import Machinery

The TIM23 complex of the mitochondrial inner membrane mediates the translocation and integration of most nuclear-encoded polypeptides that reside within the mitochondrion. The central subunit of this complex is Tim23, a voltage-gated channel-forming protein that constitutes part of the polypeptide-conducting channel. Tim23 has a bipartite domain organization with a carboxy-terminal integral membrane domain and an amino-terminal intrinsically disordered region that resides in the intermembrane space (IMS).

Energy Coupling Mechanisms and Lipid-Mediated Subunit Interactions of the Mitochondrial Protein Transport Machinery

The TIM23 complex of the mitochondrial inner membrane mediates the translocation and integration of nuclear-encoded polypeptides that are targeted to the mitochondrion. Using a host of in organello and model membrane systems, we have analyzed the conformational alterations of key TIM23 subunits and site-specific interactions that occur between subunits during different steps of the transport process. First, using a fluorescence mapping strategy, we have analyzed the structural dynamics of the central voltage-gated channel-forming subunit, Tim23. We show dramatic alterations in the conformation of Tim23 transmembrane segments of the channel region that occur in a manner coupled to the magnitude of the proton-motive force across the inner membrane. Second, using a series of model membrane systems and reductionist approaches, we have analyzed site-specific interactions that occur between the Tim23 channel and the central receptor Tim50. Using Tim23 reconstituted into nanodiscs of different lipid composition, we identify the Tim50-interactive regions within the soluble and channel domains of Tim23. We find a critical role for cardiolipin, the signature anionic phospholipid of the mitochondrion, in promoting Tim23-Tim50 interactions. Using a combination of biochemical, mass spectrometry, and molecular dynamics approaches, we identify sites of the Tim50 receptor that bind to cardiolipin-containing lipid bilayers. The headgroup and acyl chain regions of cardiolipin that are critical in mediating Tim50 binding have been addressed using mitochondria from yeast strains defective in different steps of cardiolipin remodeling as well as model membranes of different lipid composition. Finally, we use small angle x-ray scattering (SAXS) to generate ab initio models of the Tim50 receptor domain and its lipid-interactive surface. These results illuminate the early receptor-mediated stages of protein translocation by the TIM23 complex and on the role of cardiolipin in promoting subunit interactions within the translocon.

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

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.

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

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.

Signal recognition initiates reorganization of the presequence translocase during protein import

The mitochondrial presequence translocase interacts with presequence-containing precursors at the intermembrane space (IMS) side of the inner membrane to mediate their translocation into the matrix. Little is known as too how these matrix-targeting signals activate the translocase in order to initiate precursor transport. Therefore, we analysed how signal recognition by the presequence translocase initiates reorganization among Tim-proteins during import. Our analyses revealed that the presequence receptor Tim50 interacts with Tim21 in a signal-sensitive manner in a process that involves the IMS-domain of the Tim23 channel. The signal-driven release of Tim21 from Tim50 promotes recruitment of Pam17 and thus triggers formation of the motor-associated form of the TIM23 complex required for matrix transport.

Tim50’s presequence receptor domain is essential for signal driven transport across the TIM23 complex

N-terminal targeting signals (presequences) direct proteins across the TOM complex in the outer mitochondrial membrane and the TIM23 complex in the inner mitochondrial membrane. Presequences provide directionality to the transport process and regulate the transport machineries during translocation. However, surprisingly little is known about how presequence receptors interact with the signals and what role these interactions play during preprotein transport. Here, we identify signal-binding sites of presequence receptors through photo-affinity labeling. Using engineered presequence probes, photo cross-linking sites on mitochondrial proteins were mapped mass spectrometrically, thereby defining a presequence-binding domain of Tim50, a core subunit of the TIM23 complex that is essential for mitochondrial protein import. Our results establish Tim50 as the primary presequence receptor at the inner membrane and show that targeting signals and Tim50 regulate the Tim23 channel in an antagonistic manner.

Membrane Binding Mechanism of Yeast Mitochondrial Peripheral Membrane Protzein TIM44

The protein translocations across mitochondrial membranes are carried out by specialized complexes, the Translocase of Outer Membrane (TOM) and Translocase of Inner Membrane (TIM). TIM23 translocon is responsible for translocating the mitochondrial matrix proteins across the mitochondrial inner membrane. Tim44 is an essential, peripheral membrane protein in TIM23 complex. Tim44 is tightly associated with the inner mitochondrial membrane on the matrix side. The Tim44 C-Terminal Domain (CTD) functions as an Inner Mitochondrial Membrane (IMM) anchor that recruits the Presequence protein Associated Motor (PAM) to the TIM23 channel. Using X-ray crystallographic and biochemical data, we show that the N-terminal helices A1 and A2 of Tim44 - CTD are crucial for its membrane tethering function. Based on our data, we propose a model showing how the N-terminal A1 and A2 amphipathic helices can either expose their hydrophobic face during membrane binding or conceal it in the soluble form. Therefore, the A1 and A2 helices of Tim44 may function as a membrane sensor.

Role of Magmas in protein transport and human mitochondria biogenesis

Magmas, a conserved mammalian protein essential for eukaryotic development, is overexpressed in prostate carcinomas and cells exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF). Reduced Magmas expression resulted in decreased proliferative rates in cultured cells. However, the cellular function of Magmas is still elusive. In this report, we have showed that human Magmas is an ortholog of Saccharomyces cerevisiae Pam16 having similar functions and is critical for protein translocation across mitochondrial inner membrane. Human Magmas shows a complete growth complementation of Δpam16 yeast cells at all temperatures. On the basis of our analysis, we report that Magmas localizes into mitochondria and is peripherally associated with inner mitochondrial membrane in yeast and humans. Magmas forms a stable subcomplex with J-protein Pam18 or DnaJC19 through its C-terminal region and is tethered to TIM23 complex of yeast and humans. Importantly, amino acid alterations in Magmas leads to reduced stability of the subcomplex with Pam18 that results in temperature sensitivity and in vivo protein translocation defects in yeast cells. These observations highlight the central role of Magmas in protein import and mitochondria biogenesis. In humans, absence of a functional DnaJC19 leads to dilated cardiac myophathic syndrome (DCM), a genetic disorder with characteristic features of cardiac myophathy and neurodegeneration. We propose that the mutations resulting in decreased stability of functional Magmas:DnaJC19 subcomplex at human TIM23 channel leads to impaired protein import and cellular respiration in DCM patients. Together, we propose a model showing how Magmas:DnaJC19 subcomplex is associated with TIM23 complex and thus regulates mitochondrial import process.

Topogenesis of Mammalian Oxa1, a Component of the Mitochondrial Inner Membrane Protein Export Machinery

Oxa1 is a mitochondrial inner membrane protein with a predicted five-transmembrane segment (TM1 approximately 5) topology in which the N terminus and a hydrophilic loop, L2, are exposed to the intermembrane space and the C-terminal region and two loops, L1 and L3, are exposed to the matrix. Oxa1 mediates the insertion of mitochondrial DNA-encoded subunits of respiratory complexes and several nuclear DNA-encoded proteins into the inner membrane from the matrix. Compared with yeast Oxa1, little is known about the import and function of mammalian Oxa1. Here, we investigated the topogenesis of Oxa1 in HeLa cells using systematic deletion or mutation constructs and found that (i) the N-terminal 64-residue segment formed a presequence, and its deletion directed the mature protein to the endoplasmic reticulum, indicating that the presequence arrests cotranslational activation of the potential endoplasmic reticulum-targeting signal within mature Oxa1, (ii) systematic deletion of Oxa1 TM segments revealed that the presence of all five TMs is essential for efficient membrane integration, (iii) the species-conserved hexapeptide (GLPWWG) located near the N terminus of TM1 was essential for export of the N-terminal segment and L2 into the intermembrane space from the matrix, i.e. for correct topogenesis of Oxa1, and (iv) GLPWWG placed near the N terminus of TM2 or TM3 in the reporter construct also supported its membrane integration in the Nout-Cin orientation. Together, these results demonstrated that topogenesis of Oxa1 is a cooperative event of all five TMs, and GLPWWG followed immediately by TM1 is essential for correct Oxa1 topogenesis.

Step-size Analyses of the Mitochondrial Hsp70 Import Motor Reveal the Brownian Ratchet in Operation

Newly synthesized mitochondrial precursor proteins have to become unfolded by the mitochondrial Hsp70 (mtHsp70) import motor to cross the mitochondrial membranes. To assess the mechanism of unfolding of precursor proteins by mtHsp70, we designed a system to measure step sizes of the mtHsp70 import motor, which are distances at which the motor system moves along polypeptide chains during a single turnover of ATP. We made a series of fusion proteins consisting of a mitochondrial presequence containing the first mtHsp70 binding site, a spacer sequence containing an Hsp70 avoidance segment followed by the second mtHsp70 binding site, and different folded mature domains. Analyses of the dependence of the import rates of those fusion proteins on the lengths of Hsp70 avoidance segments allowed us to estimate the step sizes, which differ for different mature domains and different lengths of the spacers. These results suggest that the mtHsp70 import motor functions at least as a molecular Brownian ratchet to unfold mitochondrial precursor proteins.

Awaking TIM22, a Dynamic Ligand-gated Channel for Protein Insertion in the Mitochondrial Inner Membrane

Aqueous channels are at the core of the translocase of the outer membrane (TOM) and the translocase of the inner membrane for the transport of preproteins (TIM23), the translocases mediating the transport of proteins across the outer and inner mitochondrial membranes. Yet, the existence of a channel associated to the translocase of the inner membrane for the insertion of multitopic protein (TIM22) complex has been arguable, as its function relates to the insertion of multispanning proteins into the inner membrane. For the first time, we report conditions for detecting a channel activity associated to the TIM22 translocase in organelle, i.e. intact mitoplasts. An internal signal peptide in the intermembrane space of mitochondria is a requisite to inducing this channel, which is otherwise silent. The channel showed slightly cationic and high conductance activity of 1000 pS with a predominant half-open substate. Despite their different composition, the channels of the three mitochondrial translocases were thus remarkably similar, in agreement with their common task as pores transiently trapping proteins en route to their final destination. The opening of the TIM22 channel was a step-up process depending on the signal peptide concentration. Interestingly, low membrane potentials kept the channel fully open, providing a threshold level of the peptide is present. Our results portray TIM22 as a dynamic channel solely active in the presence of its cargo proteins. In its fully open conformation, favored by the combined action of internal signal peptide and low membrane potential, the channel could embrace the in-transit protein. As insertion progressed and initial interaction with the signal peptide faded, the channel would close, sustaining its role as a shunt that places trapped proteins into the membrane.

Tim17p Regulates the Twin Pore Structure and Voltage Gating of the Mitochondrial Protein Import Complex TIM23

The TIM23 complex mediates import of preproteins into mitochondria, but little is known of the mechanistic properties of this translocase. Here patch clamping reconstituted inner membranes allowed for first time insights into the structure and function of the preprotein translocase. Our findings indicate that the TIM23 channel has "twin pores" (two equal sized pores that cooperatively gate) thereby strikingly resembling TOM, the translocase of the outer membrane. Tim17p and Tim23p are homologues, but their functions differ. Tim23p acts as receptor for preproteins and may largely constitute the preprotein-conducting passageway. Conversely depletion of Tim17p induces a collapse of the twin pores into a single pore, whereas N terminus deletion or C terminus truncation results in variable sized pores that cooperatively gate. Further analysis of Tim17p mutants indicates that the N terminus is vital for both voltage sensing and protein sorting. These results suggest that although Tim23p is the main structural unit of the pore Tim17p is required for twin pore structure and provides the voltage gate for the TIM23 channel.

A fluorescence assay for peptide translocation into mitochondria

Translocation of the presequence is an early event in import of preproteins across the mitochondrial inner membrane by the TIM23 complex. Import of signal peptides, whose sequences mimic mitochondrial import presequences, was measured using a novel, qualitative, fluorescence assay in about 1 h. This peptide assay was used in conjunction with classical protein import analyses and electrophysiological approaches to examine the mechanisms underlying the functional effects of depleting two TIM23 complex components. Tim23p forms, at least in part, the pore of this complex while Tim44p forms part of the translocation motor. Depletion of Tim23p eliminates TIM23 channel activity, which interferes with both peptide and preprotein translocation. In contrast, depletion of Tim44p disrupts preprotein but not peptide translocation, which has no effect on TIM23 channel activity. Two conclusions were made. First, this fluorescence peptide assay was validated as two different mutants were accurately identified. Hence, this assay could provide a rapid means of screening mutants to identify those that fail an initial step in import, i.e., translocation of the presequence. Second, translocation of signal peptides required normal channel activity and disruption of the presequence translocase-associated motor complex did not modify TIM23 channel activity nor prevent presequence translocation.