Recombinant Schizosaccharomyces pombe Mitochondrial import inner membrane translocase subunit tim23 (tim23)

What is Tim23 and what is its role in mitochondrial protein import?

Tim23 is the channel-forming subunit of the TIM23 complex (Translocase of the Inner Mitochondrial membrane) in the mitochondrial inner membrane. In Schizosaccharomyces pombe, Tim23 is a 210 amino acid protein that creates a voltage-activated channel essential for importing presequence-carrying proteins into the mitochondrial matrix and inner membrane . The Tim23 channel forms a water-filled pore with a diameter of approximately 1.3-2.4 nm that allows unfolded proteins to pass while maintaining the membrane's permeability barrier . The import process is driven by the membrane potential (ΔΨ) across the inner membrane, which is necessary for insertion of inner membrane proteins . For complete protein translocation into the mitochondrial matrix, additional energy from ATP is required, provided by the presequence translocase-associated import motor PAM .

Methodological approach: To study Tim23's function, use in vitro import assays with isolated mitochondria and radiolabeled preproteins. Compare import efficiency between wild-type and Tim23 mutant mitochondria to assess Tim23's specific contribution. For more controlled studies, reconstitute purified recombinant Tim23 into liposomes to examine its channel properties independent of other mitochondrial components.

How does Tim23 structure relate to its function in the mitochondrial inner membrane?

Tim23's structure is intimately linked to its function as a protein-conducting channel. In S. pombe, Tim23 contains multiple transmembrane domains spanning the mitochondrial inner membrane . The protein consists of two main functional regions: an N-terminal domain exposed to the intermembrane space (IMS) that acts as a receptor for incoming preproteins, and a C-terminal domain forming the membrane-embedded channel . The transmembrane helices line the aqueous pore through which preproteins pass. Particularly important is the second transmembrane helix, containing highly conserved pore-lining amino acid residues that contribute to the channel's cation selectivity .

This cation selectivity is critical for Tim23 function, enabling recognition of positively charged mitochondrial targeting presequences. The positively charged presequences interact with the negatively charged surface of the channel, facilitating their translocation across the inner membrane. Mutations reducing the cation selectivity decrease substrate sensitivity and impair protein import efficiency .

Methodological approach: To investigate structure-function relationships, use site-directed mutagenesis targeting conserved residues, especially those in the second transmembrane helix. Express and purify these mutant proteins, then compare their properties to wild-type Tim23 using electrophysiological measurements in planar lipid bilayers and protein import assays with isolated mitochondria.

What are the key differences between Tim23 in S. pombe compared to S. cerevisiae?

While Tim23 is conserved across fungal species, notable differences exist between S. pombe and S. cerevisiae orthologs. S. pombe Tim23 consists of 210 amino acids , while S. cerevisiae Tim23 is slightly larger. Sequence conservation is particularly high in the transmembrane domains, especially in the pore-lining residues of the second transmembrane helix . These conserved residues include those contributing to channel cation selectivity, suggesting this property is functionally important across species.

S. pombe has been widely used as a model system for many cellular processes, though fewer studies have focused specifically on its mitochondrial import machinery compared to S. cerevisiae . One interesting aspect of S. pombe mitochondrial biology is the presence of tandem proteins, where two proteins that exist separately in other organisms are fused in S. pombe . While Tim23 itself is not described as a tandem protein, this feature highlights potential differences in protein organization and regulation between the two yeast species.

Methodological approach: Conduct comparative studies between S. pombe and S. cerevisiae Tim23 using sequence alignment, heterologous expression systems, and functional complementation assays. Express S. pombe Tim23 in S. cerevisiae tim23 deletion strains to assess functional conservation. Characterize both proteins biochemically and biophysically under identical conditions to reveal differences in stability, channel properties, or interactions with other components of the import machinery.

How is recombinant S. pombe Tim23 typically produced and purified?

Recombinant S. pombe Tim23 is typically produced in E. coli expression systems . The full-length protein (amino acids 1-210) is often fused to an N-terminal His tag to facilitate purification . The protein is commonly found in inclusion bodies after expression, requiring denaturation and refolding procedures.

Table 1: Standard Purification Protocol for Recombinant S. pombe Tim23

For reconstitution, dissolve the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, add 5-50% glycerol (with 50% being the default final concentration) and aliquot for storage at -20°C/-80°C . Avoid repeated freeze-thaw cycles to maintain protein integrity.

Methodological approach: Optimize expression conditions by testing different E. coli strains, induction temperatures (16-25°C often works best for membrane proteins), and IPTG concentrations. For purification, use denaturing conditions (8M urea or 6M guanidinium hydrochloride), followed by on-column refolding with a decreasing urea gradient to improve recovery of functional protein.

What experimental approaches are most effective for studying Tim23 channel activity?

The most effective approaches for studying Tim23 channel activity combine electrophysiological measurements with biochemical and genetic analyses, based on established protocols from S. cerevisiae Tim23 research :

Single-channel planar lipid bilayer experiments: This technique involves incorporating purified Tim23 into preformed large unilamellar vesicles (LUVs), which are then fused to a planar lipid bilayer . This allows direct measurement of channel conductance, ion selectivity, voltage-dependence, and substrate sensitivity by applying voltage across the membrane and recording current flow.

Patch-clamp analyses of inner membrane-derived vesicles: This approach uses mitochondrial inner membrane vesicles with or without specific components of the TIM23 complex . It allows measurement of channel activity in a more native-like membrane environment.

Cation selectivity measurements: The reversal potential of the channel can be measured to determine its preference for cations over anions . This property is crucial for Tim23 function in recognizing positively charged presequence peptides.

Substrate sensitivity assays: The response of the Tim23 channel to presequence peptides can be measured by observing changes in gating behavior upon peptide addition .

Methodological approach: For electrophysiological studies, carefully control lipid composition (a 3:1 mixture of POPC:POPE has been successfully used) , buffer conditions (pH and salt concentration), and protein:lipid ratios. For selectivity measurements, use asymmetric salt conditions (e.g., 250 mM KCl cis / 20 mM KCl trans) to determine reversal potential. For substrate sensitivity, use synthetic presequence peptides at concentrations of 1-50 μM.

How can researchers assess Tim23 integration into artificial membrane systems?

Proper integration of recombinant Tim23 into artificial membrane systems is crucial for functional studies. Several complementary approaches can verify successful reconstitution:

Proteoliposome flotation assays: After reconstituting Tim23 into liposomes, perform sucrose gradient centrifugation. Properly integrated protein will float with the liposomes to the top of the gradient, while non-integrated protein remains at the bottom.

Protease protection assays: Limited proteolysis of Tim23-containing proteoliposomes reveals the topology of the reconstituted protein. Transmembrane domains embedded in the lipid bilayer will be protected from proteolytic digestion, while exposed domains will be cleaved.

Dynamic light scattering (DLS): This technique assesses the size distribution of proteoliposomes and detects aggregation that might occur with improperly reconstituted protein.

Circular dichroism (CD) spectroscopy: CD evaluates the secondary structure content of reconstituted Tim23, confirming that it retains its native fold after reconstitution.

Functional assays: The ultimate test of proper integration is functional activity. Electrophysiological measurements of channel activity in planar lipid bilayers provide direct evidence of successful reconstitution.

Methodological approach: For optimal reconstitution, use detergent-mediated methods. Mix purified Tim23 in detergent (n-dodecyl-β-D-maltoside or Triton X-100) with preformed liposomes, followed by controlled detergent removal using Bio-Beads or dialysis. Use protein:lipid ratios of 1:1000 to 1:5000 (w/w) for electrophysiological studies. The lipid composition should mimic the mitochondrial inner membrane, with a mixture of phosphatidylcholine, phosphatidylethanolamine, and cardiolipin.

What are the critical buffer conditions for maintaining Tim23 stability?

Maintaining recombinant Tim23 stability requires careful attention to buffer composition:

Table 2: Optimal Buffer Conditions for S. pombe Tim23

Methodological approach: Optimize buffer conditions for specific experimental needs. For electrophysiological studies, use buffers containing 10-20 mM HEPES or Tris, 150-250 mM KCl, and 1-5 mM divalent cations (CaCl2 or MgCl2) at pH 7.4-7.6. For biochemical assays and storage, use higher buffering capacity (50 mM Tris) with stabilizing agents like glycerol or trehalose. When changing buffer conditions, make gradual transitions through dialysis or size exclusion chromatography rather than sudden changes that might destabilize the protein.

How can researchers validate the proper folding and activity of recombinant Tim23?

Validating proper folding and activity of recombinant Tim23 is essential for reliable experimental results. Several complementary approaches can be used:

Circular dichroism (CD) spectroscopy: Assess the secondary structure content, confirming that the purified protein contains the expected alpha-helical content characteristic of transmembrane domains.

Size exclusion chromatography (SEC): Evaluate the homogeneity of the protein preparation and detect aggregates or oligomers.

Thermal shift assays: Measure the thermal stability of the protein, which can indicate proper folding.

Limited proteolysis: Properly folded proteins typically show characteristic proteolytic patterns, with cleavage occurring primarily at exposed, flexible regions.

Channel activity measurements: The ultimate validation is functional activity in electrophysiological assays, measuring:

• Channel conductance

• Ion selectivity

• Voltage dependence

• Sensitivity to presequence peptides

Methodological approach: For functional validation, use electrophysiological characterization in planar lipid bilayers. Reconstitute the purified protein into liposomes and incorporate into planar lipid bilayers for single-channel recordings. Key parameters to measure include conductance (typically 100-500 pS in 150 mM KCl), cation selectivity (PK+/PCl- >5), voltage dependence (more active at negative potentials on the matrix side), and response to presequence peptides (increased gating activity) . If the recombinant protein shows these characteristics, it can be considered properly folded and functional.

How do mutations in Tim23 affect its cation selectivity and protein import function?

Mutations in Tim23, particularly in pore-lining residues of the second transmembrane helix, significantly impact its cation selectivity and consequently protein import function. Research with S. cerevisiae Tim23 provides valuable insights into structure-function relationships :

Several conserved residues in the second transmembrane helix are critical for cation selectivity, including N150, G153, L155, A156, Y159, and N160 . Mutations in these residues significantly reduce cation preference, with the strongest effect observed for N150A, which reduced selectivity to 33% of wild-type level .

Table 3: Impact of Mutations on Tim23 Function

The functional consequences of these mutations were assessed through:

• Growth phenotype analysis: Yeast expressing Tim23 with reduced selectivity showed growth defects, particularly on non-fermentable carbon sources at elevated temperatures .

• Protein import assays: Mitochondria containing Tim23 N150A showed significantly reduced import capacity for matrix proteins .

• Electrophysiological characterization: Tim23 N150A channels exhibited drastically reduced substrate sensitivity, remaining in an inactive, slowly gating state in the presence of presequence peptides .

Importantly, the N150A mutation did not affect other channel parameters, complex assembly, or membrane potential, indicating that the observed import defect was directly related to reduced cation selectivity .

Methodological approach: To study the impact of Tim23 mutations, employ a comprehensive approach combining genetics, biochemistry, and electrophysiology. Create point mutations through site-directed mutagenesis, express the mutant proteins in vivo (for growth phenotype analysis) and in vitro (for biochemical and electrophysiological characterization), and assess protein import efficiency using isolated mitochondria and radiolabeled preproteins. Measure the reversal potential under asymmetric salt conditions to calculate the selectivity ratio (PK+/PCl-), and assess substrate sensitivity by measuring channel response to presequence peptides.

What techniques can be used to study the interaction of Tim23 with other components of the TIM23 complex?

Studying interactions between Tim23 and other TIM23 complex components requires techniques that capture both stable and transient protein-protein interactions:

Co-immunoprecipitation (co-IP): This technique identifies stable interactions between Tim23 and other components like Tim17, Tim50, Tim21, and subunits of the PAM complex . Antibodies against Tim23 can pull down the entire complex, with associated proteins detected by Western blotting.

Size exclusion chromatography: This technique analyzes the integrity of the TIM23 complex and its association with the import motor under different conditions . It provides information about the size and composition of the complex.

Chemical cross-linking: This approach captures transient or weak interactions that might be missed by co-IP, using bifunctional reagents that covalently link proteins in close proximity.

Genetic interaction studies: Synthetic lethality or synthetic growth defect analyses reveal functional relationships between Tim23 and other components of the import machinery.

In vitro reconstitution: Purified components of the TIM23 complex can be reconstituted in artificial membrane systems to study their interactions and functional cooperation.

Methodological approach: For co-IP studies, optimize solubilization conditions to maintain complex integrity. Digitonin is often the preferred detergent for solubilizing mitochondrial membranes while preserving protein-protein interactions . For cross-linking studies, use membrane-permeable cross-linkers with different spacer arm lengths to map spatial relationships between complex components. When analyzing complex assembly, compare wild-type and mutant Tim23 proteins under identical conditions to accurately assess the impact of mutations on protein-protein interactions.

How can researchers measure substrate recognition by the Tim23 channel?

Measuring substrate recognition by the Tim23 channel involves assessing how the channel responds to mitochondrial targeting sequences (presequences):

Electrophysiological assays: Single-channel recordings in planar lipid bilayers directly measure Tim23 response to presequence peptides . Addition of substrate peptides typically induces increased gating activity in wild-type Tim23 channels, indicating channel activation. Quantify this response by measuring gating frequency and dwell times in open and closed states before and after peptide addition.

Peptide binding assays: Direct interaction between Tim23 (particularly its intermembrane space domain) and presequence peptides can be measured using fluorescence anisotropy, isothermal titration calorimetry, or surface plasmon resonance.

Import competition assays: The ability of synthetic presequence peptides to compete with full-length preproteins for import into isolated mitochondria can assess the specificity and affinity of substrate recognition.

Crosslinking approaches: Photoactivatable presequence peptides can identify the exact residues in Tim23 that interact with the substrate during translocation.

Methodological approach: For electrophysiological assessment, use synthetic peptides corresponding to well-characterized mitochondrial targeting sequences. Add these peptides to the cis side of the membrane (corresponding to the intermembrane space) at concentrations of 1-50 μM. Record channel activity for several minutes before and after peptide addition to quantify changes in gating behavior. Compare wild-type Tim23 with selectivity mutants like N150A to reveal how cation selectivity contributes to substrate recognition . In wild-type channels, presequence peptides typically induce rapid gating transitions, while in selectivity mutants, this response is diminished or absent.

What are the challenges in correlating in vitro channel properties with in vivo import function?

Correlating in vitro channel properties with in vivo import function presents several challenges:

Complexity of the native environment: In vivo, Tim23 functions as part of a multiprotein complex within the mitochondrial inner membrane, which has a unique lipid composition and is subject to a strong electrochemical gradient. Recreating all these conditions in vitro is challenging.

Dynamic regulation: The TIM23 complex activity is dynamically regulated by interactions with other proteins, such as Tim50, which modulates voltage-gating of the channel . These regulatory interactions may be absent or altered in simplified in vitro systems.

Substrate complexity: In vivo, Tim23 handles diverse preproteins with different properties, whereas in vitro studies often use a limited set of model substrates or synthetic peptides.

Technical limitations: Electrophysiological measurements provide detailed channel property information but are technically demanding and may introduce artifacts due to the artificial membrane environment.

Genetic background effects: When studying mutant forms of Tim23, compensatory changes in other import machinery components may occur in vivo, complicating phenotype interpretation.

Despite these challenges, research with S. cerevisiae Tim23 has successfully correlated reduced cation selectivity of Tim23 N150A with decreased substrate sensitivity in vitro and impaired protein import in vivo .

Methodological approach: To establish robust correlations between in vitro properties and in vivo function, employ a comprehensive approach:

• Create defined mutations affecting specific channel properties (selectivity, conductance)

• Characterize these mutations using purified protein in reconstituted systems

• Express the same mutations in vivo and assess impact on growth and protein import

• Isolate mitochondria from these strains for in organello import assays

• Analyze TIM23 complex integrity and potentially confounding factors (membrane potential)

This multifaceted approach can provide compelling evidence for the physiological relevance of specific channel properties, as demonstrated by the study of Tim23 N150A .