Recombinant Mouse Mitochondrial import inner membrane translocase subunit Tim23 (Timm23)

What is Recombinant Mouse Mitochondrial import inner membrane translocase subunit Tim23 (Timm23)?

Recombinant Mouse Tim23 (Timm23) is a laboratory-produced version of the native Tim23 protein found in mouse mitochondria. It is typically expressed in heterologous systems such as E. coli with modifications like His-tags to facilitate purification. The full-length mouse Timm23 protein consists of 209 amino acids and functions as an essential component of the TIM23 complex, which mediates protein translocation across the mitochondrial inner membrane . The recombinant versions are critical tools for research, allowing scientists to study the structure, function, and interactions of this protein without needing to purify it directly from mouse tissue.

How does the quaternary structure of the TIM23 complex influence its function?

The TIM23 complex has a dynamic quaternary structure that changes based on its operational state. The complex consists of membrane-integrated components (including Tim23 and Tim17) and an import motor component. How these parts associate depends on whether the complex is actively translocating proteins. Some research suggests that translocation into the matrix requires assembly of the TIM23 complex with the import motor, whereas insertion into the inner membrane is mediated by a motor-free complex .

What experimental techniques are most effective for studying Tim23 structure and interactions?

Several sophisticated experimental approaches have proven valuable for studying Tim23:

• Cysteine-scanning mutagenesis: This approach involves creating a cysteine-less variant of Tim23 (by replacing native cysteines) and then introducing single cysteine residues at specific positions. This has been used effectively to map proximities between Tim23 and other proteins in the complex .

• Chemical cross-linking combined with immunoprecipitation: By using homobifunctional thiol-reactive reagents, researchers can create covalent adducts between the introduced cysteine in Tim23 and nearby native proteins. This approach provides high-resolution positional information about protein-protein interactions within the complex .

• Translocation intermediate trapping: By using model substrate proteins like pSu9-DHFR that can be trapped during translocation (using methotrexate to tightly fold the DHFR domain), researchers can study how Tim23 engages with substrates during the translocation process .

These techniques allow researchers to determine both the static structure and dynamic changes in the TIM23 complex during different functional states.

How can researchers verify proper integration of recombinant Tim23 into functional TIM23 complexes?

Verifying proper integration and functionality of recombinant Tim23 is critical for meaningful experiments. Based on established protocols, researchers can use a combination of approaches:

• Protease protection assays: Tim23 properly integrated into the inner membrane of intact mitochondria will be protected from added proteases. This can be assessed by importing radiolabeled Tim23 into isolated mitochondria and testing its resistance to protease treatment .

• Co-immunoprecipitation: After import and membrane solubilization under non-denaturing conditions that preserve the TIM23 complex, properly assembled Tim23 should co-precipitate with antibodies against other complex components such as Tim17p. This confirms its integration into the functional complex .

• Functional translocation assays: Perhaps the most stringent test is whether the recombinant Tim23 can participate in protein translocation. This can be assessed using model substrates like pSu9-DHFR that form translocation intermediates, then testing whether the recombinant Tim23 can be cross-linked to these substrates .

What expression systems work best for producing functional recombinant Tim23?

For producing functional recombinant Tim23, the expression system choice depends on the experimental goals:

• E. coli expression system: This appears to be the most common approach for producing full-length recombinant Tim23, as seen with the mouse Tim23 protein that was successfully expressed with an N-terminal His-tag in E. coli . This system is advantageous for producing larger quantities of protein for structural studies or antibody production.

• In vitro translation systems: For studies requiring direct import into isolated mitochondria, in vitro transcription/translation systems have been successfully employed to produce radiolabeled Tim23 variants. This approach was used for creating libraries of Tim23p variants with single cysteine substitutions for interaction studies .

• Yeast expression: For studies in the native context, expressing Tim23 variants in yeast allows for assessing function in vivo, particularly when combined with depletion of the endogenous protein.

The choice between these systems should be guided by the specific experimental requirements and the intended use of the recombinant protein.

How does Tim23 contribute to sorting preproteins to different mitochondrial compartments?

The TIM23 complex has a remarkable ability to sort proteins to different mitochondrial subcompartments (matrix vs. inner membrane), but the precise mechanism remains under investigation. Initial models suggested that the TIM23 complex exists in two forms: one that directs proteins to the matrix (requiring the import motor) and another that mediates insertion into the inner membrane (motor-free but containing Tim21) .

The sorting decision appears to involve recognition of specific signals in the translocating proteins. Preproteins with only N-terminal matrix-targeting signals are typically directed completely across the inner membrane into the matrix. In contrast, preproteins containing additional hydrophobic segments (stop-transfer signals) after the matrix-targeting sequence are often laterally released into the inner membrane .

Recent research has challenged the view that Tim23 is the primary channel for translocation, suggesting instead that Tim17 contains a lateral transmembrane cavity with a negatively charged patch on the intermembrane space side that acts as a "translocation initiation site" for importing presequences across the inner membrane . This represents a significant shift in our understanding of how sorting decisions are made by the TIM23 complex.

What is the emerging understanding of the Tim17-Tim23 relationship in the TIM23 complex?

Recent research has significantly revised our understanding of the Tim17-Tim23 relationship. While Tim23 was traditionally viewed as the channel-forming component of the complex, newer evidence suggests that Tim17 plays the major role in preprotein translocation across the inner membrane .

Key findings from recent research include:

• Tim17 contains a lateral transmembrane cavity that appears to be the primary site for preprotein translocation .

• The negatively charged patch on the intermembrane space side of Tim17's lateral cavity acts as a translocation initiation site for presequences .

• Tim17 and Tim23 form a back-to-back heterodimer that functions as a unit for precursor translocation .

This represents a paradigm shift, suggesting that presequence proteins can be imported across the inner membrane at the Tim17 bilayer interface rather than through a channel formed primarily by Tim23 . The Tim17-Tim23 heterodimer appears to be the functional unit for translocation, with Tim17 playing the more direct role in guiding preproteins across the membrane.

How does membrane potential influence Tim23 function in the TIM23 complex?

The membrane potential across the mitochondrial inner membrane is crucial for Tim23 function and TIM23 complex activity. The TIM23 complex uses both the membrane potential and ATP in the mitochondrial matrix to drive protein translocation . The membrane potential provides part of the energy required for moving positively charged presequences across the inner membrane, following an electrophoretic mechanism.

Experimental designs often manipulate the membrane potential to study different states of the TIM23 complex. For instance, de-energized mitochondria (with collapsed membrane potential) prevent proper Tim23 assembly into functional TIM23 complexes . This demonstrates the membrane potential's role not just in providing energy for translocation but potentially in maintaining proper complex assembly.

Recent research suggests the membrane potential may also influence the conformation of the complex, particularly the interaction between Tim17 and Tim23, potentially affecting which mode (translocation vs. insertion) is favored under different conditions .

What is the role of Tim23 in mitochondrial pathology and disease models?

Tim23 plays critical roles beyond basic protein import that may connect to disease states. Recent research has shown that Tim23 has a role in stress-induced mitophagy (the selective degradation of damaged mitochondria) by protecting PINK1 from OMA1-mediated degradation and facilitating its accumulation at the outer mitochondrial membrane in response to depolarization . This is particularly significant because PINK1 is associated with Parkinson's disease, suggesting a potential link between Tim23 function and neurodegenerative disorders.

Dysfunction in mitochondrial protein import machinery, including the TIM23 complex, has been implicated in various human diseases, particularly neurodegenerative conditions and myopathies. Research using recombinant Tim23 can help elucidate how mutations or alterations in this protein might contribute to pathological states.

What are the latest techniques for real-time monitoring of Tim23 activity?

While the search results don't specifically mention real-time monitoring techniques, based on the methodologies described, several approaches could be adapted for dynamic studies of Tim23:

• FRET-based assays: By introducing fluorescent tags at specific positions in Tim23 and its interaction partners, Förster Resonance Energy Transfer could be used to monitor conformational changes and protein interactions in real-time.

• Site-specific crosslinking with photo-activatable crosslinkers: This would allow for temporal control over when crosslinking occurs, enabling researchers to capture specific states of the complex during the translocation process .

• Single-molecule techniques: These could potentially be used to observe individual translocation events and conformational changes in the TIM23 complex.

Development of these techniques would represent significant advances in studying the dynamic behavior of Tim23 and the TIM23 complex during protein translocation.

How can structural modifications of recombinant Tim23 enhance its utility in research applications?

Strategic modifications to recombinant Tim23 can significantly enhance its research utility:

• Affinity tags: The addition of tags like the His-tag mentioned in the product description facilitates purification and detection. Positioning these tags at different locations (N-terminal, C-terminal, or internal) can be crucial depending on the functional domain being studied.

• Fluorescent protein fusions: Creating fusion proteins with fluorescent markers can enable visualization of Tim23 localization and dynamics in live cells.

• Cysteine substitutions: As demonstrated in the research described, replacing native cysteines and introducing single cysteines at specific locations creates powerful tools for interaction studies via crosslinking .

• Domain swapping: Creating chimeric proteins by swapping domains between Tim23 and related proteins can help identify functionally important regions.

These modifications must be carefully validated to ensure they don't disrupt the native function of Tim23, using the verification techniques discussed earlier.

What are common pitfalls when working with recombinant Tim23 in experimental systems?

Based on the research methodologies described, several challenges may arise when working with recombinant Tim23:

• Proper membrane integration: As a membrane protein, Tim23 requires the appropriate environment to maintain its native structure. Ensuring proper integration into membranes (whether in isolated mitochondria or membrane mimetics) is critical and should be verified using protease protection assays .

• Cysteine reactivity issues: When using cysteine-scanning mutagenesis approaches, the reactivity of introduced cysteines can vary depending on their location in the protein structure. Some positions may be sterically hindered or exist in environments that reduce thiol reactivity.

• Assembly into functional complexes: Simply expressing and integrating Tim23 doesn't guarantee its assembly into functional TIM23 complexes. Co-immunoprecipitation with other complex components should be used to verify proper assembly .

• Maintaining native function: Modifications like tagging or amino acid substitutions can potentially alter function. Complementation assays or functional translocation assays should be used to verify that modified Tim23 retains its native activity .

How can researchers optimize crosslinking experiments to study Tim23 interactions?

Crosslinking experiments are powerful tools for studying Tim23 interactions but require careful optimization:

• Selection of crosslinking reagents: The choice between homobifunctional (e.g., thiol-reactive) or heterobifunctional crosslinkers should be based on the specific interaction being studied. The spacer arm length is also important for capturing interactions at various distances.

• Concentration and time optimization: Crosslinking reactions should be optimized for both reagent concentration and reaction time to maximize specific interactions while minimizing non-specific crosslinking.

• Two-step approach: For studying interactions with specific proteins, a two-step approach can be effective: first introducing the cysteine-specific crosslinker to the Tim23 variant, then allowing it to react with potential interaction partners.

• Controls: Proper controls are essential, including cysteine-less Tim23 variants to confirm the specificity of crosslinking to the introduced cysteine residue .

• Analysis methods: Combining crosslinking with immunoprecipitation using antibodies against suspected interaction partners can help identify specific interactions, as demonstrated in the research with Tim23p variants .

When properly optimized, these approaches can provide high-resolution information about Tim23's interactions within the TIM23 complex and with substrate proteins.