Recombinant Ashbya gossypii Mitochondrial import inner membrane translocase subunit TIM50 (TIM50)

What is the structural organization of TIM50 in Ashbya gossypii?

TIM50 in A. gossypii is a mitochondrial protein that spans the inner membrane with a single transmembrane segment. It contains two essential domains in the intermembrane space (IMS): a core domain and a presequence-binding domain (PBD). The full-length mature protein encompasses amino acids 10-476, with the transmembrane domain anchoring the protein to the inner mitochondrial membrane. The protein exposes a large hydrophilic domain in the intermembrane space that is critical for its function in protein import .

How does TIM50 contribute to mitochondrial protein import?

TIM50 functions as the central receptor of the TIM23 complex, recognizing precursor proteins in the intermembrane space. It plays a crucial role in the transfer of preproteins from the translocase of the outer membrane (TOM complex) to the TIM23 complex across the intermembrane space. The interaction between TIM50 and the IMS domain of the channel-forming subunit, Tim23, is essential for protein import across the mitochondrial inner membrane . Research has shown that both domains of TIM50 in the IMS have essential but distinct roles and together coordinate translocation of proteins across two mitochondrial membranes .

What is known about the evolutionary conservation of TIM50?

TIM50 is a highly conserved component of the mitochondrial import machinery across fungal species. Comparative studies in Saccharomyces cerevisiae, Neurospora crassa, and Ashbya gossypii have shown that TIM50 homologs share similar domain structures and functions. The essential nature of TIM50 has been demonstrated in yeast, where deletion of TIM50 is lethal, placing it in the relatively small group of mitochondrial proteins that are essential for viability .

How can recombinant A. gossypii TIM50 be expressed and purified for structural studies?

For structural studies of A. gossypii TIM50, researchers typically express the recombinant protein in E. coli expression systems with an N-terminal His-tag for purification purposes. The full-length mature protein (amino acids 10-476) can be expressed and purified to >90% homogeneity using affinity chromatography followed by size exclusion chromatography. The purified protein is commonly available as a lyophilized powder that can be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol added for long-term storage .

What experimental approaches can be used to study TIM50 interactions with other components of the TIM23 complex?

Several experimental approaches can be employed to study TIM50 interactions:

• Co-immunoprecipitation experiments: After solubilization of mitochondria with mild detergents like digitonin, affinity-purified antibodies against TIM50 can be used to isolate the protein and identify interacting partners.

• Cross-linking experiments: Chemical cross-linkers can be used to capture transient interactions between TIM50 and precursor proteins or other components of the import machinery.

• In vitro binding assays: Using recombinantly purified domains of TIM50 and potential interaction partners to study direct binding.

• Mutagenesis analysis: Random or site-directed mutagenesis can be used to identify residues important for interactions. For example, temperature-sensitive mutants have been used to map residues that affect TIM50's interaction with Tim23 .

What are the optimal storage conditions for recombinant A. gossypii TIM50 protein to maintain activity?

Based on product specifications, recombinant A. gossypii TIM50 should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use to avoid repeated freeze-thaw cycles. For long-term storage, it's recommended to add glycerol to a final concentration of 5-50% (with 50% being optimal for many applications). Working aliquots can be stored at 4°C for up to one week. The protein is typically provided in a storage buffer consisting of Tris/PBS-based buffer with 6% Trehalose at pH 8.0 or with 50% glycerol .

How can domain-specific studies of TIM50 be designed to elucidate their individual functions?

To study the individual functions of TIM50 domains, researchers can employ a domain separation approach:

Experimental design:

• Generate constructs expressing individual domains:

  • Tim50(1-365): Truncated version without the PBD

  • Tim50(366-476): Only the PBD, targeted to the IMS using targeting sequences

• Express these constructs in Tim50-depleted cells (using shuffling strains or conditional expression systems)

• Assess functionality through:

  • Viability assays

  • Protein import experiments

  • Co-immunoprecipitation to study interaction with other TIM23 components

What expression systems are most suitable for producing recombinant A. gossypii TIM50?

E. coli has been demonstrated as an effective expression system for producing recombinant A. gossypii TIM50. The full-length mature protein (amino acids 10-476) with an N-terminal His-tag can be successfully expressed and purified. Alternative expression systems might include:

• Yeast expression systems (S. cerevisiae): May provide more native post-translational modifications

• Insect cell expression systems: Useful for proteins requiring complex folding

• Cell-free protein synthesis: For rapid production and avoiding potential toxicity issues

The choice depends on research requirements, with E. coli generally providing high yields suitable for structural and biochemical studies .

How can researcher validate the functionality of recombinant TIM50 protein in vitro?

Functional validation of recombinant TIM50 can be performed using several approaches:

• In vitro binding assays: Testing the ability of purified TIM50 to bind presequence peptides or the IMS domain of Tim23

• Reconstitution experiments: Incorporating recombinant TIM50 into liposomes or nanodiscs to study its membrane interaction properties

• Import assays using isolated mitochondria: Adding recombinant TIM50 to TIM50-depleted mitochondria to test for rescue of import defects

• Structural analyses: Circular dichroism or thermal shift assays to confirm proper folding of the recombinant protein

These approaches provide complementary information about the protein's biochemical activity and structural integrity .

What controls should be included when performing experiments with recombinant TIM50?

When conducting experiments with recombinant A. gossypii TIM50, the following controls should be included:

Positive controls:

• Wild-type TIM50 protein for comparison with mutant variants

• Known interacting partners (e.g., Tim23 fragments) for binding studies

Negative controls:

• Unrelated proteins with similar size/charge properties

• Denatured TIM50 protein to control for non-specific interactions

• Buffer-only controls for background signal

Internal controls:

• For import assays: substrates that use alternative import pathways

• For binding studies: known non-binding peptides or proteins

Including these controls ensures the specificity and reliability of experimental results and helps troubleshoot potential issues .

How do the findings from domain-specific studies of TIM50 change our understanding of mitochondrial protein import?

The discovery that the two domains of TIM50 (core domain and presequence-binding domain) can function when expressed separately challenges the conventional view that TIM50 must function as a single polypeptide. Data from domain separation experiments shows that:

• The core domain of TIM50 is primarily responsible for recruitment to the TIM23 complex

• The PBD is independently capable of recognizing precursor proteins

• The two domains do not interact strongly with each other in co-immunoprecipitation experiments

These findings suggest a modular organization of TIM50 where different domains perform specialized functions that together coordinate the complex process of protein translocation across mitochondrial membranes. This modular understanding may provide opportunities for targeted manipulation of specific aspects of mitochondrial protein import .

How do mutations in TIM50 affect the import of different classes of mitochondrial proteins?

Research on TIM50 mutants has revealed differential effects on various mitochondrial protein substrates:

Table 1: Effects of TIM50 Depletion/Mutation on Different Protein Classes

This differential impact highlights the specificity of TIM50's role in the TIM23 pathway and suggests that some proteins may have alternative mechanisms for engaging the import machinery that are less dependent on TIM50 .

What are common challenges when working with recombinant TIM50 and how can they be addressed?

Researchers working with recombinant A. gossypii TIM50 may encounter several challenges:

• Protein solubility issues:

  • Problem: Aggregation or precipitation after reconstitution

  • Solution: Optimize buffer conditions (pH, salt concentration), add stabilizing agents like glycerol (5-50%), or use detergents for membrane-associated studies

• Protein degradation:

  • Problem: Loss of activity during storage

  • Solution: Add protease inhibitors, avoid repeated freeze-thaw cycles, store aliquots at -80°C

• Functional heterogeneity:

  • Problem: Variable activity between preparations

  • Solution: Establish rigorous quality control tests, use functional assays to validate each batch

• Improper folding:

  • Problem: Recombinant protein lacks native conformation

  • Solution: Explore alternative expression systems, optimize purification conditions, consider using molecular chaperones during expression

How can the experimental conditions be optimized for studying TIM50 interactions with precursor proteins?

To optimize experimental conditions for studying TIM50-precursor protein interactions:

• Buffer composition optimization:

  • Test different pH values (typically 7.0-8.0)

  • Vary salt concentrations (50-300 mM KCl or NaCl)

  • Add low concentrations of detergents (0.01-0.1% digitonin) for membrane proteins

• Sample preparation:

  • Use freshly prepared or properly stored protein samples

  • Pre-clear solutions by centrifugation to remove aggregates

  • Control temperature during experiments (typically 4-25°C)

• Detection methods:

  • For weak interactions: use chemical crosslinking with optimized crosslinker concentrations

  • For direct binding: consider fluorescence-based methods (fluorescence anisotropy or FRET)

  • For complex formation: size exclusion chromatography coupled with multi-angle light scattering

• Precursor protein design:

  • Use model precursors with strong targeting signals

  • Consider using truncated precursors that remain in a translocation-competent state

  • Include controls with mutated targeting signals