Recombinant Arabidopsis thaliana Mitochondrial import inner membrane translocase subunit TIM50 (TIM50)

What is the fundamental role of TIM50 in the mitochondrial protein import apparatus?

TIM50 functions as a critical component of the TIM23 complex in Arabidopsis thaliana mitochondria. This complex serves as the primary entry gateway for proteins destined for the matrix and inner membrane compartments. The protein spans the inner mitochondrial membrane with a single transmembrane segment and extends a large hydrophilic domain into the intermembrane space . This strategic positioning enables TIM50 to play a crucial role in the transfer of preproteins from the translocase of the outer membrane (TOM) complex to the TIM23 complex through the intermembrane space . Functional studies have demonstrated that mitochondria depleted of TIM50 display significantly reduced import kinetics of preproteins that utilize the TIM23 complex, highlighting its essential nature in mitochondrial protein import processes .

How is TIM50 expression regulated across different tissues in Arabidopsis?

Comprehensive expression analysis of the Arabidopsis TIM50 gene reveals consistent expression across all examined organs, fitting the pattern observed for most components of the mitochondrial protein import apparatus . Unlike some other import components that show tissue-specific expression patterns (such as TIM17-1, which exhibits low expression in roots, or TIM23-3 and TIM44-1, which show elevated expression in roots), TIM50 maintains relatively uniform expression levels across various tissues .

A comparative analysis of transcript abundance between different import components indicates that TIM50 maintains approximately 10-fold higher transcript levels compared to TIM44, suggesting differential regulation of import apparatus components despite their functional integration in operational complexes . This consistent expression profile across tissues aligns with TIM50's essential role in mitochondrial protein import, a process fundamental to cellular function in all tissues.

How does TIM50 expression compare to other components of the mitochondrial import apparatus?

Transcript abundance analysis reveals significant variations among different components of the mitochondrial protein import apparatus. The table below summarizes the relative expression levels of various components based on real-time RT-PCR data:

Interestingly, despite the expectation that protein levels would be similar in a functioning complex, transcript levels show approximately 10-fold differences between various components . This suggests post-transcriptional regulation mechanisms likely play an important role in establishing appropriate stoichiometry of import complex components.

What approaches can be used to study TIM50's interactions with preproteins during mitochondrial import?

To investigate TIM50's dynamic interactions with preproteins during mitochondrial import, researchers can employ several sophisticated approaches:

• Chemical Cross-linking: Using chemical cross-linkers such as DFDNB (1,5-difluoro-2,4-dinitrobenzene) at a concentration of 200 μM allows capture of transient interactions between TIM50 and preproteins . This approach is particularly effective when preproteins are halted at specific stages of import, either at the level of the TOM complex or spanning both TOM and TIM23 complexes.

• Arrested Preprotein Intermediates: Researchers can strategically arrest preproteins at different stages of import using various techniques:

  • Dissipation of membrane potential using 50 μM CCCP (carbonyl cyanide m-chlorophenylhydrazone) and 0.5 μM valinomycin to accumulate preproteins at the TOM complex

  • Use of dihydrofolate reductase (DHFR) fusion proteins with methotrexate (MTX, 2 μM) and NADPH (5 mM) to create spanning intermediates across both mitochondrial membranes

• Pulse-Chase Experiments: These can reveal the kinetics of TIM50-preprotein interactions by:

  • First accumulating precursors at the TOM complex in the absence of membrane potential

  • Re-establishing membrane potential (using 2 mM DTT and 5 mM NADH)

  • Following the time-dependent changes in cross-linking and import

• Immunoprecipitation: Following cross-linking, solubilized mitochondrial samples can be subjected to immunoprecipitation with antibodies against TIM50 to isolate and analyze the cross-linked species .

These methodologies have revealed that TIM50 interacts with preproteins as soon as they reach the trans side of the TOM complex and maintains this interaction as long as segments of the preprotein remain in the intermembrane space .

How can recombinant TIM50 be effectively prepared for in vitro studies?

Preparation of high-quality recombinant TIM50 protein is essential for in vitro reconstitution studies. The following methodological approach has proven effective:

• Expression System: Recombinant Arabidopsis thaliana TIM50 (UniProt ID: Q8VYE2) spanning residues 26-376 (the mature protein without the targeting sequence) can be efficiently expressed in E. coli with an N-terminal His tag .

• Purification Protocol:

  • Affinity chromatography using Ni-NTA resin to capture the His-tagged protein

  • Buffer optimization to maintain protein stability (typically Tris-based buffer with 50% glycerol at pH 8.0)

  • Quality control to ensure >90% purity as determined by SDS-PAGE

• Storage Conditions:

  • Lyophilization for long-term stability

  • Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Addition of 5-50% glycerol (optimally 50%) for storage at -20°C/-80°C

  • Aliquoting to avoid repeated freeze-thaw cycles

• Functional Validation:

  • Verification of proper folding through circular dichroism

  • Assessment of interaction capabilities with known binding partners

  • Confirmation of topology when reconstituted in liposomes

For researchers planning in vitro reconstitution of mitochondrial import processes, it's crucial to note that repeated freeze-thaw cycles should be avoided, and working aliquots should be stored at 4°C for no more than one week to maintain protein integrity .

How can TIM50's role in protein transfer between TOM and TIM23 complexes be experimentally verified?

Verifying TIM50's role in facilitating protein transfer between the TOM and TIM23 complexes requires sophisticated experimental approaches that capture the dynamic nature of this process:

• Two-step Import Assays: This approach involves:

  • First accumulating preproteins at the TOM complex by importing radiolabeled precursors into de-energized mitochondria (using 50 μM CCCP to dissipate membrane potential)

  • Re-isolating mitochondria and re-establishing membrane potential (with 2 mM DTT and 5 mM NADH)

  • Following the time-dependent transfer of preproteins from TOM to TIM23 complexes through processing and import analysis

• Time-resolved Cross-linking: By performing cross-linking at different time points during the import process, researchers can track the hand-off of preproteins:

  • Cross-linked products with TIM50 are observed when precursors are bound at the TOM complex (time zero)

  • These cross-links decrease as precursors are processed and imported

  • The decrease in TIM50 cross-links corresponds to the kinetics of processing and import

• Depletion Studies: Mitochondria depleted of TIM50 show significantly reduced import kinetics of preproteins that use the TIM23 complex, providing direct evidence of TIM50's role in efficient protein transfer .

These experimental approaches have collectively established that TIM50 interacts with preproteins as soon as they reach the trans side of the TOM complex, playing a crucial role in their subsequent transfer to the TIM23 complex across the intermembrane space .

What techniques are most effective for studying TIM50's interactions within the complete mitochondrial import machinery?

Investigating TIM50's interactions within the complete mitochondrial import machinery requires integration of several complementary techniques:

• Mitochondrial Subfractionation: This approach involves:

  • Isolation of highly enriched mitochondrial preparations (approximately 98% purity)

  • Fractionation into four sub-compartments: inner membrane, outer membrane, matrix, and intermembrane space

  • Enrichment rather than high-purity separation, with some acknowledged cross-contamination between fractions

• Shotgun Proteomics:

  • Trypsin digestion of protein aliquots from whole mitochondria and sub-fractions

  • Liquid chromatography tandem mass spectrometry (LC-MS/MS) for peptide identification

  • This approach has successfully identified peptides from 17 different import components, including TIM50

• Correlation with Transcript Analysis:

  • Integration of proteomic data with transcript abundance determined by real-time RT-PCR, microarray, massively parallel signature sequencing (MPSS), and EST numbers

  • This integrated approach revealed a close correlation between highly expressed family members and the presence of the encoded protein in mitochondria

• Purification of Native Complexes:

  • Isolation of intact TIM23 complex containing TIM50 along with other components

  • Blue native electrophoresis to assess complex integrity and composition

  • Functional reconstitution of purified complexes in liposomes

This multi-layered approach has confirmed TIM50's localization in the inner membrane fraction and its association with the TIM23 complex, validating its predicted role based on comparative studies with yeast and mammalian systems .

How does Arabidopsis thaliana TIM50 compare to its homologs in other organisms?

While the search results don't provide extensive comparative data, several key points about TIM50 conservation and divergence can be inferred:

• Functional Conservation: The fundamental role of TIM50 in the transfer of preproteins from the TOM complex to the TIM23 complex appears to be conserved between plants (Arabidopsis) and fungi (Neurospora crassa), suggesting evolutionary conservation of this critical function .

• Structural Organization: Across different organisms, TIM50 maintains its characteristic topology with:

  • A single transmembrane segment anchoring it in the inner membrane

  • A large hydrophilic domain extending into the intermembrane space

• Essential Nature: TIM50 has been demonstrated to be essential for viability in yeast, suggesting its fundamental importance across eukaryotic lineages .

• System Integration: In both plant and fungal systems, TIM50 functions as part of the TIM23 complex, the main entry gate for proteins destined for the matrix and inner membrane .

While specific sequence comparisons between Arabidopsis TIM50 and its homologs in other organisms would provide more detailed evolutionary insights, the available data suggests conservation of the core functionality and structural organization of this protein across diverse eukaryotic lineages.

What unique features distinguish plant TIM50 from its fungal and mammalian counterparts?

Although the search results don't provide comprehensive comparative data between plant, fungal, and mammalian TIM50 proteins, several distinguishing features of the Arabidopsis TIM50 can be noted:

• Genetic Designation: In Arabidopsis, TIM50 is also known as EMB1860 (EMBRYO DEFECTIVE 1860), suggesting its essential role in embryonic development, which may represent a plant-specific functional adaptation .

• Expression Pattern: Unlike some other components of the mitochondrial import apparatus in Arabidopsis that show tissue-specific expression patterns, TIM50 maintains relatively uniform expression levels across various tissues . This expression pattern may differ from that observed in other organisms.

• Protein Sequence: The mature Arabidopsis TIM50 protein (residues 26-376) contains regions rich in proline residues (PPPNQPPPPPPP), which may represent plant-specific structural adaptations .

• Genetic Context: In Arabidopsis, TIM50 is encoded by the At1g55900 gene (also known as F14J16.15), situated in a genomic context that may differ from its fungal and mammalian counterparts .

A more comprehensive comparative analysis would require detailed sequence alignments, structural comparisons, and functional studies across different organisms, which would help elucidate the evolutionary adaptations of TIM50 in the plant lineage.

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

Based on the information available in the search results, researchers working with recombinant Arabidopsis TIM50 may encounter several challenges:

• Protein Stability Issues:

  • Challenge: TIM50 may exhibit limited stability during storage and experimental manipulation.

  • Solution: Store as lyophilized powder; reconstitute in deionized sterile water to 0.1-1.0 mg/mL; add 5-50% glycerol (optimally 50%) for storage at -20°C/-80°C; avoid repeated freeze-thaw cycles; keep working aliquots at 4°C for no more than one week .

• Expression and Purification Optimization:

  • Challenge: Achieving high yield and purity of recombinant protein.

  • Solution: Express in E. coli with N-terminal His tag; use optimized Tris-based buffer at pH 8.0; verify purity (>90%) by SDS-PAGE .

• Maintaining Native Conformation:

  • Challenge: Ensuring the recombinant protein adopts its native conformation.

  • Solution: Carefully optimize buffer conditions; consider adding 6% trehalose to stabilize protein structure; validate proper folding through circular dichroism or functional assays .

• Reconstitution in Membrane Systems:

  • Challenge: TIM50 is a membrane protein, making reconstitution in membrane-mimetic systems challenging.

  • Solution: Consider reconstitution in liposomes or nanodiscs to maintain the native membrane environment; validate proper topology of the reconstituted protein.

These methodological considerations are crucial for researchers planning to work with recombinant TIM50 to ensure reliable and reproducible experimental outcomes.

How can cross-linking approaches be optimized to capture TIM50's transient interactions during protein import?

Optimizing cross-linking approaches to capture the dynamic interactions of TIM50 during protein import requires careful consideration of several methodological aspects:

• Cross-linker Selection:

  • DFDNB (1,5-difluoro-2,4-dinitrobenzene) at 200 μM concentration has proven effective for capturing TIM50 interactions with preproteins .

  • The cross-linker should be carefully selected based on spacer arm length, chemical reactivity, and compatibility with downstream analyses.

• Timing Optimization:

  • Import reactions should be chilled on ice before adding cross-linker to capture the state of interest .

  • A 30-minute cross-linking period followed by quenching with 1/10 volume of 1 M glycine pH 8.8 has been successfully employed .

• Preprotein Accumulation Strategies:

  • De-energize mitochondria with 50 μM CCCP and 0.5 μM valinomycin to accumulate preproteins at the TOM complex .

  • Use DHFR fusion proteins with 2 μM MTX and 5 mM NADPH to create spanning intermediates across both membranes .

• Analysis of Cross-linked Products:

  • Solubilize mitochondria in SDS-containing buffer after cross-linking

  • Analyze cross-linked products by immunoprecipitation with antibodies against the N-terminal peptide of TIM50

  • Use SDS-PAGE and autoradiography for detection of radiolabeled preproteins

• Time-resolved Approach:

  • For studying dynamic interactions, implement a chase protocol:

    • Accumulate preproteins at the TOM complex in de-energized mitochondria

    • Re-establish membrane potential with 2 mM DTT and 5 mM NADH

    • Take time-point samples for cross-linking and immunoprecipitation