Recombinant Phytophthora infestans Mitochondrial import inner membrane translocase subunit TIM50 (TIM50)

What is the basic structure and function of Phytophthora infestans TIM50?

Phytophthora infestans TIM50 is a mitochondrial import inner membrane translocase subunit that plays a critical role in protein translocation across the inner membrane. Structurally, the mature protein (amino acids 64-409) contains distinct domains that facilitate different aspects of protein import . TIM50 functions primarily as a receptor for preproteins in the intermembrane space (IMS) after they emerge from the TOM complex . The protein contains at least two distinct functional domains: a core domain that serves as the main recruitment point to the TIM23 complex and contains the primary presequence-binding site, and a presequence-binding domain (PBD) that supports receptor function and coordinates protein translocation across the TOM and TIM23 complexes . This domain architecture allows TIM50 to efficiently interact with incoming precursor proteins and direct them to the appropriate translocation pathway.

How does P. infestans TIM50 differ from TIM50 in other organisms?

P. infestans TIM50 shares the fundamental function of mitochondrial protein import with its orthologs in other organisms, but exhibits distinct characteristics reflecting the evolutionary adaptation of this oomycete pathogen. While the core functional domains remain conserved, sequence analysis reveals specific variations in the presequence-binding regions that may optimize protein import for the unique proteome and metabolic requirements of P. infestans . Unlike the well-characterized yeast Tim50, which has been extensively studied through genetic manipulation, P. infestans TIM50 exists in an organism with more limited genetic tractability, making comparative analyses particularly valuable . Additionally, as a component of an essential cellular process in a major plant pathogen, P. infestans TIM50 represents a potential target for selective inhibition that might not affect host plant mitochondrial import machinery, a distinction of particular interest to researchers developing novel control strategies for late blight disease .

What are the optimal expression conditions for recombinant P. infestans TIM50 in E. coli?

For optimal expression of recombinant P. infestans TIM50 in E. coli, researchers should consider several critical parameters. The expression system described in the literature utilizes E. coli as the host organism for the production of His-tagged recombinant TIM50 (amino acids 64-409) . To maximize protein yield while maintaining proper folding, expression should be induced at mid-log phase (OD600 of 0.6-0.8) with IPTG concentrations between 0.1-0.5 mM. Lower induction temperatures (16-20°C) are recommended over standard 37°C expression to reduce inclusion body formation and increase the proportion of soluble protein . The addition of specific chaperones may further improve folding efficiency. For purification, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is effective due to the N-terminal His-tag, followed by size exclusion chromatography to improve purity. Buffer optimization is crucial, with Tris/PBS-based buffers at pH 8.0 containing 6% trehalose showing good stability for the final product . These conditions balance protein yield with proper folding to maintain the functional characteristics of TIM50.

What methods are used to assess the interaction between TIM50 and presequence peptides?

Multiple complementary techniques can be employed to assess TIM50-presequence interactions. In vitro binding assays using recombinantly purified intermembrane space segment of TIM50 and synthetic presequence peptides provide direct evidence of receptor function . Researchers typically employ fluorescence anisotropy with fluorescently labeled presequence peptides to measure binding affinity and kinetics. Isothermal titration calorimetry (ITC) offers thermodynamic parameters of the interaction. Cross-linking approaches are particularly valuable, as demonstrated in studies showing that TIM50 can be cross-linked to precursors as they emerge from the TOM complex, with the cross-linking efficiency decreasing as the precursor is imported . For structural characterization, NMR spectroscopy using isotopically labeled TIM50 constructs can map the binding interface at atomic resolution. Complementary in vivo and in organello approaches include genetic manipulation to create domain-specific mutants, followed by import assays with radiolabeled precursor proteins to assess functional consequences . Surface plasmon resonance (SPR) can provide real-time kinetic measurements, while hydrogen-deuterium exchange mass spectrometry (HDX-MS) offers insights into conformational changes upon binding. Together, these methods provide a comprehensive understanding of the mechanistic basis for presequence recognition by TIM50.

How can researchers effectively reconstitute lyophilized P. infestans TIM50 protein while maintaining activity?

Effective reconstitution of lyophilized P. infestans TIM50 requires careful attention to several parameters to maintain protein activity. The recommended protocol begins with brief centrifugation of the vial to bring contents to the bottom prior to opening . Reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with gentle mixing rather than vigorous vortexing to prevent protein denaturation . The addition of 5-50% glycerol (final concentration) is recommended as a cryoprotectant, with 50% being the default concentration for optimal stability . Following reconstitution, the solution should be aliquoted to minimize freeze-thaw cycles, which can significantly impact protein integrity and activity. For storage, keep working aliquots at 4°C for up to one week, while long-term storage should be at -20°C/-80°C . When assessing activity after reconstitution, researchers should employ functional assays such as presequence peptide binding or interaction with other components of the import machinery. If activity is suboptimal, buffer optimization may be necessary, potentially including the addition of stabilizing agents such as reducing agents (DTT or β-mercaptoethanol) to maintain cysteine residues in their reduced state, or low concentrations of non-ionic detergents to prevent aggregation.

What is the current understanding of TIM50's role in P. infestans pathogenicity and virulence?

The relationship between TIM50 and P. infestans pathogenicity remains an emerging area of research, with indirect evidence suggesting potentially significant connections. As a component of the essential mitochondrial protein import machinery, TIM50 likely plays a critical role in maintaining mitochondrial function during the energy-intensive processes of host infection and colonization . While TIM50 has not been directly implicated in pathogenicity, proteomic studies of P. infestans have identified several mitochondrial proteins that show differential expression during appressorium formation and host infection stages, suggesting that mitochondrial function adaptation is important during pathogenesis . Additionally, certain cell wall-associated proteins (Piacwp1-3) that undergo mitochondrial processing have been shown through silencing experiments to reduce infection severity, highlighting the potential importance of properly functioning mitochondrial import machinery for virulence . The historical significance of P. infestans as the causative agent of potato late blight, including the Irish Potato Famine, underscores the importance of understanding all aspects of its cellular machinery that could contribute to its aggressive pathogenicity . Further targeted studies examining the effects of TIM50 modulation on infection processes would provide more direct evidence of its role in virulence.

How might TIM50 function differ during various life stages of P. infestans?

TIM50 function likely undergoes significant adaptation during different life stages of P. infestans to accommodate stage-specific metabolic and developmental requirements. During sporulation and germination, increased energy demands and rapid protein synthesis would require upregulated mitochondrial import capacity, potentially involving modified TIM50 activity or expression levels . Proteomic analyses have revealed stage-specific protein expression patterns during appressorium formation, with proteins involved in amino acid biosynthesis, cellulose synthesis, and cell wall modification showing differential abundance . As TIM50 mediates import of many of these proteins, its activity must be regulated to support these stage-specific proteome changes. During host invasion, P. infestans likely experiences oxidative stress and altered nutrient availability, requiring mitochondrial adaptation and potentially modified import pathways . Comparative proteomic studies of the cell wall during sporulating mycelium, nonsporulating mycelium, and appressoria stages have identified unique proteins in each stage, suggesting that the mitochondrial import machinery, including TIM50, must accommodate these changing protein localization requirements . Additional research specifically examining TIM50 expression, modification, and interaction patterns across life stages would provide greater insight into how this essential component of mitochondrial import adapts to support the complex lifecycle of this devastating plant pathogen.

How can researchers distinguish between the functional domains of P. infestans TIM50 experimentally?

Researchers can distinguish between the functional domains of P. infestans TIM50 through a combination of bioinformatic prediction and experimental validation approaches. Initial domain identification should employ sequence analysis tools and structural prediction algorithms, comparing the P. infestans TIM50 sequence with well-characterized orthologs from model organisms . Experimentally, limited proteolysis coupled with mass spectrometry can identify stable domains based on differential protease sensitivity. For functional characterization, researchers should generate recombinant constructs expressing individual domains, such as the core domain and the presequence-binding domain (PBD), similar to the approach used in yeast studies . Binding assays using fluorescently labeled presequence peptides can then determine which domains retain presequence recognition capability. Cross-linking experiments with isolated domains and precursor proteins can identify specific interaction regions . Domain-specific antibodies can be developed for immunoprecipitation studies to identify domain-specific interaction partners. In vitro reconstitution experiments combining different domain constructs can test for complementation of function, revealing whether domains can work in trans as demonstrated in yeast systems . For in vivo validation, expressing domain-specific constructs in P. infestans (if transformation protocols are available) followed by phenotypic analysis would provide physiological relevance. Together, these approaches would establish a comprehensive map of domain-specific functions in P. infestans TIM50.

What proteomic approaches have been successful in studying P. infestans mitochondrial proteins?

Several proteomic approaches have proven effective for studying P. infestans mitochondrial proteins, providing insights into their expression, localization, and function. Early studies employed 2D gel-based experiments that allowed identification of approximately 200 spots, revealing proteins that show differential abundance during appressorium formation and preinfection stages, including those involved in amino acid biosynthesis and cellulose synthesis . More advanced LC-MS/MS techniques have been applied to analyze proteins from specific subcellular fractions, such as the cell wall of sporulating mycelium, nonsporulating mycelium, and appressoria, identifying unique proteins in each condition . These approaches revealed four proteins unique to the P. infestans appressorium cell wall . For comprehensive mitochondrial proteome analysis, subcellular fractionation followed by high-resolution mass spectrometry has been effective, though specific data for P. infestans mitochondrial proteome has been limited compared to model organisms. Stable isotope labeling approaches (such as SILAC or TMT labeling) could provide quantitative insights into mitochondrial protein dynamics during infection processes. Targeted proteomics using selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) would allow precise quantification of specific mitochondrial proteins, including TIM50, across different conditions. Integration of proteomics with transcriptomics and metabolomics would provide a systems-level understanding of mitochondrial function in this important plant pathogen. The ProteomeXchange database contains relevant datasets, including the PXD002446 dataset mentioned in the literature .

How can CRISPR-Cas9 or RNAi approaches be optimized for studying TIM50 function in P. infestans?

Optimizing genetic manipulation approaches for studying TIM50 in P. infestans requires careful consideration of this organism's unique biology and the essential nature of mitochondrial import. For RNAi approaches, which have been successfully applied to P. infestans, design of specific siRNAs targeting TIM50 mRNA should avoid regions with similarity to other genes to minimize off-target effects . Since complete silencing of TIM50 would likely be lethal, inducible or partial silencing systems are preferable, such as using the pSTORA vector with a dexamethasone-inducible promoter . Transient silencing experiments, similar to those used for the Piacwp1-3 proteins that demonstrated reduced infection severity, would be appropriate initial approaches . For CRISPR-Cas9 editing, which has been more challenging in oomycetes, codon-optimized Cas9 for P. infestans and specific sgRNAs with minimal off-target effects are essential. Given TIM50's likely essential function, CRISPR interference (CRISPRi) or approaches that create conditional or hypomorphic alleles rather than complete knockouts may be more informative. Domain-specific modifications that maintain minimal TIM50 function while disrupting specific interactions would be particularly valuable, similar to the domain separation approach used in yeast studies . Delivery methods must be optimized for P. infestans, with protoplast transformation or Agrobacterium-mediated transformation being potential approaches. Phenotypic assessment should include growth rates, mitochondrial morphology and function, protein import efficiency, and virulence in plant infection assays, providing a comprehensive understanding of TIM50's role in this important plant pathogen.

What computational approaches can predict interactions between TIM50 and other components of the protein import machinery?

Advanced computational approaches can provide valuable predictions of TIM50 interactions with other components of the mitochondrial protein import machinery in P. infestans. Homology modeling based on better-characterized orthologs can generate structural models of P. infestans TIM50, which can then be used for protein-protein docking simulations with modeled structures of TIM23, TOM components, and presequence peptides . Molecular dynamics simulations can assess the stability of predicted interaction interfaces and identify key residues involved in binding. Sequence-based approaches including co-evolution analysis can identify residues that have evolved together in TIM50 and its interaction partners, often indicating functional interactions. Machine learning algorithms trained on known protein-protein interactions can predict novel interaction partners based on sequence and structural features. Integrative approaches combining multiple data types (structural, evolutionary, expression, and literature-derived information) through network analysis can provide a systems-level view of the import machinery. Virtual screening of peptide libraries against the presequence-binding site could identify optimal binding motifs and potential inhibitors. For experimental validation of predicted interactions, the split-protein approach demonstrated in yeast studies offers a powerful framework, where individual domains expressed in trans can support TIM50 function through their predicted interactions . These computational predictions would guide targeted experimental studies, accelerating our understanding of the molecular mechanisms underlying protein import in this important plant pathogen.

What are the latest methodologies for investigating the real-time dynamics of TIM50-mediated protein translocation?

Cutting-edge methodologies for investigating real-time dynamics of TIM50-mediated protein translocation combine advanced imaging, biophysical, and biochemical techniques. Single-molecule FRET (smFRET) approaches using fluorescently labeled preproteins and TIM50 can track interaction dynamics at nanometer resolution, revealing transient states during translocation . This can be complemented by optical tweezers to measure forces involved in protein unfolding and translocation through the import channels. Time-resolved cross-linking experiments, similar to those showing that TIM50 interacts with precursors as they reach the trans side of the TOM complex, provide snapshots of translocation intermediates . Reconstituted proteoliposome systems containing purified TIM50 and other translocase components enable controlled biochemical studies of translocation kinetics. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can identify conformational changes in TIM50 during different stages of the translocation process. For cellular studies, super-resolution microscopy techniques such as PALM or STORM can visualize translocase components within mitochondria at nanoscale resolution. Correlative light and electron microscopy (CLEM) can connect functional observations with ultrastructural context. Recent developments in cryo-electron tomography could potentially visualize the translocase machinery in near-native states within mitochondria. Time-course proteomics using pulsed SILAC labeling can track the kinetics of protein import on a global scale. Integration of these complementary approaches would provide unprecedented insights into the dynamic process of TIM50-mediated protein translocation, potentially revealing unique features of the P. infestans import machinery that could be exploited for targeted intervention strategies.

What comparative analyses of TIM50 across Phytophthora species could reveal about host adaptation and pathogenicity evolution?

Comparative analyses of TIM50 across diverse Phytophthora species could provide valuable insights into host adaptation and pathogenicity evolution. Phylogenetic analysis of TIM50 sequences from multiple Phytophthora species with different host ranges (P. infestans affecting potatoes, P. sojae affecting soybeans, P. ramorum affecting oak trees, etc.) could reveal selection signatures associated with host specialization . Molecular evolution analyses including dN/dS ratios and tests for positive selection would identify specific domains and residues under selective pressure, potentially indicating adaptations to different host environments or defense mechanisms. Structural bioinformatics comparing homology models of TIM50 from different species could identify variations in binding pockets or interaction surfaces that might influence mitochondrial import efficiency under different host conditions. Expression pattern comparisons across species during infection of their respective hosts would reveal conserved and divergent regulatory mechanisms. Functional complementation experiments, where TIM50 from one species is expressed in another, could test the degree of functional conservation and host-specific adaptations. Integration with genomic data on mitochondrial-targeted proteomes across species would provide context for TIM50 evolution relative to its substrates. Correlation of TIM50 sequence variations with virulence traits, host range, or metabolic capabilities could establish links between mitochondrial import and pathogenicity. Archaeological genomics approaches using herbarium specimens, similar to those used to study the Phytophthora lineage that triggered the Irish potato famine, could provide historical context for TIM50 evolution . These comparative approaches would not only advance our understanding of oomycete evolution but could also identify conserved features of TIM50 that might serve as broad-spectrum targets for disease management strategies.

What are the most significant unanswered questions about P. infestans TIM50 that warrant future research?

Several critical questions about P. infestans TIM50 remain unanswered and represent important directions for future research. The three-dimensional structure of P. infestans TIM50 has not been determined, leaving uncertainty about how its molecular architecture enables its diverse functions in protein recognition and translocation . The regulatory mechanisms controlling TIM50 expression and activity during different life stages and infection processes remain largely unexplored, despite evidence that mitochondrial proteins show differential expression during appressorium formation and infection . The specific contribution of TIM50 to P. infestans virulence has not been directly assessed through targeted genetic manipulation, leaving its potential as a pathogenicity factor unconfirmed . The complete interactome of P. infestans TIM50 with other components of the mitochondrial import machinery and potentially with host factors during infection is unknown . The evolution of TIM50 across oomycete species and its relationship to host adaptation and speciation remains an open question with implications for understanding pathogen evolution . The potential for species-specific inhibitors of TIM50 as novel control agents has not been systematically investigated despite the need for new management strategies for late blight disease . The adaptation of the mitochondrial import machinery to the unique metabolic demands of different life stages, particularly during host colonization, represents another significant knowledge gap. Addressing these questions would not only advance our fundamental understanding of this important plant pathogen but could also reveal novel approaches for disease management.

How can collaborative research approaches accelerate progress in understanding TIM50 function in P. infestans?

Collaborative research approaches can significantly accelerate progress in understanding TIM50 function through integration of diverse expertise and technologies. Interdisciplinary teams combining structural biologists, molecular geneticists, plant pathologists, and computational biologists would bring complementary perspectives to the complex questions surrounding TIM50 function . International consortia connecting researchers studying different Phytophthora species could facilitate comparative analyses that reveal evolutionary patterns and host-specific adaptations . Public-private partnerships between academic institutions and agricultural technology companies could accelerate translation of basic research into applied disease management strategies. Standardized protocols and materials sharing, such as recombinant protein constructs, antibodies, and genetic modification tools optimized for P. infestans, would enhance reproducibility and enable more rapid progress. Centralized data repositories integrating proteomics, genomics, and functional studies related to mitochondrial import in oomycetes would facilitate meta-analyses and systems-level insights. Collaborative field testing networks could evaluate the relevance of laboratory findings to real-world agricultural contexts. Engagement with potato growers and industry stakeholders would ensure research priorities align with practical needs for disease management. Educational initiatives training early-career researchers in the interdisciplinary skills needed for this research would build capacity for future discovery. Open science approaches with preprint sharing and open access publication would accelerate knowledge dissemination. Together, these collaborative strategies would transform our understanding of TIM50 function in P. infestans more rapidly than traditional siloed research approaches, potentially leading to innovative solutions for managing this devastating plant pathogen.