Recombinant Schizosaccharomyces pombe Mitochondrial import inner membrane translocase subunit tim50 (tim50)

What is the structure and localization of Tim50 in S. pombe?

Tim50 in S. pombe, like its counterparts in other organisms, is a component of the mitochondrial inner membrane translocase (TIM23) complex. It spans the inner mitochondrial membrane with a single transmembrane segment and exposes a large hydrophilic domain in the intermembrane space (IMS) . The protein contains an N-terminal mitochondrial targeting signal that directs it to the mitochondria. The C-terminal domain is exposed to the intermembrane space, where it functions as a receptor for presequence-carrying proteins . Unlike the Tim50 homolog in Saccharomyces cerevisiae which contains only one cysteine residue (C268) in the presequence-binding groove, Tim50 in other organisms including S. pombe contains multiple cysteine residues distributed throughout the protein, though their functional significance remains to be fully determined .

What is the primary function of Tim50 in mitochondrial protein import?

Tim50 serves as a critical receptor component of the TIM23 complex, playing a crucial role in facilitating the transfer of precursor proteins from the translocase of the outer membrane (TOM) complex to the TIM23 complex through the intermembrane space . It recognizes and binds to the presequences of nuclear-encoded mitochondrial proteins as they emerge from the TOM complex into the intermembrane space. This initial recognition is essential for the subsequent translocation of these proteins into the mitochondrial matrix or their insertion into the inner membrane . In cells depleted of Tim50, mitochondria display significantly reduced import kinetics of preproteins using the TIM23 complex, highlighting its essential role in protein import .

How is Tim50 essential for cellular viability?

Tim50 is essential for viability in various organisms, including yeast . The crucial nature of Tim50 stems from its fundamental role in mitochondrial protein import, a process indispensable for mitochondrial function and consequently cellular survival. When Tim50 is depleted in cells, the import of nuclear-encoded mitochondrial proteins is severely compromised . In zebrafish embryos, downregulation of Tim50 during early development causes neurodegeneration, dysmorphic heart features, and reduced motility due to apoptosis . These observations underscore that the absence of functional Tim50 leads to severe mitochondrial dysfunction, triggering cellular stress responses and ultimately cell death, explaining its essentiality for organismal viability.

What experimental approaches are used to study Tim50 depletion effects?

To study the effects of Tim50 depletion, researchers typically employ several methodologies:

How does S. pombe Tim50 compare with Tim50 in other organisms?

S. pombe Tim50 shares the fundamental function of mitochondrial protein import with Tim50 homologs in other organisms, but there are notable differences:

• Cysteine Content: While Saccharomyces cerevisiae Tim50 (ScTim50) contains only one cysteine residue (C268) located in the presequence-binding groove, S. pombe Tim50, like other Tim50 homologs, contains multiple cysteine residues distributed throughout the protein .

• Phosphatase Activity: Similar to human TIMM50 (hTIMM50) but unlike ScTim50, Trypanosoma brucei Tim50 (TbTim50) possesses a dual specificity phosphatase activity with greater affinity for protein tyrosine phosphate than for protein serine/threonine phosphate . This suggests that S. pombe Tim50 might also possess phosphatase activity, though this requires direct experimental verification.

• Additional Functions: While the protein import function is conserved, the non-canonical functions of Tim50 vary between species. In humans, TIMM50 is linked to cancer cell growth and apoptosis regulation . In T. brucei, Tim50 is involved in regulating VDAC expression . The specific non-canonical functions of S. pombe Tim50 remain to be fully characterized.

What methodologies are recommended for recombinant expression of S. pombe Tim50?

For successful recombinant expression of S. pombe Tim50, researchers should consider the following approaches:

What approaches can be used to study Tim50's phosphatase activity in S. pombe?

Based on findings that TbTim50 possesses dual specificity phosphatase activity , researchers investigating potential phosphatase activity in S. pombe Tim50 should consider:

• Substrate Specificity Assays:

  • Use synthetic phosphopeptides containing phosphotyrosine, phosphoserine, or phosphothreonine residues

  • Employ para-nitrophenyl phosphate (pNPP) as a general phosphatase substrate

  • Compare activity levels against different substrates to determine preference (TbTim50 shows greater affinity for tyrosine phosphate)

• Mutational Analysis:

  • Create point mutations in the conserved C-terminal domain phosphatase motif (particularly targeting the conserved aspartic acid residues which are critical for phosphatase activity in TbTim50)

  • Analyze how these mutations affect both phosphatase activity and protein import function

• Identification of Physiological Substrates:

  • Perform pull-down assays using substrate-trapping mutants of Tim50

  • Use phosphoproteomics approaches to identify changes in the phosphorylation status of proteins in Tim50-depleted or overexpressing cells

  • Investigate known phosphoproteins of the mitochondrial import machinery as potential substrates

• In Vivo Functional Studies:

  • Complement Tim50-depleted cells with phosphatase-deficient Tim50 mutants to determine if phosphatase activity is essential for viability

  • Analyze the impact of phosphatase activity on mitochondrial protein import efficiency

  • Investigate potential connections between phosphatase activity and other cellular functions

How can researchers distinguish between Tim50's roles in protein import versus its other cellular functions?

To differentiate between Tim50's canonical role in protein import and its non-canonical functions, researchers can employ several strategic approaches:

• Domain-Specific Mutations:

  • Generate mutations that specifically affect either the protein import function or other activities (such as phosphatase activity)

  • For example, mutations in the presequence-binding region would specifically affect protein import, while mutations in the phosphatase domain would target that function

  • Complementation assays with these mutants can reveal which functions are essential for viability

• Temporal Separation of Functions:

  • Use rapid depletion systems (like the urg1 promoter system in S. pombe ) to observe immediate versus delayed effects

  • Immediate effects following depletion likely reflect direct roles in protein import

  • Delayed effects may indicate secondary consequences or non-canonical functions

• Biochemical Separation of Activities:

  • Perform in vitro assays that specifically measure protein import (using isolated mitochondria and radiolabeled precursors)

  • Separately measure phosphatase activity using appropriate substrates

  • Compare how different mutations or conditions affect these distinct activities

• Interaction Partner Analysis:

  • Identify proteins that interact with Tim50 through co-immunoprecipitation and mass spectrometry

  • Categorize partners as related to either protein import (e.g., other TIM23 components) or other functions

  • Use proximity labeling techniques like BioID or APEX to identify transient or context-specific interactions

What is the significance of Tim50 mutations in disease models and how can S. pombe be used to study them?

Human TIMM50 mutations have been linked to severe diseases, including rapidly progressing encephalopathy . S. pombe provides an excellent model to study these mutations due to its genetic tractability and the conservation of mitochondrial import machinery.

• Creating Disease-Relevant Models in S. pombe:

  • Introduce mutations corresponding to human disease mutations into the S. pombe Tim50 gene

  • For example, mutations analogous to the human S112* (N-terminal truncation) and G190A (in the transmembrane domain) could be created

  • Use complementation assays to determine if these mutants can rescue Tim50-depleted cells

• Functional Characterization of Disease Mutations:

  • Analyze protein import efficiency using in vitro import assays with isolated mitochondria

  • Measure effects on membrane potential and ROS production

  • Assess impacts on respiratory function and ATP production

  • Determine effects on the stability of other TIM23 complex components

• S. pombe-Specific Advantages:

  • S. pombe has a growth rate of approximately 108 minutes , allowing for relatively rapid experimental cycles

  • The well-characterized mitotic recombination system in S. pombe enables sophisticated genetic manipulations

  • S. pombe cells have well-defined mitochondrial networks that can be visualized and quantified

• Translating Findings to Human Systems:

  • Compare phenotypes observed in S. pombe with those in patient-derived cells

  • Use complementation studies with human TIMM50 in S. pombe tim50 mutants to assess functional conservation

  • Identify potential therapeutic targets or strategies based on mechanisms elucidated in the S. pombe model

What cutting-edge techniques can be applied to study dynamic interactions of Tim50 in living cells?

Several advanced methodologies can help researchers study the dynamic interactions and functions of Tim50 in live S. pombe cells:

• Live-Cell Imaging Techniques:

  • Fluorescence Resonance Energy Transfer (FRET) to monitor protein-protein interactions between Tim50 and other TIM23 components

  • Fluorescence Recovery After Photobleaching (FRAP) to assess the mobility and dynamics of Tim50 within the mitochondrial inner membrane

  • Split fluorescent protein systems (e.g., split GFP) to visualize specific interactions in living cells

• Proximity-Based Labeling Approaches:

  • BioID or TurboID: Fusion of biotin ligase to Tim50 to identify proximal proteins

  • APEX2: Peroxidase-based proximity labeling for electron microscopy visualization and proteomic identification of neighbors

  • These approaches are particularly valuable for capturing transient interactions that might be missed in conventional co-immunoprecipitation experiments

• Single-Molecule Tracking:

  • Use photoactivatable or photoswitchable fluorescent proteins fused to Tim50 to track individual molecules

  • This approach can reveal the dynamics and heterogeneity of Tim50 behavior in the mitochondrial inner membrane

• Cryo-Electron Tomography:

  • Visualize the TIM23 complex architecture in situ in S. pombe cells

  • Immunogold labeling of Tim50 can help locate it precisely within the complex

  • This technique can reveal structural changes associated with different functional states

• Optogenetic Tools:

  • Light-inducible dimerization systems to control Tim50 interactions or localizations

  • Allows for precise temporal control to study immediate versus long-term effects of Tim50 function disruption

What controls should be included when studying recombinant S. pombe Tim50?

When designing experiments with recombinant S. pombe Tim50, researchers should include the following controls:

• Expression Controls:

  • Empty vector controls to account for effects of the expression system itself

  • Wild-type Tim50 expression as a positive control alongside any mutant variants

  • Expression of a known unrelated mitochondrial protein to control for general effects on mitochondrial function

• Functionality Controls:

  • For protein import studies, use well-characterized substrate proteins that depend on the TIM23 pathway

  • Include substrates that use alternative import pathways (e.g., TIM22-dependent proteins) as negative controls

  • When studying phosphatase activity, include both positive controls (known phosphatases) and negative controls (phosphatase-dead mutants)

• Localization Controls:

  • Verify correct mitochondrial localization using mitochondrial markers

  • Ensure proper membrane insertion and topology using protease protection assays

  • For IMS domain studies, confirm correct submitochondrial localization

• Interaction Controls:

  • Include both known interaction partners (e.g., TbTim50 interacts with TbTim17 ) and non-interacting proteins

  • Use crosslinking controls with varying linker lengths or without crosslinkers

  • For co-immunoprecipitation, include antibody-only controls and irrelevant protein controls

How should researchers address data discrepancies when comparing Tim50 from different species?

When confronted with discrepancies in Tim50 data across different species, researchers should:

• Systematic Comparative Analysis:

  • Create a detailed alignment of Tim50 sequences from multiple species to identify conserved versus divergent regions

  • Generate a table comparing known functions across species, as shown below:

  Species	Protein Import Function	Phosphatase Activity	Other Known Functions	Key Structural Features
  S. pombe	Yes	Unknown (predicted)	To be determined	Multiple cysteine residues
  S. cerevisiae	Yes	No	Substrate for Pptc7 phosphatase	Single cysteine in presequence-binding groove
  T. brucei	Yes	Yes (dual specificity)	VDAC regulation	Six cysteine residues
  Human	Yes	Yes	Cancer cell growth, apoptosis regulation	Four cysteine residues

• Direct Experimental Comparison:

  • Express Tim50 from different species in a single host (e.g., S. pombe tim50Δ cells)

  • Compare their ability to complement essential functions

  • Perform side-by-side biochemical assays using recombinant proteins prepared under identical conditions

• Domain Swap Experiments:

  • Create chimeric proteins combining domains from Tim50 of different species

  • Test which domains confer which specific functions

  • This approach can help identify the structural basis for functional differences

• Consider Evolutionary Context:

  • Analyze the evolutionary relationships between the species being compared

  • Consider differences in mitochondrial biology and protein import requirements

  • Account for potential adaptations to different cellular environments

What are the critical parameters for optimizing crosslinking experiments with Tim50?

Crosslinking experiments are valuable for studying Tim50's interactions with preproteins and other components of the protein import machinery . Key optimization parameters include:

• Crosslinker Selection:

  • Choose appropriate crosslinking agents based on the distance between interaction partners:

    • Short-range crosslinkers (e.g., DSS, BS3) for tight interactions

    • Longer crosslinkers for more distant interactions

    • Heterobifunctional crosslinkers (e.g., SMCC) when specific chemistry is needed

  • Consider photocrosslinking approaches for capturing transient interactions

• Reaction Conditions:

  • Optimize crosslinker concentration (typically 0.1-2 mM) to maximize specific crosslinking while minimizing non-specific reactions

  • Determine optimal reaction time (usually 15-60 minutes) to capture interactions without excessive non-specific crosslinking

  • Test various pH conditions, as crosslinking efficiency is often pH-dependent

  • Control temperature based on the stability of the interaction (4°C for stable complexes, room temperature for transient ones)

• Sample Preparation:

  • For studying interactions with preproteins, generate translocation intermediates by:

    • Using preproteins with folded domains that block complete translocation

    • Depleting ATP to stall the import process

    • Dissipating the membrane potential to trap preproteins at specific stages

  • For Tim50-Tim23 interactions, use gentle solubilization conditions to preserve the complex

• Detection Methods:

  • Use antibodies against both Tim50 and potential interaction partners

  • For unidentified crosslinking products, employ mass spectrometry techniques

  • Consider radiolabeled preproteins for highly sensitive detection of crosslinked species

How should researchers interpret changes in Tim50 phosphorylation status?

Tim50 phosphorylation can significantly impact its function, as demonstrated for ScTim50 where phosphorylation reduces protein import capability . When analyzing Tim50 phosphorylation data, researchers should:

• Phosphorylation Site Mapping:

  • Use mass spectrometry to identify specific phosphorylation sites

  • Compare identified sites with known functional domains to predict potential effects

  • Create site-specific phosphomimetic (S/T→D/E) and phospho-null (S/T→A) mutants

• Functional Correlation Analysis:

  • Measure protein import efficiency in relation to phosphorylation status

  • Assess phosphatase activity (if present) in relation to phosphorylation state

  • Evaluate how phosphorylation affects interactions with other TIM23 components

• Regulatory Mechanism Identification:

  • Search for kinases that may phosphorylate Tim50 (currently unknown for fungal, human, and T. brucei Tim50 )

  • Investigate whether S. pombe Tim50, like ScTim50, is a substrate for mitochondrial phosphatase Pptc7

  • Determine if phosphorylation is constitutive or regulated by specific cellular conditions

• Physiological Significance Assessment:

  • Create a correlation table of phosphorylation status versus various cellular parameters:

  Phosphorylation State	Protein Import Efficiency	Interaction with Tim23	Cell Growth Rate	Mitochondrial Membrane Potential
  Hyperphosphorylated	(Measured value)	(Measured value)	(Measured value)	(Measured value)
  Normal phosphorylation	(Measured value)	(Measured value)	(Measured value)	(Measured value)
  Hypophosphorylated	(Measured value)	(Measured value)	(Measured value)	(Measured value)

What bioinformatic approaches are useful for analyzing S. pombe Tim50?

Computational analyses can provide valuable insights into S. pombe Tim50 structure, function, and evolution:

• Sequence Analysis Tools:

  • Multiple sequence alignment (using CLUSTAL, MUSCLE, T-Coffee) to identify conserved regions

  • Hidden Markov Models to detect distant homologs and functional domains

  • Conservation analysis to identify functionally important residues (ConSurf, Evolutionary Trace)

• Structural Prediction and Analysis:

  • AlphaFold2 or RoseTTAFold for predicting Tim50 structure, particularly the IMS domain

  • Molecular dynamics simulations to study conformational dynamics

  • Protein-protein docking to model interactions with presequences and other TIM23 components

  • Electrostatic surface potential analysis to identify potential presequence-binding sites

• Systems Biology Approaches:

  • Network analysis to position Tim50 within the mitochondrial protein import interactome

  • Gene expression correlation analysis using existing S. pombe transcriptome data

  • Flux balance analysis to predict metabolic consequences of Tim50 dysfunction

• Evolutionary Analysis:

  • Phylogenetic tree construction to understand Tim50 evolution

  • Selection pressure analysis (dN/dS ratio) to identify sites under positive or negative selection

  • Ancestral sequence reconstruction to trace the evolution of specific functions

How can researchers accurately quantify mitochondrial protein import defects in Tim50 mutants?

Accurate quantification of protein import defects is essential for characterizing Tim50 mutants. Researchers should consider:

• In Vitro Import Assays:

  • Use radiolabeled precursor proteins and isolated mitochondria

  • Quantify import by measuring the appearance of mature (processed) protein or protease-protected protein

  • Calculate import efficiency as a percentage relative to wild-type mitochondria

  • Plot import kinetics over time (0-30 minutes) to determine both rate and extent of import

• In Vivo Import Measurements:

  • Use reporter constructs with mitochondrial targeting signals fused to fluorescent proteins

  • Measure the ratio of mitochondrial to cytosolic fluorescence

  • Employ flow cytometry for high-throughput quantification of large cell populations

  • Use pulse-chase experiments with inducible reporters to measure import dynamics

• Substrate-Specific Analysis:

  • Test multiple substrates with different dependencies on the TIM23 pathway

  • Create a comprehensive table of import efficiencies for different substrates:

  Substrate	Import Pathway	Wild-type Import (%)	Tim50 Mutant Import (%)	Fold Reduction
  Matrix protein 1	TIM23	100	(Measured value)	(Calculated)
  Matrix protein 2	TIM23	100	(Measured value)	(Calculated)
  Inner membrane protein	TIM23/Stop-transfer	100	(Measured value)	(Calculated)
  Carrier protein	TIM22	100	(Measured value)	(Calculated)

• Normalization and Controls:

  • Normalize import data to mitochondrial protein content

  • Control for changes in mitochondrial membrane potential using potential-independent import substrates

  • Include positive controls (known import-defective mutants) and negative controls (unrelated mitochondrial protein mutants)

What strategies can address low expression or solubility of recombinant S. pombe Tim50?

Researchers frequently encounter expression and solubility issues with mitochondrial membrane proteins like Tim50. Effective solutions include:

• Expression System Optimization:

  • Try multiple expression systems (E. coli, S. cerevisiae, S. pombe, insect cells)

  • For E. coli, use specialized strains designed for membrane proteins (C41, C43) or those with additional tRNAs (Rosetta)

  • Consider cell-free expression systems which can accommodate detergents during translation

• Construct Design Refinement:

  • Express only the soluble IMS domain (approximately residues 133-476 based on homology)

  • Use solubility-enhancing fusion partners (MBP, SUMO, TrxA) rather than just affinity tags

  • Create a panel of constructs with different N- and C-terminal boundaries to identify optimal soluble fragments

• Solubilization Optimization:

  • For full-length Tim50, test different detergents:

    • Mild detergents: DDM, LMNG, Digitonin

    • Zwitterionic detergents: CHAPS, LDAO

    • Novel solubilizing agents: SMALPs, nanodiscs, amphipols

  • Optimize detergent concentration, temperature, and time for extraction

• Refolding Strategies:

  • Express as inclusion bodies and develop a refolding protocol

  • Use gradual dialysis with decreasing denaturant concentration

  • Add lipids or detergents during refolding to stabilize the transmembrane domain

How can researchers verify that recombinant Tim50 maintains its native structure and function?

Ensuring that recombinant Tim50 retains its native conformation and activity is crucial. Verification approaches include:

• Structural Integrity Assessment:

  • Circular dichroism (CD) spectroscopy to verify secondary structure content

  • Thermal shift assays to measure protein stability

  • Limited proteolysis to probe for well-folded domains resistant to digestion

  • Dynamic light scattering to check for aggregation

• Functional Assays:

  • Presequence binding assays using synthetic presequence peptides

  • In vitro reconstitution with other TIM23 components to test complex formation

  • For phosphatase activity: enzyme kinetics with standard substrates like pNPP

  • Integration into proteoliposomes to test membrane insertion and orientation

• Interaction Validation:

  • Pull-down assays with known interaction partners (e.g., Tim23)

  • Surface plasmon resonance to measure binding affinities

  • Native gel electrophoresis to assess complex formation

  • Crosslinking experiments to verify specific interactions

• In Vivo Complementation:

  • The ultimate test: Can the recombinant protein complement Tim50 depletion in S. pombe?

  • Express recombinant Tim50 in Tim50-depleted cells and assess restoration of:

    • Cell growth and viability

    • Mitochondrial protein import

    • Mitochondrial membrane potential

    • Respiratory function

What are the key considerations when designing CRISPR-based genome editing for S. pombe Tim50 studies?

CRISPR-Cas9 technology offers powerful approaches for manipulating the S. pombe Tim50 gene. Important considerations include:

• Guide RNA Design:

  • Select target sites with minimal off-target potential using S. pombe-specific prediction tools

  • Consider the GC content (40-60% ideal) and avoid repetitive sequences

  • Target conserved functional domains for structure-function studies

  • Design gRNAs for both N- and C-terminal tagging strategies

• Repair Template Design:

  • Include homology arms of appropriate length (40-80 bp typically sufficient in S. pombe)

  • For tagging, ensure tags do not interfere with critical functional domains

  • For point mutations, introduce silent mutations near the edit site to prevent re-cutting

  • Consider including selection markers that can later be removed

• Delivery Method Optimization:

  • Transform CRISPR components as plasmids, RNPs, or a combination

  • Use appropriate S. pombe-specific promoters for Cas9 and gRNA expression

  • Consider temperature-sensitive or inducible Cas9 systems for essential genes like Tim50

• Validation Strategy:

  • Design PCR primers outside the homology arms to verify integration

  • Sequence the entire modified locus to check for unwanted mutations

  • Verify protein expression levels to ensure they match wild-type levels

  • Confirm functional integrity through complementation assays

What emerging technologies might advance our understanding of S. pombe Tim50?

Several cutting-edge technologies hold promise for elucidating new aspects of Tim50 biology:

• Cryo-EM Studies:

  • High-resolution structures of the entire TIM23 complex with bound preproteins

  • Visualizing conformational changes during protein translocation

  • Comparative structural biology between Tim50 from different species

• Single-Molecule Techniques:

  • Optical tweezers to measure forces during protein translocation

  • Single-molecule FRET to track conformational changes during substrate binding

  • Nanopore recordings to study preprotein translocation in real-time

• Spatiotemporal Proteomics:

  • Proximity labeling combined with quantitative proteomics to map the dynamic Tim50 interactome

  • Pulse-SILAC to determine protein turnover rates and assembly dynamics

  • Thermal proteome profiling to identify ligands and interaction partners

• Advanced Genome Editing:

  • Base editing or prime editing for precise manipulation without double-strand breaks

  • Multiplexed CRISPR screens to identify genetic interactions

  • CRISPR interference/activation to modulate Tim50 expression levels with temporal control

What are the most promising applications of S. pombe Tim50 research in disease models?

S. pombe Tim50 research has potential applications for understanding and addressing human diseases:

• Neurodegenerative Disorders:

  • TIMM50 mutations have been linked to severe encephalopathy

  • S. pombe models can help elucidate mechanisms underlying mitochondrial dysfunction in neurodegeneration

  • Screening approaches in S. pombe could identify compounds that restore function to mutant Tim50

• Cardiac Pathologies:

  • TIM50 has been shown to attenuate pathological cardiac hypertrophy by reducing oxidative stress

  • S. pombe models could help dissect the molecular mechanisms connecting Tim50 to redox regulation

  • The relatively fast growth rate of S. pombe (108 minutes ) enables rapid screening of potential therapeutic interventions

• Cancer Biology:

  • Tim50 levels correlate with growth and proliferation of various cancer cells

  • S. pombe studies could reveal how alterations in Tim50 function contribute to metabolic reprogramming in cancer

  • Identify potential vulnerabilities in cancer cells with altered Tim50 expression or function

• Metabolic Disorders:

  • S. pombe mitochondrial studies can provide insights into fundamental aspects of energy metabolism

  • Connections between protein import efficiency and metabolic adaptation can be explored

  • The well-characterized S. pombe system allows for precise genetic manipulations to model metabolic disease states

How might the dual functions of Tim50 be exploited for biotechnological applications?

The multifunctional nature of Tim50, combining protein import and potential phosphatase activities, offers interesting biotechnological opportunities:

• Protein Targeting Systems:

  • Engineer Tim50-based systems for targeted protein delivery to mitochondria

  • Develop switchable protein import systems controlled by phosphorylation

  • Create synthetic protein translocation pathways with modified specificity

• Biosensors:

  • Design Tim50-based biosensors that detect changes in mitochondrial function

  • Develop FRET-based reporters using Tim50's presequence binding to monitor import activity

  • Create phosphorylation-sensitive reporters based on Tim50's phosphatase domain

• Protein Production Platforms:

  • Optimize mitochondrial import for enhanced production of recombinant proteins in yeast

  • Engineer Tim50 to improve import efficiency of specific cargo proteins

  • Develop mitochondrial targeting systems for difficult-to-express proteins

• Drug Discovery Platforms:

  • Use S. pombe Tim50 systems to screen for compounds that modulate protein import

  • Identify inhibitors or activators of Tim50's phosphatase activity

  • Develop yeast-based screens for compounds that can rescue disease-associated Tim50 mutations