Recombinant Dictyostelium discoideum Mitochondrial import inner membrane translocase subunit tim17 (timm17)

What is the structural composition of Recombinant D. discoideum tim17 protein?

Recombinant Full Length Dictyostelium discoideum Mitochondrial import inner membrane translocase subunit tim17 (timm17) is a 183 amino acid protein with UniProt ID Q54K35. The full amino acid sequence is: MEAPCPDKIWQDAGGAFAIGYVLMGVVNIGLGFKRSPPNKRVLYTFALLRKKSPKFGGNFAIWGSLFSGFDCTLSYIRKTEDTVNPIAAGALTGGILAARSGWKHSVQAAAFGGIFIGIIEAFQHMMQKRMQAQQEEMTQQHLEERKRYEEERKQREGERKKLNENGKSKKNKQQQNGENDLD . The commercially available recombinant protein is typically expressed in E. coli with an N-terminal His-tag to facilitate purification and experimental applications .

What role does tim17 play in mitochondrial protein import in D. discoideum?

Tim17 serves as a core component of the TIM23 complex responsible for translocating presequence-containing proteins across the mitochondrial inner membrane. In D. discoideum, as in other eukaryotes, tim17 forms part of the essential machinery that recognizes and facilitates the movement of proteins destined for the mitochondrial matrix or inner membrane . The protein is positioned in close proximity to both matrix-targeted and laterally sorted preproteins during their translocation, suggesting a direct role in guiding these proteins to their final destination . Tim17's function is particularly dependent on conserved negatively charged residues within its transmembrane domains that create a lateral cavity essential for protein translocation .

How is recombinant D. discoideum tim17 protein typically stored and reconstituted for laboratory use?

For optimal stability, recombinant tim17 protein is typically stored at -20°C/-80°C in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 . Prior to use, the lyophilized protein should be briefly centrifuged and reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, it's recommended to add glycerol to a final concentration of 5-50% (standard recommendation is 50%) and divide into working aliquots to avoid repeated freeze-thaw cycles, which can compromise protein integrity . For short-term use, working aliquots can be stored at 4°C for up to one week .

What are the optimal conditions for assessing tim17 function in mitochondrial protein import assays?

When designing experiments to assess tim17 function in mitochondrial protein import, researchers should consider both in vivo and in vitro approaches. For in vitro assays, isolated mitochondria containing wild-type or mutant tim17 can be used to assess import of radiolabeled or fluorescently tagged preproteins . The import buffer should maintain physiological pH (typically 7.2-7.4) and contain ATP and NADH to energize mitochondria. The assay should be performed at 25°C for D. discoideum mitochondria .

To distinguish between matrix-targeting and membrane insertion, researchers can use established matrix-destined precursors (such as the b2-DHFR construct) with dihydrofolate reductase (DHFR) as a passenger domain. The addition of methotrexate (MTX) causes DHFR folding, which prevents complete translocation and creates import intermediates that can be analyzed by chemical crosslinking or co-immunoprecipitation with tim17 . Temperature-sensitive tim17 mutants can be particularly useful for dissecting specific steps in the import process, as demonstrated by reduced crosslinking efficiency in tim17-4 and tim17-5 mutants .

How can researchers effectively express and purify functional tim17 for in vitro studies?

Expression and purification of functional tim17 requires careful consideration of its hydrophobic nature as a membrane protein. Based on established protocols for similar proteins:

• Expression system selection: E. coli BL21(DE3) is commonly used for tim17 expression with an N-terminal His-tag . The protein is typically cloned into a pET vector under control of the T7 promoter.

• Culture conditions: Expression should be induced with IPTG (0.1-0.5 mM) at lower temperatures (16-18°C) overnight to reduce inclusion body formation.

• Purification protocol:

  • Lyse cells in a buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and appropriate protease inhibitors

  • Solubilize membranes with mild detergents (0.5-1% DDM or LDAO)

  • Purify using Ni-NTA affinity chromatography

  • Consider size exclusion chromatography as a final polishing step

• Quality control: Assess protein purity using SDS-PAGE (>90% purity is desired) and functional integrity through circular dichroism or limited proteolysis.

• Storage: Store in buffer containing 10-20% glycerol and appropriate detergent at concentrations above the CMC .

What crosslinking strategies can be used to identify interaction partners of tim17 during protein translocation?

Crosslinking approaches provide valuable insights into the dynamic interactions of tim17 during protein import. Several methodologies have proven effective:

• Chemical crosslinking: Homobifunctional reagents like DSS (disuccinimidyl suberate) or heterobifunctional crosslinkers like EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) can be used to capture transient interactions . In studies with arrested precursor proteins, chemical crosslinking has successfully demonstrated that tim17 is in close proximity to both matrix-targeted and laterally sorted preproteins .

• Site-specific crosslinking: Introducing cysteine residues at strategic positions in tim17 (such as D76C or E126C) allows for thiol-specific crosslinking to precursor proteins containing engineered cysteines . This approach has confirmed that precursors are translocated near conserved negatively charged residues of tim17 .

• Photo-crosslinking: Incorporation of photo-activatable amino acids (like p-benzoyl-L-phenylalanine) followed by UV irradiation can capture even very transient interactions.

• Analysis methods: Crosslinked products can be analyzed by western blotting using antibodies against tim17 and potential interaction partners. For comprehensive identification, mass spectrometry following immunoprecipitation is recommended.

The choice of crosslinking strategy should be determined by the specific research question, considering factors such as the distance between interacting molecules and the chemical environment of the interaction site.

How can mutagenesis of tim17 be utilized to dissect its functional domains in D. discoideum mitochondrial import?

Strategic mutagenesis of tim17 provides powerful insights into structure-function relationships. Based on research findings, a systematic approach would include:

• Charge-altering mutations: The conserved negatively charged residues in tim17's transmembrane domains (D17, D76, and E126) are critical targets . Single alanine substitutions (D17A, D76A, E126A) produce mild to moderate phenotypes, while double mutants show severe growth defects and the triple mutant (D17A_D76A_E126A) is non-viable . This demonstrates these residues' essential role in presequence protein translocation.

• Cysteine scanning mutagenesis: Converting specific residues to cysteines (e.g., Tim17 N16C, D76C, E126C) enables disulfide bond formation and crosslinking studies that reveal spatial relationships between tim17 domains and interaction partners .

• Hydrophilic cavity mutations: Substituting hydrophilic residues within the lateral cavity (e.g., N64L in TM2, S114L in TM4) affects precursor protein translocation, revealing the importance of the cavity's hydrophilic nature .

• Interspecies chimeras: Creating chimeric proteins with tim17 domains from different species can identify evolutionarily conserved functional elements and species-specific adaptations.

Results should be assessed through complementation tests, protein import assays, and analysis of mitochondrial function in vivo.

What are the methods for analyzing how tim17 cooperates with other TIM23 complex components?

Investigating tim17's interactions within the TIM23 complex requires multifaceted approaches:

• Co-immunoprecipitation with cross-stabilization analysis: Using antibodies against tim17 to pull down associated proteins, then analyzing how depletion or mutation of tim17 affects stability of other components . This approach has revealed that tim17 and tim23 form a stable core complex.

• Disulfide bond formation: Engineered cysteine residues in tim17 (e.g., N16C) can form disulfide bonds with similarly positioned cysteines in tim23, demonstrating their spatial proximity and orientation . Such studies have shown that tim17 and tim23 adopt a back-to-back orientation rather than having their cavities face each other .

• Blue native gel electrophoresis: This technique preserves native protein complexes and can reveal how mutations in tim17 affect assembly of the TIM23 complex .

• Genetic interaction studies: Synthetic lethality or suppressor screens can identify functional relationships between tim17 and other import components.

• Structural analysis: Recent advances combining co-evolution analysis with ColabFold have generated structural models of the Tim17-Tim23 core complex . Cryo-EM studies of the purified complex provide direct visualization of component organization.

These approaches have demonstrated that tim17 and tim23 form a back-to-back orientation in the inner membrane, with tim17's negatively charged residues creating an essential binding site for translocating preproteins .

How can D. discoideum tim17 be utilized in comparative studies with mammalian systems?

D. discoideum serves as an excellent model for comparative studies with mammalian mitochondrial import systems due to evolutionary conservation of core machinery . Research approaches include:

• Heterologous expression: D. discoideum tim17 can be expressed in mammalian cells to assess functional complementation and identify conserved mechanisms. Studies with dynamin B presequence regions have shown that targeting sequences functional in D. discoideum are similarly efficient in mammalian mitochondria, suggesting conservation of import mechanisms .

• Chimeric protein analysis: Creating fusion proteins with domains from D. discoideum and mammalian tim17 can identify functionally equivalent regions and species-specific adaptations.

• Structural comparison: Alignment of D. discoideum tim17 with mammalian homologs reveals that higher eukaryotes contain two directly adjacent negatively charged residues at positions equivalent to residues 16 and 17 in yeast, highlighting the crucial role of these charges in presequence protein import .

• Evolutionary analysis: Sequence alignment of Tim17 family proteins shows highest conservation of negative charges within transmembrane domains, particularly on the intermembrane space side within the lateral cavity . This suggests an ancient, conserved mechanism for presequence recognition.

• Pathogen response studies: As D. discoideum is a model for cell-autonomous defenses with conserved pathways for antimicrobial responses , studies of tim17's role during infection can provide insights into mitochondrial functions during host-pathogen interactions.

These comparative approaches leverage D. discoideum's experimental tractability while generating findings relevant to human health and disease.

What are common challenges in working with recombinant tim17 and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant tim17:

• Protein aggregation: As a membrane protein, tim17 is prone to aggregation during expression and purification.

  • Solution: Use mild detergents (DDM, LDAO) at concentrations above CMC throughout purification

  • Add 6% trehalose to storage buffer to enhance stability

  • Express at lower temperatures (16-18°C) to improve folding

• Low expression yields: Hydrophobic membrane proteins often express poorly in heterologous systems.

  • Solution: Optimize codon usage for expression host

  • Use specialized E. coli strains (C41, C43) designed for membrane protein expression

  • Consider fusion partners (MBP, SUMO) to enhance solubility

• Protein misfolding: Ensuring native-like folding of recombinant tim17 is challenging.

  • Solution: Validate protein functionality through binding assays with known partners

  • Assess secondary structure using circular dichroism

  • Incorporate tim17 into liposomes or nanodiscs to provide a lipid environment

• Reconstitution difficulties: Transferring purified tim17 into functional assays can be problematic.

  • Solution: Ensure complete solubilization in reconstitution buffer before adding to assays

  • Follow recommended reconstitution protocol in deionized sterile water

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots with 50% glycerol

• Non-specific binding: His-tagged proteins can exhibit non-specific interactions.

  • Solution: Include low concentrations of imidazole (10-20 mM) in binding buffers

  • Consider removing the His-tag after purification if it interferes with function

  • Validate interactions using multiple methods (pull-down, crosslinking, functional assays)

How can researchers distinguish between tim17's roles in matrix protein import versus lateral membrane insertion?

Discriminating between tim17's dual functions requires specialized experimental approaches:

• Substrate selection strategy: Use well-characterized model substrates:

  • For matrix targeting: Employ matrix-destined precursors like b2(84)-DHFR

  • For lateral insertion: Use inner membrane proteins like b2(110)Δ-DHFR that contain stop-transfer signals

• Import intermediate trapping: Generate stable import intermediates by:

  • Adding methotrexate (MTX) to cause DHFR domain folding, preventing complete translocation

  • Using temperature-sensitive tim17 mutants and performing heat shock prior to import

• Crosslinking approach: Apply position-specific crosslinking:

  • Site-specific photocrosslinking at different positions along the tim17 protein

  • Cysteine-specific crosslinking between engineered cysteines in tim17 and the substrate

• Mutational analysis: Target specific domains:

  • Mutations in the lateral cavity (N64L, S114L) primarily affect lateral release

  • Mutations in negative charges (D17A, D76A, E126A) affect both pathways but with different severities

• Kinetic discrimination: Monitor import kinetics:

  • Matrix targeting typically shows different kinetics than lateral insertion

  • Pulse-chase experiments can distinguish between transient versus stable interactions

Research has demonstrated that tim17 is in close proximity to both matrix-targeted and laterally sorted preproteins accumulated in TOM-TIM23 import sites, confirming its dual role in both pathways .

What controls should be included when assessing tim17 function in mitochondrial protein import assays?

Rigorous controls are essential for reliable interpretation of tim17 functional studies:

• Positive and negative substrate controls:

  • Known TIM23-dependent substrates (matrix proteins with presequences)

  • TIM23-independent substrates (carrier proteins that use the TIM22 pathway)

  • Substrates with mutated/deleted targeting sequences

• Membrane potential controls:

  • Include samples with uncouplers (CCCP or valinomycin) to dissipate Δψ

  • Verify membrane potential integrity using potential-sensitive dyes

• Processing controls:

  • Inhibit the matrix processing peptidase (MPP) to distinguish import from processing

  • Include precursors with mutated MPP cleavage sites

• Protease accessibility:

  • Perform protease protection assays with increasing protease concentrations

  • Include detergent controls to verify complete protease accessibility when membranes are solubilized

• Genetic controls:

  • Wild-type tim17 as positive control

  • Temperature-sensitive tim17 mutants as functional controls

  • Tim17 with mutations in key residues (D17A, D76A, E126A) as negative controls

• Import pathway specificity:

  • Verify TIM23 pathway dependence using tim23 mutants or depletion

  • Test import via alternative pathways (TIM22, SAM/TOB) to confirm specificity

• Detection method controls:

  • For fluorescent proteins, include photobleaching controls

  • For crosslinking experiments, include no-crosslinker and no-UV controls

  • For immunoprecipitation, include isotype control antibodies

These comprehensive controls ensure that observed effects can be specifically attributed to tim17 function rather than to experimental artifacts or indirect effects.

What are promising approaches for studying the dynamic interactions between tim17 and translocating precursor proteins?

Several cutting-edge approaches show promise for elucidating the dynamic interactions of tim17:

• Single-molecule techniques: Apply methods such as single-molecule FRET (smFRET) to monitor real-time conformational changes in tim17 during protein translocation. This requires strategic placement of fluorophores at non-disruptive positions in both tim17 and precursor proteins.

• Time-resolved crosslinking: Develop systems for rapidly inducible crosslinking triggered at defined stages of translocation. This could involve photocaged crosslinkers activated by light pulses at specific timepoints during import.

• Cryo-electron tomography: Apply this technique to visualize the native arrangement of tim17 within the TIM23 complex in situ, potentially capturing different states of the translocase during protein import.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Utilize HDX-MS to identify regions of tim17 that undergo conformational changes upon precursor binding, providing insights into the molecular mechanism of translocation.

• Native mass spectrometry: Apply this technique to intact TIM23 complexes to determine the stoichiometry and dynamics of different components during various stages of precursor protein engagement.

• Integrative structural biology: Combine computational modeling based on co-evolution analysis (as has been initiated for the Tim17-Tim23 core complex ) with experimental constraints from crosslinking, HDX-MS, and cryo-EM to generate comprehensive structural models.

These approaches would help resolve outstanding questions about how tim17 recognizes, orients, and facilitates the movement of diverse precursor proteins across or into the inner mitochondrial membrane.

How might comparative studies between D. discoideum and other species inform our understanding of tim17 evolution and function?

Comparative evolutionary studies offer significant potential for understanding tim17 function:

• Phylogenetic approach: Comprehensive sequence analysis across evolutionary distant organisms could identify absolutely conserved residues likely essential for core functions versus lineage-specific adaptations. The observation that higher eukaryotes contain two adjacent negatively charged residues at positions equivalent to residues 16-17 in yeast represents an example of evolutionary refinement of tim17 function .

• Model organism comparisons: Systematic functional studies in diverse models (D. discoideum, yeast, mammals) could reveal:

  • Conservation of negatively charged residues in transmembrane domains

  • Species-specific adaptations in the lateral cavity

  • Differences in interaction with other TIM23 components

• Pathogen responses: As D. discoideum is a model for cell-autonomous defenses , comparative studies of tim17 function during host-pathogen interactions across species could reveal evolutionary adaptations in mitochondrial function during infection.

• Pre-LECA evolution: Investigation of tim17 homologs in diverse eukaryotes could help reconstruct the evolution of the mitochondrial import machinery before the last eukaryotic common ancestor (LECA), providing insights into the endosymbiotic origin of mitochondria.

• Horizontal gene transfer: Analysis of potential horizontal gene transfer events involving tim17 or related proteins could reveal unexpected evolutionary relationships and functional convergence.

These comparative approaches would contextualize findings from D. discoideum within broader evolutionary patterns, potentially identifying conserved mechanisms critical for mitochondrial function across eukaryotes.

What potential applications exist for manipulating tim17 function in biomedical or biotechnological contexts?

Manipulation of tim17 function offers several promising applications:

• Targeted mitochondrial therapeutics: Engineering modified presequences based on understanding tim17-presequence interactions could improve delivery of therapeutic molecules specifically to mitochondria in diseases with mitochondrial dysfunction.

• Synthetic biology applications: Creating designer tim17 variants with altered substrate specificity could enable novel mitochondrial engineering approaches, such as:

  • Controlling mitochondrial protein composition in a targeted manner

  • Creating synthetic organelle-targeting pathways for biotechnological applications

  • Developing switchable import systems regulated by small molecules

• Biomarker development: Alterations in tim17 expression or function have been linked to various pathological conditions. Understanding these connections could lead to new mitochondrial-based biomarkers.

• Drug discovery platforms: The detailed understanding of tim17's essential negatively charged residues provides potential targets for developing compounds that modulate mitochondrial protein import in a controlled manner.

• Agricultural applications: Exploiting differences between plant, fungal and animal tim17 proteins could inform the development of selective agents that disrupt protein import in plant pathogens while sparing beneficial organisms.

• Enhanced recombinant protein production: Optimizing mitochondrial import through tim17 engineering could improve the production of mitochondrially-targeted recombinant proteins in biotechnological applications.

These applications highlight how fundamental research on tim17 structure and function in model organisms like D. discoideum can ultimately translate into practical innovations with broad impacts across multiple fields.