Recombinant Bovine Mitochondrial import inner membrane translocase subunit Tim17-B (TIMM17B)

What is TIMM17B and what is its fundamental role in mitochondrial biology?

TIMM17B (Translocase of Inner Mitochondrial Membrane 17B) is a multipass transmembrane protein that forms an integral component of the mitochondrial translocase TIM23 complex. This complex facilitates the transport of mitochondrial proteins from the cytosol across the mitochondrial inner membrane and into the mitochondrion . TIMM17B is also known by several alternative names including JM3, TIM17B, and DXS9822 .

The protein functions as part of the core translocation channel through which precursor proteins with mitochondrial targeting sequences pass. Recent research indicates that TIMM17B plays a more central role in forming the protein-conducting channel than previously thought, working in conjunction with TIMM23 and other complex components .

What are the genomic and molecular characteristics of TIMM17B?

TIMM17B is encoded by a gene located on the X chromosome at position Xp11.23 in humans . The genomic and molecular details of human TIMM17B are summarized in the following table:

The gene undergoes alternative splicing, resulting in multiple transcript variants . Additionally, there is a pseudogene for TIMM17B on chromosome 12 in humans .

How does TIMM17B differ from TIMM17A in structure and function?

Humans have two orthologs of yeast Tim17: TIMM17A and TIMM17B . Both proteins interact with TIMM23 and ROMO1 (reactive oxygen species modulator 1, a homolog of yeast Mgr2) to form two populations of highly similar complexes . Computational structure prediction analyses have revealed that both TIMM17A and TIMM17B complexes exhibit high structural conservation with their yeast counterparts .

Despite their structural similarities, the evolutionary conservation of both TIMM17A and TIMM17B in higher eukaryotes suggests distinct functional roles or regulatory mechanisms. This raises an interesting question about the mechanistic and functional differences that justify the existence of two variants of TIM23 in mammals compared to the single variant in yeast .

How does the structure of TIMM17B contribute to the formation of the protein-conducting channel?

Recent computational structural analyses have provided insights into the architecture of the human core TIM23 complex. TIMM17B forms a critical part of this complex along with TIMM23 and ROMO1 . Together, these proteins create a channel-like structure that allows proteins to pass through the mitochondrial inner membrane.

TIMM17B and ROMO1 can create a channel-like structure similar to what has been observed in yeast models . The high structural conservation between yeast and human TIM23 components suggests a similar mechanism of protein translocation across species. The transmembrane domains of TIMM17B are particularly important for forming the protein-conducting channel, with specific residues likely contributing to channel gating and substrate recognition.

How do inhibitors like stendomycin affect TIMM17B function and mitochondrial protein import?

Stendomycin, a natural product produced by Streptomyces hygroscopicus, has been identified as a potent and specific inhibitor of the TIM23-dependent mitochondrial protein import pathway . While the exact molecular target of stendomycin within the TIM23 complex is still being investigated, its inhibitory effect demonstrates the critical importance of this pathway.

By specifically inhibiting TIM23-dependent import, stendomycin and similar compounds provide valuable tools for studying the function of complex components like TIMM17B. These inhibitors can help differentiate between TIM23-dependent and independent import pathways and provide insights into the structural features necessary for successful protein translocation.

What are the recommended antibodies and detection methods for studying TIMM17B?

For researchers studying TIMM17B, several validated antibodies and detection methods are available. The table below summarizes information about a commercially available antibody with documented reactivity against TIMM17B:

For western blotting applications, TIMM17B typically appears as a band between 17-20 kDa, consistent with its calculated molecular weight of 18 kDa . When designing experiments to detect TIMM17B, mitochondrial fractionation prior to analysis can enhance sensitivity by enriching for mitochondrial proteins.

How can researchers effectively knock down TIMM17B expression for functional studies?

Based on methods used for studying other components of the TIM23 complex, RNA interference using short hairpin RNA (shRNA) delivered via lentiviral vectors represents an effective approach for TIMM17B knockdown studies. The methodology can be adapted from successful protocols used for TIMM50 knockdown :

• Design multiple shRNA sequences targeting different regions of TIMM17B mRNA

• Clone shRNA sequences into a lentiviral vector (such as pLL3.7) that allows for expression under control of the U6 promoter

• For neuronal studies, utilize vectors that co-express a fluorescent reporter (e.g., EGFP) under control of a neuron-specific promoter like hSyn

• Produce lentiviral particles by co-transfecting the shRNA vector with helper constructs (pMDLg-pRRE, pRSV-REV, CMV-VSVG) in HEK293T cells

• Transduce cells with the generated lentiviruses (for neuronal cultures, transduction typically occurs at 4 days in vitro)

• Validate knockdown efficiency by immunoblotting with anti-TIMM17B antibodies, targeting a reduction of approximately 70-80% for functional studies

For control experiments, include untransduced cells, vector-only controls (expressing the vector without shRNA sequence), and scrambled shRNA sequences to control for non-specific effects .

What experimental approaches can be used to assess the impact of TIMM17B on cellular physiology?

To comprehensively assess the impact of TIMM17B on cellular physiology, researchers can employ multiple complementary approaches:

• Mitochondrial protein import assays: Measure the import efficiency of radiolabeled or fluorescently-tagged mitochondrial precursor proteins in cells with normal or reduced TIMM17B levels.

• Electrophysiological measurements: For neuronal studies, utilize whole-cell patch clamp techniques to assess:

  • Action potential frequency and characteristics

  • Response to channel-specific inhibitors (e.g., α-dendrotoxin for potassium channels)

  • Spontaneous excitatory and inhibitory activity

• Mitochondrial function analyses:

  • Measure mitochondrial membrane potential using fluorescent dyes

  • Assess ATP production rates

  • Evaluate respiratory chain complex activities

  • Analyze mitochondrial morphology using confocal or electron microscopy

• Proteomics approaches: Quantitative proteomics can reveal global changes in protein expression, particularly focusing on mitochondrial proteins that may be differentially imported in the absence of fully functional TIMM17B.

How can researchers differentiate between the functions of TIMM17A and TIMM17B experimentally?

To distinguish between the specific functions of TIMM17A and TIMM17B, several experimental strategies can be employed:

• Isoform-specific knockdown or knockout: Use RNA interference or CRISPR-Cas9 to selectively deplete either TIMM17A or TIMM17B, followed by comprehensive phenotypic analysis.

• Rescue experiments: In cells depleted of both isoforms, reintroduce either TIMM17A or TIMM17B individually to determine which phenotypes can be rescued by which isoform.

• Domain swapping: Create chimeric proteins containing domains from both TIMM17A and TIMM17B to identify regions responsible for isoform-specific functions.

• Substrate specificity analysis: Use in vitro import assays with reconstituted TIM23 complexes containing either TIMM17A or TIMM17B to determine if they show different preferences for various mitochondrial preproteins.

• Tissue expression profiling: Analyze the expression patterns of both isoforms across different tissues and developmental stages to identify potential tissue-specific functions.

What are the key considerations for expressing and purifying recombinant bovine TIMM17B for structural studies?

Expressing and purifying recombinant bovine TIMM17B presents several challenges due to its hydrophobic nature as a transmembrane protein. Based on approaches used for similar mitochondrial membrane proteins, researchers should consider:

• Expression system selection:

  • Bacterial systems like E. coli often lead to inclusion body formation, requiring refolding protocols

  • Eukaryotic systems such as insect cells or yeast typically provide better folding but with lower yields

  • Cell-free expression systems may offer advantages for membrane proteins

• Construct design:

  • Include a small affinity tag (His6 or Strep-tag) at the N- or C-terminus to facilitate purification

  • Consider fusion proteins (e.g., MBP, SUMO) to enhance solubility

  • Determine whether to express full-length protein or specific domains

• Solubilization strategy:

  • Test multiple detergents (DDM, digitonin, LMNG) to identify optimal extraction conditions

  • Consider newer technologies like nanodiscs, SMALPs, or amphipols for maintaining native-like membrane environments

• Co-expression approach:

  • For structural studies, co-express with interaction partners (TIMM23, ROMO1) to enhance stability

  • Design multi-protein expression systems if studying the intact complex

• Functional validation:

  • Develop assays to confirm that purified protein retains native structure and function

  • Consider reconstitution into liposomes for functional studies

For structural studies specifically, cryo-electron microscopy has emerged as the method of choice for membrane protein complexes like the TIM23 complex, potentially allowing visualization of TIMM17B in its native conformation within the larger translocase assembly.