Recombinant Human Mitochondrial import receptor subunit TOM70 (TOMM70A)

Basic Research Questions

• What is the structure and functional organization of TOMM70A?

TOMM70A is a component of the translocase of outer mitochondrial membrane (TOM) complex with a complex domain structure. It consists of an N-terminal transmembrane domain that anchors it to the mitochondrial outer membrane, followed by three soluble domains exposed to the cytosol: the Clamp (C1), Core (C2), and C-tail (C3) domains. All three domains are formed by tetratricopeptide repeats (TPRs) .

The C1 domain forms a binding groove that recruits the C-terminal EEVD tetrapeptides of Hsp70 and Hsp90 chaperones, while the C2 and C3 domains bind internal targeting signals of precursor proteins . This structural organization enables TOMM70A to coordinate both chaperone and precursor protein interactions, facilitating efficient mitochondrial protein import.

• How can researchers measure TOMM70A protein levels in experimental systems?

Researchers can quantify TOMM70A levels using several methods:

• ELISA: Using commercially available sandwich ELISA kits specific for human TOMM70A. These kits employ antibodies pre-coated onto microplates that capture TOMM70A from samples, followed by detection with biotin-conjugated antibodies and streptavidin-HRP .

• Western blotting: Using specific antibodies against TOMM70A for detection after protein separation.

• Mass spectrometry: For more comprehensive proteomic analysis, tandem mass spectrometry (LC-MS) can be employed following tryptic digestion .

• Imaging: Single-cell resolution of TOMM70A can be achieved using fluorescently-tagged versions (GFP-tagged TOMM70A) or antibody-based immunofluorescence .

• What experimental methods are used to assess TOMM70A function in protein import?

Several complementary approaches can be used:

• In vitro import assays: Isolated mitochondria from wild-type and TOMM70A-depleted cells are incubated with radioactively labeled precursor proteins. The import efficiency is assessed by protection from externally added proteases and analysis by gel electrophoresis .

• Genetic interaction screens: Crossing TOMM70A deletion strains with genome-wide knockout collections to identify synthetic genetic interactions that provide functional insights .

• Proximity labeling approaches: Using APEX2 or TurboID fused to TOMM70A to identify proteins in its vicinity through biotinylation, followed by pulldown and mass spectrometry analysis .

• Fluorescence microscopy: Using GFP-tagged mitochondrial proteins to assess their localization and abundance in cells with manipulated TOMM70A levels .

• How do TOMM70A expression levels vary during aging, and what are the implications?

TOMM70A undergoes age-dependent reduction in both yeast models and mammalian systems. In yeast, this reduction is associated with loss of mitochondrial membrane potential, mtDNA, and other mitochondrial proteins .

Methodologically, researchers can investigate this by:

• Purifying replicatively old cells using age-based separation techniques

• Employing imaging and single-photon-sensitive APDs (avalanche photodiodes) to achieve single cell/age resolution

• Quantifying mitochondrial proteins and membrane potential in different age groups

Importantly, overexpressing TOMM70A can rescue mitochondrial membrane potential in aged cells and prevent the age-associated reduction of other mitochondrial proteins . This suggests TOMM70A could be a potential target for interventions aimed at mitigating age-related mitochondrial dysfunction.

Advanced Research Questions

• What is the dual role of TOMM70A in transcriptional regulation and protein import, and how can researchers investigate this connection?

TOMM70A has been discovered to have two interconnected functions:

Protein Import Function: Acts as a receptor for mitochondrial precursor proteins, particularly hydrophobic carriers, facilitating their transfer to the general import pore.

Transcriptional Regulation: Influences the expression of nuclear-encoded mitochondrial genes through interaction with transcription factors like Fkh1/2 .

To investigate this dual role, researchers can employ:

• Transcriptomics and proteomics comparison: RNA-seq and quantitative proteomics to identify genes/proteins affected by TOMM70A overexpression or deletion

• ChIP-seq of transcription factors: To identify binding sites of TFs that respond to TOMM70A levels

• Import defect rescue experiments: Testing if the transcriptional repression during import defects requires TOMM70A

Experimental data has shown that TOMM70A overexpression increases the abundance of mitochondrial proteins from all four sub-compartments of the organelle . This effect is partially mediated through transcription factors like Fkh1/2, as their deletion blocks some of the induced expression of mitochondrial proteins in TOMM70A-overexpressing cells .

• How does the oligomeric state of TOMM70A affect its function, and what experimental approaches can resolve this controversy?

The oligomeric state of TOMM70A has been controversial, with evidence for both monomeric and homodimeric forms. Human TOMM70A exists in an equilibrium between monomer and dimer, with research suggesting the monomeric form may be more functionally active .

To investigate this, researchers can use:

• Analytical ultracentrifugation: To determine the sedimentation profile of purified TOMM70A

• Size-exclusion chromatography combined with multi-angle light scattering: To assess molecular weight distribution

• Cross-linking experiments: To capture transient protein-protein interactions

• Mutagenesis of the dimerization interface: Point mutations (like YS585AA) can shift the equilibrium toward the monomeric form

• Functional assays with different oligomeric states: Compare preprotein targeting efficiency

Research has shown that a point mutation at the predicted dimer interface increased the percentage of monomeric TOMM70A and enhanced preprotein targeting, but not chaperone docking . Cross-linking of endogenous human TOMM70A on isolated mitochondria failed to generate homodimeric cross-links, further supporting the functional relevance of the monomeric form .

• How can researchers identify TOMM70A-dependent mitochondrial proteins on a proteome-wide scale?

To identify TOMM70A-dependent proteins, researchers can employ:

• Comparative proteomics: Measure protein abundance in wild-type versus TOMM70A-depleted cells using quantitative mass spectrometry with techniques like TMT (tandem mass tag) labeling

• In vitro import assays with total cellular mRNA: Import translation products of total RNA into isolated mitochondria from wild-type and TOMM70A-deficient cells, followed by two-dimensional electrophoresis to identify affected proteins

• GFP-fusion libraries: Screening libraries of GFP-tagged mitochondrial proteins for altered localization or abundance in TOMM70A mutants

Research has revealed that TOMM70A-dependent proteins share specific characteristics:

• Enrichment of internal mitochondrial targeting sequences (iMTS-Ls)

• Higher aggregation propensities

• Inclination to form hydrophobic carriers that are particularly aggregation-prone

The table below summarizes key features of TOMM70A-dependent proteins:

• What proximity labeling strategies can be employed to identify TOMM70A interaction partners, and how do they compare?

Proximity labeling approaches provide crucial insights into the TOMM70A interactome. Several strategies include:

• APEX2-based proximity labeling: Fusion of TOMM70A with APEX2 enzyme allows biotinylation of proximal proteins in living cells when exposed to biotin-phenol and hydrogen peroxide. This approach identified distinct sets of associated proteins for TOMM70A versus other TOM components like TOMM20 .

• TurboID-based proximity labeling: An alternative approach using TurboID fusion proteins that can biotinylate neighboring proteins without requiring hydrogen peroxide.

• BioID-based proximity labeling: Uses a biotin ligase fusion that works more slowly but with less background.

Methodological considerations include:

• Control experiments with matrix-targeted APEX2 (Mito-APEX2) to distinguish MOM-specific from matrix interactions

• Comparison with other TOM components (e.g., TOMM20-APEX2) to identify component-specific interactions

• Quantitative proteomics with label-free quantification (LFQ) to measure enrichment

Research using TOMM70A-APEX2 has revealed fewer MOM-annotated proteins (6) compared to TOMM20-APEX2 (14), but additional interactors belonging to IMS, MIM, and matrix protein groups . These approaches also revealed that TOMM70A and TOMM20 remodel their interactomes differently in response to translation stress.

• How can researchers manipulate TOMM70A expression to study its role in age-related mitochondrial dysfunction?

TOMM70A levels decline with age, contributing to mitochondrial dysfunction. Researchers can investigate this through:

• Promoter replacement strategies: Replacing TOMM70A's native promoter with inducible promoters (e.g., GAL promoter in yeast) to prevent age-associated reduction

• Conditional expression systems: Using tetracycline-inducible or estradiol-inducible systems for temporal control of expression

• Viral vector delivery: Adeno-associated viral vectors for overexpressing TOMM70A in specific tissues of aging animal models

• CRISPR-based approaches: For precise genomic editing to control TOMM70A expression

Experimental data demonstrates that overexpressing TOMM70A can rescue mitochondrial membrane potential in aged cells and prevent age-associated reduction of other mitochondrial proteins . This effect is specific to TOMM70A, as overexpressing other TOM proteins cannot fully prevent the loss of mitochondrial membrane potential during aging.

The table below summarizes the effects of TOMM70A manipulation on aging-related parameters:

• What role does TOMM70A play in pathological cardiac hypertrophy, and how can it be studied experimentally?

TOMM70A has been implicated as a molecular switch that determines pathological cardiac hypertrophy by integrating hypertrophic stresses and mitochondrial responses . Researchers can investigate this through:

• Animal models of cardiac hypertrophy: Using pressure overload models (e.g., transverse aortic constriction) or angiotensin II infusion in wild-type, TOMM70A-knockout, and TOMM70A-overexpressing mice

• In vitro cardiomyocyte hypertrophy models: Treating isolated cardiomyocytes with pro-hypertrophic stimuli (e.g., phenylephrine, angiotensin II) and manipulating TOMM70A expression

• Human heart tissue analysis: Comparing TOMM70A expression in heart samples from patients with pathological hypertrophy versus healthy controls

Research has shown that TOMM70A is downregulated in pathological hypertrophic hearts from both humans and experimental animals . Reduction in TOMM70A expression produces distinct pathological cardiomyocyte hypertrophy both in vivo and in vitro, while increased TOMM70A levels provide cardiomyocytes with resistance to diverse pro-hypertrophic insults.

The mechanistic link appears to involve defective mitochondrial import of TOMM70A-targeted optic atrophy-1 (Opa1), triggering intracellular oxidative stress that leads to pathological cellular responses .

• What methodological approaches can determine if a specific mitochondrial protein depends on TOMM70A for import?

To determine TOMM70A-dependency of a specific mitochondrial protein, researchers can use:

• In vitro import assays: Compare import efficiency of the protein into isolated mitochondria from wild-type and TOMM70A-deficient cells

• Domain swapping experiments: Create chimeric constructs by fusing presequences and mature domains from TOMM70A-dependent and independent proteins to identify regions conferring TOMM70A dependency

• Aggregation assays: Test if the protein forms aggregates in the cytosol when TOMM70A is absent

• Direct binding assays: Use purified components to test if the protein directly interacts with TOMM70A

Research has shown that for presequence-containing proteins, TOMM70A dependency often resides in the mature part rather than the presequence. The presequences of TOMM70A-dependent precursor proteins are recognized by TOMM20, whereas their mature parts exhibit TOMM70A-dependent import when attached to the presequence of TOMM70A-independent precursor proteins .

This indicates that TOMM70A plays a role in maintaining the solubility of aggregate-prone mature domains rather than in targeting signal recognition, providing a methodological framework for identifying and characterizing TOMM70A-dependent proteins.

• How does the interplay between chaperones and TOMM70A affect mitochondrial protein import, and what experimental designs can elucidate this relationship?

TOMM70A functions as a docking site for cytosolic chaperones including Hsp70/Hsc70 and Hsp90, which deliver preproteins to mitochondria. To study this interplay, researchers can use:

• Chaperone inhibition: Using specific inhibitors like geldanamycin (Hsp90) or VER-155008 (Hsp70) to block chaperone function and assess import

• Mutational analysis: Creating TOMM70A variants with mutations in the TPR domain (e.g., R192A) that disrupt chaperone binding without affecting other functions

• Artificial chaperone tethering: Experiments showing that tethering an unrelated chaperone-binding domain (like Tah1) to the mitochondrial surface can complement defects caused by TOMM70A deletion

• Co-immunoprecipitation: To identify specific chaperone-preprotein-TOMM70A complexes

Research has demonstrated that cytosolic chaperones HSP90 and HSP70 dock onto the specialized tetratricopeptide domain in TOMM70A at the outer mitochondrial membrane. This interaction delivers preproteins to the receptor for subsequent membrane translocation dependent on the HSP90 ATPase. Disruption of the chaperone/TOMM70A recognition inhibits the import of these preproteins into mitochondria .

Surprisingly, artificial tethering of the chaperone-binding TPR protein Tah1 to the mitochondrial surface can almost fully replace TOMM70A in promoting cell growth at increased temperature, stabilizing the mitochondrial proteome, and providing resistance against overexpressed inner membrane proteins . This suggests that the physiologically relevant function of TOMM70A is primarily that of a chaperone-binding factor rather than a direct substrate receptor.

• What is the significance of TOMM70A in maintaining cytosolic proteostasis, and how can this be experimentally demonstrated?

TOMM70A plays a crucial role in maintaining cytosolic proteostasis by preventing the aggregation of mitochondrial precursor proteins in the cytosol. Researchers can investigate this through:

• Protein aggregation assays: Monitoring the formation of protein aggregates in cells with normal or reduced TOMM70A levels using techniques like filter trap assays or fluorescence microscopy of aggregation-prone reporter proteins

• Cytosolic stress response analysis: Measuring activation of cytosolic stress responses like the heat shock response or the cytosolic protein response in TOMM70A-deficient cells

• Combined import and transcription defects: Inducing mitochondrial import defects (e.g., with temperature-sensitive TIM23 mutants) in wild-type or TOMM70A-deficient backgrounds to assess proteostasis collapse

• Chaperone sequestration analysis: Examining the availability of cytosolic chaperones when TOMM70A is absent

Research has shown that TOMM70A's dual roles in both transcription/biogenesis and import of mitochondrial proteins allow cells to accomplish mitochondrial biogenesis without compromising cytosolic proteostasis . When mitochondrial import is perturbed (e.g., by Tim23 inactivation), unimported nascent mitochondrial proteins accumulate on the surface of mitochondria. This activates a stress response that represses mitochondrial protein biogenesis to relieve cytosolic accumulation of nascent mitochondrial proteins, a response that requires TOMM70A .