Recombinant Caenorhabditis elegans Mitochondrial import receptor subunit TOM7 homolog (tomm-7), partial

What is TOMM-7 and what role does it play in C. elegans mitochondrial function?

TOMM-7 is one of the three evolutionarily conserved regulatory subunits (along with TOMM-5 and TOMM-6) of the TOM complex that directly associates with TOMM-40, the central pore-forming β-barrel protein of the complex. These regulatory subunits are crucial as they modulate the assembly and stability of the TOM complex, thereby controlling its function in protein import . The TOM complex serves as the main entry gate for nuclear-encoded proteins destined for the mitochondria, making TOMM-7 essential for maintaining mitochondrial proteostasis and function.

How does C. elegans TOMM-7 compare structurally and functionally to human TOMM7?

While both C. elegans TOMM-7 and human TOMM7 serve as regulatory subunits of the TOM complex, several notable differences exist in their structure and function. Human TOMM7 promotes the disassembly of higher-order oligomeric TOM complexes, whereas in fungi (a relationship that may extend to C. elegans), TOM7 is critical for the disassembly process that allows incorporation of new components into the complex . This functional divergence suggests species-specific adaptations in TOM complex regulation.

Structurally, human TOM7 contains a helical domain that bends in a clockwise fashion around the β-barrel, positioning it close enough to suggest an interaction with TOM22 . The C. elegans homolog likely adopts a similar structural arrangement, though specific structural data for C. elegans TOMM-7 is less well-characterized than its human counterpart. One significant functional difference is that human TOM7 exhibits a distinct interactome compared to other β-barrel-associated subunits, suggesting specialized roles beyond basic structural support .

What experimental models are available for studying TOMM-7 in C. elegans?

C. elegans offers several advantageous experimental models for studying TOMM-7 function. The nematode aligns with the 3Rs ('Replacement, Reduction and Refinement') principles for ethical animal research while providing a powerful system for genetic and drug screens . Several specific experimental approaches are particularly valuable:

The transparency of C. elegans facilitates direct observation of mitochondrial morphology and function in vivo, making it an excellent model for studying the consequences of TOMM-7 manipulation on mitochondrial network dynamics and cellular health.

How does the interactome of C. elegans TOMM-7 compare with its human counterpart?

The interactome of C. elegans TOMM-7 likely shares core conserved interactions with its human counterpart but also exhibits nematode-specific protein-protein interactions. Human TOM7 displays a distinct interactome from other β-barrel-associated subunits (TOM5 and TOM6), suggesting specialized functions beyond structural support of the TOM complex . In humans, TOM7 interacts with the core TOM complex components, particularly TOM40, and functionally impacts PINK1 stabilization during mitophagy.

Unlike the fungi-to-human comparison where TOM7 assembly follows different pathways (in humans through a TOM22-free intermediate complex), C. elegans TOMM-7 assembly pathways are not fully characterized . Research suggests that similar to human TOMM7, C. elegans TOMM-7 likely participates in the PINK1/Parkin pathway for mitochondrial quality control, though nematode-specific interacting partners may modulate this function differently.

An important technical consideration for interactome studies is that fusion of reporter tags to the C-terminus of human TOM7 completely abrogates proper targeting, which may also apply to C. elegans TOMM-7 . This suggests that experimental approaches to map the C. elegans TOMM-7 interactome should employ N-terminal tagging strategies or alternative methods such as proximity labeling techniques to avoid disruption of targeting sequences.

What role does TOMM-7 play in mitochondrial quality control pathways in C. elegans?

TOMM-7 likely plays a crucial role in mitochondrial quality control in C. elegans, particularly through its involvement in mitophagy pathways. In human cells, TOMM7 is implicated in PINK1/Parkin-mediated mitophagy as a critical factor for PINK1 stabilization, Parkin recruitment, and outer mitochondrial membrane accumulation . This function appears conserved across species, suggesting C. elegans TOMM-7 may similarly facilitate PINK1 stabilization upon mitochondrial membrane depolarization.

The mechanistic basis for this role likely stems from TOMM-7's position at the outer mitochondrial membrane and its influence on TOM complex assembly dynamics. By regulating the stability and conformation of the import channel, TOMM-7 may control the retrotranslocation or accumulation of PINK1 when mitochondrial membrane potential is compromised. In human cells, TOMM7-deficient conditions prevent PINK1 accumulation even under depolarization conditions, a phenotype rescued by knockdown of inner mitochondrial membrane protease OMA1 .

C. elegans provides an excellent model for studying these quality control functions due to its transparent body, which allows for direct visualization of mitochondrial networks and mitophagy events in vivo. Additionally, the well-characterized genetics of C. elegans facilitates examination of genetic interactions between TOMM-7 and other mitochondrial quality control factors, such as PINK-1, PDR-1 (Parkin homolog), and DCT-1 (NIX/BNIP3 homolog).

What are the optimal methods for expressing and purifying recombinant C. elegans TOMM-7?

Expressing and purifying recombinant C. elegans TOMM-7 presents several challenges due to its membrane-associated nature and small size. Based on experiences with TOM complex components in structural studies, the following methodological approach is recommended:

Expression Systems:

Expression Constructs:

• The most successful expression strategy should focus on identifying the core structural elements of TOMM-7 .

• N-terminal tags (His6, GST, or MBP) are preferable as C-terminal tags may interfere with proper folding and function, similar to observations with human TOM7 where C-terminal GFP fusion abrogated proper targeting .

• TEV or PreScission protease cleavage sites should be incorporated between the tag and TOMM-7 sequence to allow tag removal.

Purification Protocol:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged constructs)

• Tag cleavage followed by reverse affinity chromatography

• Size exclusion chromatography as a final polishing step

• For membrane-integrated studies, purification in the presence of appropriate detergents (DDM, LMNG) or direct incorporation into nanodiscs is recommended

Verification Methods:

• Mass spectrometry to confirm protein identity

• Circular dichroism to verify secondary structure

• Functional assays measuring interaction with other TOM complex components

This approach has proven effective for structural studies of TOM complex components and should be adaptable for C. elegans TOMM-7 with appropriate modifications based on its specific properties.

How can recombinant TOMM-7 be used to study mitochondrial protein import mechanisms?

Recombinant C. elegans TOMM-7 provides a valuable tool for dissecting the mechanisms of mitochondrial protein import through reconstitution experiments and interaction studies. Several experimental approaches can leverage purified TOMM-7 for mechanistic investigations:

Reconstitution of TOM Complex Components:
Purified recombinant TOMM-7 can be combined with other recombinant TOM complex components to reconstruct partial or complete complexes in vitro. This approach allows researchers to study how TOMM-7 influences the assembly, stability, and dynamic rearrangements of the TOM complex. By systematically adding or removing components, the specific contribution of TOMM-7 to complex structure and function can be determined.

The human cryo-EM studies of the TOM complex revealed that TOM7 occupies a specific position on the β-barrel and interacts with other components in a conformation that influences complex stability . Similar structural studies with C. elegans components could reveal nematode-specific arrangements that may inform functional differences.

In Vitro Import Assays:
Proteoliposomes containing reconstituted TOM complexes with or without TOMM-7 can be used to study protein import efficiency. By comparing import rates of various mitochondrial-targeted proteins between TOMM-7-containing and TOMM-7-deficient proteoliposomes, researchers can quantify its direct contribution to import functionality. This approach can also identify substrate-specific effects of TOMM-7 on import efficiency.

Interaction Mapping:
Surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or microscale thermophoresis (MST) can be employed to characterize the binding affinities and kinetics between TOMM-7 and other TOM complex components. These quantitative measurements can reveal how TOMM-7 interactions differ from those of its human counterpart, potentially explaining functional divergences between species.

What techniques are effective for studying TOMM-7's role in PINK1/Parkin-mediated mitophagy?

Investigating TOMM-7's role in PINK1/Parkin-mediated mitophagy requires approaches that can detect and quantify mitophagy events while manipulating TOMM-7 function. Several effective techniques include:

Genetic Complementation Studies:
Recombinant wild-type or mutant TOMM-7 can be expressed in TOMM-7-deficient C. elegans to assess rescue of mitophagy defects. This approach allows structure-function analysis by testing specific TOMM-7 mutations for their ability to restore normal mitophagy. Human studies have shown that TOMM7 is critical for PINK1 stabilization during mitophagy, and knockdown of TOMM7 prevents PINK1 accumulation even under depolarization conditions . Similar complementation studies in C. elegans can determine if this function is conserved.

Mitophagy Flux Assays:
Dual-fluorescent reporters, where mitochondria are labeled with both pH-sensitive and pH-insensitive fluorophores, can track mitophagy flux in vivo. This technique allows quantification of how TOMM-7 manipulation affects the rate and extent of mitochondrial degradation under basal or stress conditions.

In Vitro PINK1 Stabilization Assays:
Purified mitochondria from wild-type or TOMM-7-deficient C. elegans can be subjected to membrane depolarization, followed by immunoblotting for PINK1 accumulation. This approach directly tests whether C. elegans TOMM-7, like its human counterpart, is necessary for PINK1 stabilization. The finding that human TOMM7-deficient phenotypes can be rescued by knockdown of the inner mitochondrial membrane protease OMA1 suggests potential conserved mechanisms that can be explored in C. elegans.

Quantitative Proteomics:
Mass spectrometry-based proteomics of purified mitochondria from wild-type versus TOMM-7-deficient worms, under basal and stress conditions, can identify proteins whose levels are affected by TOMM-7 disruption. This approach can reveal additional mitophagy-related pathways influenced by TOMM-7 beyond the canonical PINK1/Parkin pathway.

How can C. elegans TOMM-7 be used to model human mitochondrial diseases?

C. elegans provides an excellent platform for modeling human mitochondrial diseases related to TOMM7 dysfunction. The conservation of mitochondrial import machinery between nematodes and humans makes C. elegans a valuable model organism for studying disease mechanisms and potential therapeutic approaches . Several specific applications include:

Modeling TOMM7 Variants:
Human disease-associated TOMM7 variants, such as the hypomorphic variant reported in a patient with short stature and growth failure , can be introduced into C. elegans TOMM-7 using CRISPR-Cas9 genome editing. These modified worms can then be assessed for phenotypes related to mitochondrial function, development, and lifespan. The transparency of C. elegans facilitates direct observation of mitochondrial morphology and function in vivo, allowing researchers to visualize the consequences of these mutations on mitochondrial networks.

High-Throughput Screening:
C. elegans is amenable to genetic and drug screens , making it an ideal model for identifying genetic modifiers or small molecules that can rescue TOMM-7-related phenotypes. This approach could identify potential therapeutic targets for human mitochondrial diseases associated with TOMM7 dysfunction. The ability to rapidly generate large populations of genetically identical animals enables robust statistical analysis of screening results.

Investigating Disease Mechanisms:
The genetic tractability of C. elegans allows researchers to investigate how TOMM-7 dysfunction affects various cellular processes relevant to human disease. For example, studies can examine the impact of TOMM-7 mutations on:

• Mitochondrial protein import efficiency

• Mitochondrial network morphology and distribution

• Cellular energy metabolism and ATP production

• Reactive oxygen species generation and oxidative stress

• Cell death pathways and tissue degeneration

The conservation of the PINK1/Parkin pathway in C. elegans makes it particularly useful for studying how TOMM-7 variants affect mitophagy, which has been implicated in various neurodegenerative diseases .

What methodological considerations are important when using TOMM-7 as a target for mitochondrial disease research?

When utilizing C. elegans TOMM-7 for mitochondrial disease research, several methodological considerations are crucial for obtaining reliable and translatable results:

Expression Level Control:
When expressing recombinant TOMM-7 (wild-type or mutant variants), it is essential to maintain physiologically relevant expression levels. Overexpression may mask subtle phenotypes or create artificial effects not relevant to disease conditions. Endogenous promoters or carefully calibrated inducible systems should be employed to achieve appropriate expression levels.

• Protein import assays measuring the efficiency of mitochondrial protein translocation

• Co-immunoprecipitation studies assessing interactions with other TOM complex components

• Analyses of TOM complex assembly using blue native PAGE

• Assessment of PINK1 stabilization upon mitochondrial depolarization

Environmental Considerations:
C. elegans phenotypes can be influenced by environmental factors such as temperature, food source, and population density. Standardizing these conditions is essential for reproducibility. Additionally, certain phenotypes may only manifest under specific stress conditions, such as oxidative stress or mitochondrial toxin exposure, which should be systematically evaluated.

Tissue-Specific Effects:
TOMM-7 dysfunction may affect different tissues to varying degrees, similar to how mitochondrial diseases often present with tissue-specific symptoms. Techniques for tissue-specific knockdown or expression of TOMM-7 variants can help dissect these differential effects and may reveal why certain tissues are more vulnerable to mitochondrial protein import defects.

Translational Relevance: When modeling human TOMM7 variants in C. elegans, it is important to consider structural conservation and ensure that the introduced mutations accurately reflect the human condition. Combining C. elegans studies with parallel analyses in human cells can strengthen the translational relevance of findings.