Recombinant Saccharomyces cerevisiae Mitochondrial chaperone TCM62 (TCM62)

What is TCM62 and what is its primary function in yeast cells?

TCM62 is a gene in Saccharomyces cerevisiae that encodes a mitochondrial membrane protein of approximately 64 kDa (64,211 Da). Its primary function is as a molecular chaperone necessary for the assembly of mitochondrial succinate dehydrogenase (SDH), also known as respiratory complex II . The protein forms a complex containing at least three SDH subunits and facilitates their proper assembly into the functional enzyme complex .

While initially identified for its role in SDH assembly, further research has revealed that Tcm62p also functions as a general chaperone that ensures the stability of various mitochondrial proteins, including other respiratory chain complexes, particularly under heat stress conditions . This dual role—specific assembly factor for SDH and general mitochondrial protein stabilizer—makes TCM62 an important factor in yeast mitochondrial homeostasis.

How is TCM62 structurally related to other molecular chaperones?

Tcm62p shares notable sequence homology with established molecular chaperones. Specifically, it exhibits 17.3% sequence identity with yeast hsp60 (heat shock protein 60) and approximately 16% identity with E. coli GroEL . Both hsp60 and GroEL are well-characterized molecular chaperones that form large oligomeric complexes to assist protein folding.

Despite these similarities, TCM62 appears to be relatively unique in the evolutionary landscape. No close homologs have been reported in other organisms, suggesting that this specific chaperone may be yeast-specific . This evolutionary distinction raises interesting questions about the specialized nature of mitochondrial protein assembly mechanisms in Saccharomyces cerevisiae compared to other eukaryotes.

When not associated with SDH subunits, Tcm62p forms a complex of approximately 450 kDa, which researchers hypothesize may adopt a heptameric ring-like structure similar to that observed in E. coli GroEL . This structural arrangement would be consistent with its chaperone function, providing surfaces for binding unfolded or partially folded protein substrates.

What is the subcellular localization and topology of Tcm62p?

Tcm62p is a mitochondrial membrane-spanning protein that exhibits a specific topological orientation within the mitochondrial compartments. The protein is initially synthesized as a precursor in the cytoplasm and subsequently transported into the mitochondria, where its pre-sequence is proteolytically cleaved to form the mature, functional protein .

Within the mitochondria, Tcm62p adopts a specific orientation: its amino terminus (N-terminus) is located in the mitochondrial matrix, while its carboxyl terminus (C-terminus) is accessible from the intermembrane space . This transmembrane topology is significant because it allows Tcm62p to interact with components in both mitochondrial compartments, potentially facilitating the coordination of protein assembly processes that span the inner membrane.

This specific localization is crucial for its function, as it positions Tcm62p to interact with both the membrane-embedded and matrix-exposed components of the succinate dehydrogenase complex during its assembly.

What expression systems are most effective for producing recombinant Tcm62p for structural studies?

For structural studies of Tcm62p, heterologous expression in E. coli systems has proven effective, though with important modifications to account for the protein's mitochondrial nature. When expressing recombinant Tcm62p, researchers should consider:

• Codon optimization: The codon usage of S. cerevisiae differs from E. coli, necessitating optimization for efficient expression. Utilizing strains like Rosetta (DE3) that supply rare tRNAs can help overcome codon bias issues.

• Expression construct design: For optimal expression, constructs should exclude the mitochondrial targeting sequence (first ~20-30 amino acids), as this sequence can interfere with proper folding in bacterial systems. Additionally, incorporating a cleavable affinity tag (His6 or GST) at the N-terminus facilitates purification.

• Expression conditions: Induction at lower temperatures (16-18°C) overnight with reduced IPTG concentrations (0.1-0.5 mM) minimizes inclusion body formation, as Tcm62p has a tendency to form insoluble aggregates when overexpressed .

• Buffer optimization: During purification, including stabilizing agents such as glycerol (10%), reducing agents (DTT or β-mercaptoethanol), and appropriate salt concentrations (typically 150-300 mM NaCl) enhances protein stability.

For researchers specifically interested in studying Tcm62p in its native oligomeric state (approximately 450 kDa complex), native gel electrophoresis combined with size exclusion chromatography provides valuable insights into assembly states. When studying interactions with SDH subunits, co-expression systems that simultaneously produce Tcm62p and SDH components allow for reconstruction of the assembly process in vitro.

How can researchers effectively study the interaction between Tcm62p and SDH subunits?

Multiple complementary approaches can be employed to characterize Tcm62p-SDH subunit interactions:

• Co-immunoprecipitation (Co-IP): This remains a gold standard for verifying protein-protein interactions in near-native conditions. Using antibodies against Tcm62p or epitope-tagged versions, researchers can precipitate complexes and identify interacting partners through immunoblotting or mass spectrometry. Evidence shows that Tcm62p forms complexes with at least three SDH subunits .

• Yeast two-hybrid assays: While useful for initial screening, these may yield false negatives for mitochondrial proteins due to improper localization. Modified versions like the split-ubiquitin system are more appropriate for membrane-associated proteins like Tcm62p.

• Bimolecular Fluorescence Complementation (BiFC): By fusing complementary fragments of fluorescent proteins to Tcm62p and putative binding partners, researchers can visualize interactions in living cells through reconstitution of fluorescence when proteins interact.

• Cross-linking coupled with mass spectrometry: This approach can map specific interaction domains. Chemical cross-linkers with varying spacer arm lengths can capture both stable and transient interactions, which is particularly valuable given the chaperone nature of Tcm62p.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique can identify regions of Tcm62p that undergo conformational changes upon binding SDH subunits, revealing the dynamic nature of chaperone-substrate interactions.

When Tcm62p is overexpressed, it forms insoluble large aggregates that specifically trap Sdh1 and Sdh2 subunits but not unrelated proteins, indicating specificity in its interactions . This property can be experimentally leveraged to identify the minimal interaction domains through truncation analysis.

What methodologies are appropriate for studying the role of Tcm62p in iron-sulfur cluster assembly?

Investigating Tcm62p's hypothesized role in [3Fe-4S] cluster formation or insertion into apo-Sdh2p requires specialized approaches:

• Electron Paramagnetic Resonance (EPR) spectroscopy: This technique provides direct information about the iron-sulfur clusters' formation, allowing researchers to determine whether Tcm62p facilitates proper cluster assembly by comparing spectra from wild-type and Tcm62-deficient mitochondria.

• In vitro reconstitution assays: Combining purified apo-Sdh2p with Tcm62p and iron-sulfur cluster assembly machinery components allows for direct assessment of Tcm62p's contribution to cluster insertion. Key readouts include:

  • Spectroscopic changes (UV-visible absorption at characteristic wavelengths)

  • Activity measurements of reconstituted SDH

  • Iron content quantification using colorimetric assays or ICP-MS

• Time-resolved studies: Using pulse-chase experiments with radiolabeled iron (55Fe) allows tracking of iron incorporation into Sdh2p in the presence or absence of functional Tcm62p, revealing kinetic aspects of the process.

• Targeted mutagenesis: Introducing mutations in Tcm62p residues predicted to interact with Sdh2p or influence iron-sulfur cluster handling can identify critical functional regions. Complementation assays using these mutants in tcm62Δ strains provide functional validation.

• Cryo-electron microscopy: For structural characterization of Tcm62p-Sdh2p complexes at different stages of iron-sulfur cluster insertion, providing molecular insights into the mechanism.

Researchers studying this aspect should account for the oxidation-sensitive nature of iron-sulfur clusters by conducting experiments under anaerobic conditions or with appropriate reductants present to prevent artifactual results .

What phenotypic assays are most informative when studying tcm62Δ mutants?

Several phenotypic assays provide valuable information about the functional consequences of TCM62 deletion:

• Respiratory competence assays:

  • Growth assessment on non-fermentable carbon sources (glycerol, ethanol, lactate) reveals respiratory deficiency

  • Oxygen consumption measurements using Clark-type electrodes or more advanced respirometry approaches (Seahorse XF analyzers) provide quantitative data on respiratory capacity

  • Succinate-dependent respiration measurements specifically evaluate SDH functionality

• Complex II activity assays:

  • Spectrophotometric measurement of succinate:ubiquinone oxidoreductase activity in isolated mitochondria

  • Blue Native PAGE followed by in-gel activity staining allows visualization of assembled, functional complex II

  • Succinate:DCPIP (2,6-dichlorophenolindophenol) reduction assays measure electron transfer capability

• Temperature sensitivity tests:

  • Growth comparisons at normal (30°C) versus elevated temperatures (37°C, 39°C) reveal the general chaperone function of Tcm62p at heat stress conditions

  • Recovery assessment after acute heat shock provides insights into mitochondrial protein stability

• Mitochondrial protein aggregation analysis:

  • Differential centrifugation followed by detergent solubility tests to quantify aggregated proteins

  • Monitoring levels of specific mitochondrial proteins via immunoblotting to identify those dependent on Tcm62p for stability

• Iron homeostasis assessment:

  • Measuring total mitochondrial iron content

  • Assessing activity of other iron-sulfur containing enzymes (aconitase, respiratory complex I) to determine specificity of effects

When designing these experiments, it's essential to include appropriate controls, particularly strains with selective defects in other SDH assembly factors. This allows researchers to distinguish Tcm62p-specific effects from general consequences of SDH deficiency .

How can researchers distinguish between the specific role of Tcm62p in SDH assembly versus its general chaperone function?

Distinguishing between Tcm62p's specific role in SDH assembly and its general chaperone function requires careful experimental design:

• Temperature-dependent complementation studies: Introducing targeted mutations in TCM62 may selectively disrupt either the SDH-specific functions or general chaperone activity. By conducting complementation assays at normal and elevated temperatures, researchers can identify separation-of-function mutants.

• Substrate specificity analysis: Comprehensive proteomic profiling of proteins that interact with Tcm62p under normal versus stress conditions can reveal distinct substrate sets. Techniques like proximity-based biotinylation (BioID) coupled with mass spectrometry are particularly valuable for capturing both stable and transient interactions.

• Domain mapping experiments: Creating chimeric proteins by swapping domains between Tcm62p and other mitochondrial chaperones (like Hsp60) can identify regions responsible for substrate specificity versus general chaperone function.

• Stress-specific induction: While many chaperones show increased expression under stress conditions, monitoring Tcm62p levels in response to different stressors (heat, oxidative stress, respiratory inhibition) provides insights into its primary role.

• In vitro activity assays: Reconstitution experiments comparing Tcm62p's ability to prevent aggregation of SDH subunits versus unrelated proteins under varying conditions can directly assess substrate specificity.

Research has shown that Tcm62p overexpression leads to the formation of insoluble aggregates that specifically trap Sdh1 and Sdh2 but not unrelated proteins . This selective interaction suggests a specific recognition mechanism beyond general chaperone activity. Conversely, the finding that Tcm62p ensures stability of various mitochondrial proteins at high temperatures indicates a broader role under stress conditions .

What are the primary technical challenges in purifying functional Tcm62p and how can they be overcome?

Purifying functional Tcm62p presents several technical challenges that researchers should anticipate:

• Membrane association: As a membrane-spanning protein, Tcm62p requires careful solubilization. Recommended approaches include:

  • Using mild detergents like digitonin (0.5-1%), DDM (0.05-0.1%), or LMNG (0.01-0.05%)

  • Step-wise detergent screening to identify optimal conditions

  • Incorporating lipids or amphipols during purification to maintain native-like environment

• Oligomeric state preservation: Tcm62p forms a complex of approximately 450 kDa, which researchers hypothesize may be a heptameric ring structure similar to GroEL . To maintain this oligomeric state:

  • Avoid harsh denaturants and extreme pH conditions

  • Include stabilizing agents (glycerol, trehalose)

  • Consider chemical cross-linking to stabilize complexes for structural studies

  • Use native gel filtration to separate oligomeric forms

• Aggregation tendency: When overexpressed, Tcm62p tends to form insoluble aggregates . Strategies to minimize this include:

  • Expressing at lower temperatures (16-18°C)

  • Using weaker promoters to reduce expression level

  • Co-expressing with substrate proteins or co-chaperones

  • Including molecular crowding agents (PEG, Ficoll) in buffers

• Maintaining activity: Functional assays should be incorporated throughout purification to ensure the purified protein retains its chaperone activity:

  • Substrate protein aggregation prevention assays

  • ATPase activity measurements (if ATP-dependent)

  • Binding assays with known substrates (SDH subunits)

• Reconstitution with cofactors: If Tcm62p requires cofactors for full functionality, these should be identified and included during purification or added during activity assays.

For structural studies requiring highly pure, homogeneous protein, researchers might consider nanobody-assisted purification or protein engineering approaches like surface entropy reduction to enhance crystallizability while preserving function.

How does Tcm62p compare to other known SDH assembly factors?

Tcm62p represents one of several assembly factors crucial for SDH biogenesis, but with distinct characteristics compared to other known factors:

Key functional distinctions of Tcm62p include:

• Membrane integration: Unlike many other assembly factors that are soluble matrix proteins, Tcm62p spans the mitochondrial membrane with domains in both matrix and intermembrane space .

• Dual functionality: Tcm62p serves both as a specific SDH assembly chaperone and as a general mitochondrial protein stabilizer under heat stress, a versatility not described for other SDH assembly factors .

• Structural homology: Tcm62p shows sequence similarity to general chaperones like Hsp60 and GroEL (17.3% and 16% identity, respectively), suggesting a evolutionary relationship to these broad-specificity folding assistants .

• Complex formation: In the absence of SDH subunits, Tcm62p forms a 450 kDa complex that may adopt a ring-like structure, reminiscent of chaperonin complexes—a unique feature among SDH assembly factors .

• Iron-sulfur cluster handling: While SDHAF1/LYRM8 and SDHAF3/SDH7 have established roles in iron-sulfur cluster insertion or protection, Tcm62p has been hypothesized to participate in [3Fe-4S] cluster formation/insertion into apo-Sdh2p, suggesting potential functional overlap with these factors .

Understanding these distinctions is crucial for developing comprehensive models of SDH assembly and for designing targeted experiments that distinguish between the roles of various assembly factors.

What is known about the ATP dependence of Tcm62p chaperone activity?

While the search results do not explicitly address the ATP dependence of Tcm62p, structural and functional comparisons with other chaperones allow for informed hypotheses:

To experimentally determine ATP dependence, researchers should consider:

• Sequence analysis for ATP-binding motifs: Examining Tcm62p for conserved Walker A and B motifs or other ATP-binding signatures through bioinformatic analysis.

• ATP binding assays:

  • Fluorescent ATP analogs (TNP-ATP, MANT-ATP) to detect direct binding

  • Equilibrium dialysis with radiolabeled ATP

  • Isothermal titration calorimetry (ITC) to measure binding thermodynamics

• ATPase activity measurements:

  • Colorimetric assays (malachite green) to detect inorganic phosphate release

  • Coupled enzyme assays (pyruvate kinase/lactate dehydrogenase system)

  • Comparing activity rates in the presence of SDH subunits versus general substrates

• Functional dependence studies:

  • Assessing chaperone activity with ATP versus non-hydrolyzable analogs (AMP-PNP)

  • Testing effects of ATP depletion on Tcm62p-substrate interactions

  • Introducing mutations in predicted ATP-binding residues and assessing functional consequences

• Structural studies:

  • Hydrogen-deuterium exchange mass spectrometry to detect conformational changes upon ATP binding

  • Cryo-EM structures in different nucleotide-bound states

How does environmental stress affect Tcm62p expression and function?

Research indicates that Tcm62p exhibits distinct functional characteristics under different environmental conditions, particularly in response to temperature stress:

To comprehensively characterize stress effects, researchers should investigate:

• Transcriptional regulation: Analyzing TCM62 mRNA levels under various stressors (heat, oxidative stress, respiratory inhibition, nutrient limitation) using RT-qPCR or RNA-seq approaches.

• Protein abundance changes: Quantifying Tcm62p levels through western blotting or targeted proteomics under various stress conditions to determine if protein levels increase similar to canonical heat shock proteins.

• Post-translational modifications: Examining whether stress conditions induce modifications (phosphorylation, acetylation, etc.) that might regulate Tcm62p activity using mass spectrometry-based proteomics.

• Substrate profile changes: Identifying stress-specific interaction partners through co-immunoprecipitation or proximity labeling techniques under various conditions.

• Subcellular redistribution: Monitoring potential changes in Tcm62p localization or complex formation under stress using fluorescence microscopy or native gel electrophoresis.

Understanding these adaptive responses is crucial for interpreting experimental results and for developing a complete picture of Tcm62p's role in mitochondrial homeostasis under varying environmental conditions.

What are the most promising future research directions for TCM62 studies?

Several promising research directions could significantly advance our understanding of TCM62:

• Structural characterization: Determining the high-resolution structure of Tcm62p, both alone and in complex with SDH subunits, would provide crucial insights into its mechanism of action. Cryo-electron microscopy appears particularly suited for this purpose given the large size of the Tcm62p complex (approximately 450 kDa) and its membrane association.

• Evolutionary relationships: Investigating why TCM62 appears to be yeast-specific and identifying functional equivalents in other organisms would illuminate evolutionary aspects of mitochondrial chaperone systems. Comparative genomics and functional complementation studies could reveal whether its function has been adopted by other proteins in higher eukaryotes.

• Regulatory network mapping: Characterizing how TCM62 expression and activity are regulated in response to cellular needs and stress conditions would place it within the broader context of mitochondrial quality control systems. This includes identifying transcription factors that control its expression and potential post-translational modifications that modulate its activity.

• Substrate recognition mechanisms: Elucidating how Tcm62p specifically recognizes SDH subunits for assembly while also functioning as a general chaperone under stress conditions would reveal fundamental principles of chaperone-substrate interactions and specificity determinants.

• Therapeutic implications: Exploring whether manipulation of TCM62 homologs or functionally related proteins in human cells could enhance mitochondrial function in diseases associated with respiratory chain defects represents a translational direction with potential clinical relevance.

These research avenues would collectively contribute to a more comprehensive understanding of mitochondrial protein quality control and respiratory complex assembly, with implications beyond the specific functions of TCM62.

How can insights from TCM62 research inform our understanding of mitochondrial disease mechanisms?

Research on TCM62 provides valuable insights into fundamental processes that, when disrupted, contribute to mitochondrial diseases:

• Assembly mechanisms of respiratory complexes: The study of Tcm62p illuminates the intricate process of SDH/Complex II assembly , potentially informing our understanding of pathogenic mechanisms in conditions like hereditary paraganglioma and pheochromocytoma, which are linked to SDH deficiency. The principles learned may extend to assembly processes of other respiratory complexes implicated in mitochondrial diseases.

• Protein quality control systems: As both a specific assembly chaperone and general stabilizer of mitochondrial proteins under stress conditions , Tcm62p exemplifies the sophisticated quality control mechanisms protecting mitochondrial proteostasis. Disruptions in these systems are increasingly recognized as contributing factors in neurodegenerative diseases with mitochondrial involvement, such as Parkinson's disease.

• Stress response adaptations: The functional shift of Tcm62p under heat stress highlights how mitochondrial systems adapt to environmental challenges. This adaptability (or its failure) may influence disease progression and treatment responses in mitochondrial disorders.

• Iron-sulfur cluster biogenesis: Tcm62p's hypothesized role in iron-sulfur cluster handling connects to a critical aspect of mitochondrial function that, when disrupted, leads to conditions like Friedreich's ataxia and multiple mitochondrial dysfunction syndromes.

• Evolutionary conservation of essential functions: Despite TCM62 appearing to be yeast-specific , the functions it performs are likely essential across species. Identifying functional equivalents in humans could reveal new candidate genes for unexplained mitochondrial disorders.