Recombinant Bovine Transmembrane protein 65 (TMEM65)

What is Transmembrane Protein 65 (TMEM65) and where is it localized?

TMEM65 is an inner mitochondrial membrane protein that plays a significant role in mitochondrial respiratory chain function. Subcellular fractionation studies have confirmed its presence in the inner mitochondrial membrane . Additionally, researchers have developed a TMEM65 reporter mouse that confirms TMEM65 as a mitochondrial protein . While earlier studies suggested potential localization in intercalated discs, recent evidence points to its primary function being in mitochondria, where it participates in crucial processes like calcium homeostasis and respiratory chain function .

What experimental approaches can be used to study TMEM65 function?

Several complementary approaches have proven effective for studying TMEM65 function:

• RNA interference: Knocking down TMEM65 expression using specific siRNAs to study the effects on mitochondrial content and respiration rate .

• CRISPR-based knockout models: Generation of TMEM65 knockout mice through disruption of specific exons (such as exon1 and/or exon4) .

• Conditional knockout models: Creating floxed Tmem65 mouse lines (Tmem65flox/flox) that can be crossed with tissue-specific Cre-expressing lines for targeted deletion .

• Protein interaction studies: Immunoprecipitation and super-resolution microscopy to investigate physical interactions between TMEM65 and other proteins .

• Mitochondrial function assays: Measurement of mitochondrial membrane potential, NADH redox status, and respiration rates in cells with altered TMEM65 expression .

How does TMEM65 regulate mitochondrial calcium homeostasis and what methodologies can be used to investigate this function?

TMEM65 plays a critical role in mitochondrial calcium efflux, particularly through its interaction with NCLX (sodium/calcium exchanger). Loss of TMEM65 results in greater accumulation of mitochondrial calcium and a lack of sodium-stimulated calcium efflux .

Methodological approaches to study TMEM65's role in calcium homeostasis:

• Calcium imaging: Using fluorescent calcium indicators to measure mitochondrial calcium levels in wild-type versus TMEM65-deficient cells.

• Protein interaction studies: AlphaFold structural prediction modeling has been used to investigate the interaction between TMEM65 and NCLX. The modeling shows that TMEM65's transmembrane domains align within a cleft next to NCLX's longest transmembrane helix .

• Genetic rescue experiments: Crossing TMEM65 knockout mice with MCU (mitochondrial calcium uniporter) knockout mice has demonstrated that rebalancing mitochondrial calcium dynamics by removing MCU can rescue the lethality associated with TMEM65 loss .

• Electrophysiological measurements: Patch-clamp techniques to measure calcium currents in cells with modified TMEM65 expression .

• Functional complementation assays: Overexpressing TMEM65 activates mitochondrial sodium/calcium exchange, while removing it blocks this process .

What phenotypes emerge when TMEM65 is knocked out in animal models, and how can these be quantified?

TMEM65 knockout in mice produces severe and quantifiable phenotypes:

These phenotypes are consistent with mitochondrial encephalomyopathy, supporting the clinical relevance of TMEM65 knockout models for human disease research .

How can researchers address challenges in interpreting contradictory findings regarding TMEM65 localization in different tissues?

Early research on TMEM65 presented conflicting data about its localization, with some studies suggesting presence in intercalated discs while others found it in mitochondria . To address such contradictions:

• Use multiple localization techniques: Combine immunofluorescence, subcellular fractionation, and reporter constructs. When antibody specificity is an issue, as noted in some TMEM65 studies , generate reporter mice that express fluorescently tagged TMEM65.

• Tissue-specific expression analysis: Perform RNA-seq and proteomics across different tissues to identify tissue-specific expression patterns.

• Super-resolution microscopy: Employ techniques like STORM or STED microscopy to precisely localize TMEM65 at the nanoscale level in different cellular compartments.

• Biochemical validation: Confirm localization through protease protection assays, membrane integration analyses, and co-immunoprecipitation with known markers of specific cellular compartments.

• Tissue-specific knockout models: Generate conditional knockouts to evaluate TMEM65 function in specific tissues, which can help resolve contradictory findings between cardiac and neuronal tissues.

What controls are essential when working with recombinant TMEM65 in functional studies?

For robust experimental design with recombinant TMEM65:

• Expression controls:

  • Western blot validation of recombinant protein purity and integrity

  • Size exclusion chromatography to confirm proper folding and oligomeric state

• Functional controls:

  • Inactive mutant versions of TMEM65 (e.g., transmembrane domain mutants)

  • Heat-denatured TMEM65 protein as negative control

  • Reconstitution experiments with known interacting partners (e.g., NCLX)

• System-specific controls:

  • Untransfected cells/mock transfections when introducing recombinant TMEM65

  • Heterozygous TMEM65 knockouts to assess dose-dependent effects

  • Rescue experiments with wild-type TMEM65 in knockout models

• Specificity controls:

  • Other mitochondrial membrane proteins of similar size/structure

  • Related TMEM family members to assess specificity of observed effects

What are the primary technical challenges in purifying functional recombinant TMEM65 and how can they be overcome?

As a membrane protein, TMEM65 presents several purification challenges:

• Solubility issues:

  • Challenge: Transmembrane domains tend to aggregate during extraction.

  • Solution: Use mild detergents like DDM, LMNG, or digitonin; screen detergent combinations for optimal solubilization.

• Protein misfolding:

  • Challenge: E. coli expression systems may not provide proper folding environment.

  • Solution: Consider mammalian or insect cell expression systems; use fusion partners that enhance solubility (MBP, SUMO); employ low temperature induction.

• Purification efficiency:

  • Challenge: Low yield and purity when using standard methods.

  • Solution: Implement two-step purification strategies combining affinity chromatography with size exclusion or ion exchange; optimize buffer conditions for stability.

• Functional validation:

  • Challenge: Confirming that purified protein retains native function.

  • Solution: Develop activity assays specific to TMEM65's role in calcium transport; use proteoliposome reconstitution to test membrane integration.

How do mutations in TMEM65 contribute to human disease, and what cellular models best recapitulate these conditions?

Mutations in TMEM65 have been linked to severe mitochondrial encephalomyopathy . A homozygous splice variant (c.472+1G>A) in the TMEM65 gene results in mitochondrial dysfunction and a severe clinical phenotype .

Optimal cellular models for studying TMEM65-related diseases:

• Patient-derived fibroblasts: Primary cells from patients with TMEM65 mutations provide direct access to disease-relevant phenotypes .

• CRISPR-engineered cell lines: Introduction of specific patient mutations into relevant cell types (neurons, cardiomyocytes) using precise genome editing.

• iPSC-derived models: Patient-specific induced pluripotent stem cells differentiated into affected cell types (neurons, cardiac cells) to study tissue-specific manifestations.

• Conditional knockout mice: Tissue-specific deletion of TMEM65 to model organ-specific aspects of the disease .

• Organoid models: Brain or cardiac organoids with TMEM65 mutations to study three-dimensional tissue effects.

The phenotypic similarity between Tmem65 knockout mouse models and patients with TMEM65 mutations highlights the clinical relevance of these models for understanding this protein's function in disease contexts .

What methodological approaches can assess the impact of TMEM65 deficiency on mitochondrial respiratory chain complexes?

TMEM65 deficiency significantly affects mitochondrial function. To comprehensively assess these effects:

• Respirometry analysis:

  • Oxygen consumption rate measurements using Seahorse XF or Oroboros technologies

  • Substrate-specific respiration rates to identify affected complexes

  • Coupled vs. uncoupled respiration to assess membrane potential dependence

• Enzyme activity assays:

  • Spectrophotometric measurements of individual complex activities

  • In-gel activity assays following blue native PAGE

  • ATP synthesis rate determination

• Protein abundance analysis:

  • Western blotting for respiratory chain complex subunits

  • Proteomic analysis of mitochondrial fractions

  • Immunocytochemistry to visualize complex distribution

• Assembly analysis:

  • Blue native PAGE to assess complex assembly

  • Pulse-chase experiments to track complex formation kinetics

  • Co-immunoprecipitation to identify affected assembly intermediates

• Functional imaging:

  • Live-cell imaging of membrane potential using JC-1 or TMRM

  • NADH/FAD autofluorescence to assess redox state

  • Mitochondrial morphology analysis using MitoTracker or targeted fluorescent proteins

These approaches have revealed that TMEM65 ablation in primary fibroblasts affects mitochondrial structure and function with lower mitochondrial membrane potential, altered NADH redox status, increased NADH flux, fragmented mitochondrial networks, and increased mitophagy .

What are the emerging hypotheses about TMEM65's evolutionary conservation and its potential roles beyond mitochondrial calcium regulation?

TMEM65's high conservation across species suggests fundamental biological roles that extend beyond currently established functions. Emerging research directions include:

• Evolutionary analysis: Comparative genomics studies to trace TMEM65's evolution and identify conserved domains that might indicate additional functions.

• Tissue-specific roles: Investigation of TMEM65's function in tissues beyond brain and heart, particularly in tissues with high mitochondrial content like skeletal muscle and liver.

• Interaction networks: Comprehensive interactome studies to identify novel binding partners of TMEM65 beyond NCLX and MCU.

• Bioenergetic adaptation: Exploration of TMEM65's potential role in mitochondrial adaptation to different metabolic states, particularly in tissues that undergo metabolic remodeling.

• Signaling functions: Investigation of TMEM65's possible role in retrograde signaling from mitochondria to the nucleus, potentially influencing nuclear gene expression patterns.

Current research indicates that adult-onset muscle atrophy and tissue remodeling upon muscle-specific TMEM65 deletion provides a unique window to probe TMEM65's primary functions without the confounding effects of pathological adaptations seen in whole-body knockouts .

How can structural biology approaches contribute to understanding TMEM65 function and designing targeted interventions?

Structural biology offers powerful tools for understanding TMEM65's molecular mechanism:

• AlphaFold prediction integration: Building on existing AlphaFold structural predictions to guide experimental approaches. Current modeling has already provided insights into how TMEM65's transmembrane domains might interact with NCLX .

• Cryo-EM analysis: Determination of TMEM65's structure alone and in complex with interaction partners like NCLX to understand the structural basis of calcium efflux regulation.

• Site-directed mutagenesis guided by structure: Systematic mutation of predicted interaction interfaces to define functional domains and critical residues.

• Small molecule screening: Structure-based virtual screening to identify compounds that might modulate TMEM65 function, potentially leading to therapeutic interventions for mitochondrial diseases.

• Protein engineering: Design of modified versions of TMEM65 with enhanced stability or altered function for research applications and potential therapeutic delivery.

Current structural models suggest that TMEM65's soluble N-terminal 'matrix hook domain' may interact with both the soluble 'regulatory loop' of NCLX and the C-terminal end of NCLX's longest transmembrane helix , providing a foundation for targeted intervention strategies.