Recombinant Human Transmembrane protein 11, mitochondrial (TMEM11)

What is TMEM11 and what is its subcellular localization?

TMEM11 (Transmembrane Protein 11) is a mitochondrial membrane protein with multiple transmembrane domains. While early studies suggested TMEM11 was an inner mitochondrial membrane (IMM) protein, recent research has definitively established it as an outer mitochondrial membrane (OMM) protein .

The protein contains approximately 190 amino acids and three putative transmembrane domains. The exact topology has been determined through multiple complementary approaches, including:

• Protease protection assays showing TMEM11 is degraded by proteinase K in intact mitochondria, similar to the OMM marker TOMM20

• APEX2-mediated proximity labeling revealing OMM localization in electron microscopy studies

• Super-resolution microscopy showing TMEM11 distribution patterns distinct from IMM markers like MIC60

Methodologically, researchers can confirm TMEM11's localization by:

• Isolating intact mitochondria through differential centrifugation

• Treating with proteinase K before or after disruption of the OMM

• Comparing degradation patterns with known OMM (e.g., TOMM20) and IMM (e.g., MIC60) markers

How does TMEM11 affect mitochondrial morphology?

TMEM11 plays a critical role in maintaining normal mitochondrial morphology. Depletion of TMEM11 using CRISPRi in multiple cell types leads to dramatic alterations in mitochondrial network architecture :

• Mitochondria become enlarged and/or bulbous compared to the normal tubular structure

• More than half of TMEM11-depleted cells exhibit these morphological changes

• The phenotype is distinct from that caused by depletion of other mitochondrial dynamics proteins:

  • DRP1 knockdown causes hyper-elongated tubules with some spherical entities

  • OPA1 depletion results in smaller, more numerous fragmented mitochondria

To assess these morphological changes, researchers typically:

• Deplete TMEM11 using CRISPRi or siRNA approaches

• Stain mitochondria with vital dyes like MitoTracker

• Perform fluorescence microscopy to visualize the network

• Quantify the percentage of cells showing morphological alterations

These morphological effects appear to be functionally significant, as reintroduction of TMEM11 can rescue the phenotype .

What is the mechanism by which TMEM11 regulates cardiomyocyte proliferation?

TMEM11 functions as a negative regulator of cardiomyocyte proliferation and cardiac regeneration through a complex molecular pathway . The mechanism involves:

• TMEM11 directly interacts with METTL1 (methyltransferase-like protein 1)

• This interaction enhances m7G methylation of Atf5 mRNA

• Increased methylation elevates ATF5 protein expression

• ATF5 promotes transcription of Inca1 (inhibitor of cyclin-dependent kinase interacting with cyclin A1)

• Inca1 suppresses cardiomyocyte proliferation by inhibiting cell cycle progression

This TMEM11-METTL1-ATF5-INCA1 axis represents a novel regulatory pathway in cardiac biology. Experimental evidence shows:

• TMEM11 deletion enhances cardiomyocyte proliferation and restores heart function after myocardial injury

• TMEM11 overexpression inhibits neonatal cardiomyocyte proliferation and regeneration in mouse hearts

Methodologically, researchers can investigate this pathway by:

• Analyzing protein-protein interactions through co-immunoprecipitation

• Measuring m7G methylation levels of Atf5 mRNA

• Assessing ATF5 and Inca1 expression levels following TMEM11 manipulation

• Quantifying cardiomyocyte proliferation using markers like Ki67, EdU incorporation, or phospho-histone H3

How does TMEM11 interact with BNIP3/BNIP3L mitophagy receptors?

TMEM11 forms a complex with BNIP3 and BNIP3L (NIX), two key mitophagy receptors located at the outer mitochondrial membrane . This interaction has significant implications for mitophagy regulation:

• TMEM11 directly interacts and stably forms a complex with BNIP3 and BNIP3L

• Co-enrichment occurs at sites of mitophagosome formation

• TMEM11 functions as a negative regulator of BNIP3/BNIP3L-dependent mitophagy

• Depletion of TMEM11 enhances both basal and hypoxia-induced mitophagy

The interaction can be investigated through:

• Proximity labeling approaches to identify interaction partners

• Co-immunoprecipitation studies to confirm direct binding

• BN-PAGE analysis to characterize complex formation and stability

• Fluorescence microscopy to visualize co-localization at mitophagy initiation sites

Importantly, BNIP3 and BNIP3L are primarily responsible for the mitochondrial morphology defects observed in TMEM11-depleted cells, suggesting their activation contributes to the phenotype .

What are the most effective methods for studying TMEM11 localization and function?

Multiple complementary approaches can be employed to thoroughly characterize TMEM11:

For comprehensive analysis, researchers should combine:

• Genetic manipulation (CRISPR, RNAi) to alter TMEM11 expression

• Biochemical approaches to study protein interactions and complexes

• Microscopy techniques to visualize localization and morphological effects

• Functional assays to measure biological outcomes like mitophagy or proliferation

How can researchers effectively generate and validate recombinant TMEM11 proteins?

Producing high-quality recombinant TMEM11 presents challenges common to membrane proteins. Based on available data , researchers should consider:

• Expression Systems:

  • HEK-293 cells for mammalian expression (preserves post-translational modifications)

  • E. coli for higher yield (may require optimization for membrane proteins)

  • Cell-free protein synthesis systems for difficult constructs

• Tags and Fusion Partners:

  • His-tag for purification via immobilized metal affinity chromatography

  • Fluorescent protein fusions (GFP, CFP) for localization studies

  • APEX2 fusions for electron microscopy visualization

• Validation Methods:

  • Western blot with anti-tag antibodies or TMEM11-specific antibodies

  • Bis-Tris PAGE or SDS-PAGE for purity assessment (>90% purity is achievable)

  • Analytical SEC (HPLC) for homogeneity analysis

  • Functional validation by complementation in TMEM11-depleted cells

• Buffer Considerations:

  • PBS with potential additives like urea for stability

  • Detergents may be necessary for solubilization

  • Storage at -80°C with avoidance of freeze-thaw cycles

Commercially available recombinant TMEM11 proteins typically achieve >80% purity as determined by SDS-PAGE and Coomassie blue staining , providing a benchmark for in-house production efforts.

How can TMEM11 be targeted for therapeutic cardiac regeneration?

Based on the TMEM11-METTL1-ATF5-INCA1 axis identified in cardiac regeneration studies , several therapeutic strategies could be explored:

• Direct TMEM11 Inhibition:

  • Small molecule inhibitors targeting TMEM11-METTL1 interaction

  • Antisense oligonucleotides or siRNAs to reduce TMEM11 expression

  • CRISPR-based approaches for targeted knockout in cardiomyocytes

• Pathway Modulation:

  • Inhibitors of m7G methylation to reduce ATF5 expression

  • Direct ATF5 antagonists

  • Inca1 inhibitors to promote cell cycle reentry

• Experimental Considerations:

  • Timing of intervention is critical (acute vs. chronic heart failure)

  • Delivery methods to target cardiomyocytes specifically

  • Potential off-target effects on mitochondrial function

• Validation Approaches:

  • Ex vivo cardiac slice cultures for initial screening

  • Mouse models of myocardial infarction

  • Assessment of cardiac function using echocardiography

  • Histological analysis of cardiomyocyte proliferation

The potential therapeutic value is supported by evidence that TMEM11 deletion enhances cardiomyocyte proliferation and restores heart function after myocardial injury in mouse models .

What is the relationship between TMEM11 and mitochondrial dynamics proteins?

TMEM11's role in mitochondrial morphology regulation appears to be mechanistically distinct from canonical fission/fusion machinery :

• Comparison with Fission/Fusion Phenotypes:

  • TMEM11 depletion: enlarged, bulbous mitochondria

  • DRP1 knockdown (fission defect): hyper-elongated tubules

  • OPA1 depletion (fusion defect): fragmented, smaller mitochondria

  • MFN knockdown (fusion defect): fragmented mitochondria

• Pathway Independence:

  • Genetic interaction studies suggest TMEM11 functions independently of DRP1

  • TMEM11 likely regulates mitochondrial shape through a novel mechanism

• Connection to BNIP3/BNIP3L:

  • BNIP3/BNIP3L are primarily responsible for mitochondrial morphology defects in TMEM11-depleted cells

  • This suggests a pathway connecting mitophagy receptors to mitochondrial morphology

• Experimental Approaches:

  • Double knockdown experiments with TMEM11 and dynamics proteins

  • Analysis of dynamics protein localization in TMEM11-depleted cells

  • Live-cell imaging to assess mitochondrial dynamics (fission/fusion events)

The evidence indicates TMEM11 represents a novel regulatory mechanism for mitochondrial morphology, potentially linking morphology to mitophagy regulation through interaction with BNIP3/BNIP3L .

How can conflicting reports about TMEM11's submitochondrial localization be reconciled?

The literature contains apparently contradictory findings regarding TMEM11's localization . Early studies suggested IMM localization, while more recent work demonstrates OMM localization. These discrepancies can be reconciled by:

• Methodological Differences:

  • Early studies relied primarily on fractionation and electron microscopy

  • Recent studies employed multiple complementary approaches:

    • Protease protection assays

    • APEX2-mediated proximity labeling

    • Super-resolution microscopy

    • Biochemical membrane association tests

• Technical Considerations:

  • Overexpression artifacts in some studies

  • Differences in epitope accessibility

  • Cross-contamination in submitochondrial fractionation

• Interpretation Framework:

  • The preponderance of recent evidence strongly supports OMM localization

  • TMEM11 may have dynamic associations with multiple compartments

  • Some TMEM11 populations might associate with contact sites between OMM and IMM

• Model Organisms:

  • Original studies in Drosophila suggested IMM localization

  • Mammalian studies consistently show OMM localization

  • Potential evolutionary differences in TMEM11 localization

The current scientific consensus based on multiple lines of evidence is that mammalian TMEM11 is primarily an OMM protein , which explains its ability to directly interact with the OMM-localized BNIP3/BNIP3L mitophagy receptors.

What explains the variability in mitochondrial phenotypes observed after TMEM11 manipulation?

Different studies report variations in mitochondrial phenotypes following TMEM11 depletion . These variations can be understood through:

• Cell Type Specificity:

  • Different cell types have varying baseline mitochondrial network architecture

  • Expression levels of BNIP3/BNIP3L vary among cell types

  • Metabolic status affects mitochondrial morphology responses

• Knockdown Efficiency:

  • Complete vs. partial depletion yields different phenotypes

  • Acute vs. chronic depletion allows for compensatory mechanisms

• Experimental Conditions:

  • Culture conditions (confluency, medium composition) affect mitochondrial morphology

  • Fixation methods can alter apparent morphology

  • Imaging methods and resolution affect phenotype classification

• Genetic Background:

  • Expression levels of other mitochondrial dynamics proteins

  • Presence of genetic modifiers

For consistent results, researchers should:

• Use multiple siRNAs or sgRNAs targeting different regions

• Confirm knockdown efficiency by Western blot

• Quantify phenotypes using standardized methods

• Include appropriate controls (scrambled siRNA, non-targeting sgRNA)

• Test multiple cell types to determine generalizability

The involvement of BNIP3/BNIP3L in the TMEM11 phenotype suggests that baseline expression levels of these proteins may contribute significantly to the observed variability.

What are the most promising unexplored aspects of TMEM11 biology?

Several underexplored areas warrant further investigation:

• Physiological Regulation:

  • How is TMEM11 expression and function regulated under different stress conditions?

  • Does TMEM11 respond to metabolic cues or cellular stress?

  • Are there post-translational modifications that regulate TMEM11 activity?

• Tissue-Specific Functions:

  • Beyond cardiac tissue, what roles does TMEM11 play in other highly metabolic tissues?

  • Are there tissue-specific interaction partners?

  • Do tissue-specific isoforms exist with unique functions?

• Disease Relevance:

  • What is TMEM11's role in neurodegenerative diseases with mitochondrial dysfunction?

  • Does TMEM11 contribute to metabolic disorders?

  • Are there disease-associated TMEM11 variants with functional consequences?

• Evolutionary Conservation:

  • How has TMEM11 function evolved across species?

  • What core functions are conserved from invertebrates to mammals?

  • Do homologs in model organisms share similar interaction partners?

• Therapeutic Targeting:

  • Can TMEM11 inhibition be achieved pharmacologically?

  • Would tissue-specific TMEM11 modulation provide therapeutic benefits?

  • What are potential off-target effects of TMEM11 manipulation?

Experimental approaches to address these questions might include:

• Single-cell analyses to identify cell type-specific functions

• Proteomics to identify condition-specific interaction partners

• CRISPR-based screening for genetic interactors

• Analysis of patient samples to identify disease associations

How might TMEM11's role in mitophagy connect to its function in cardiac regeneration?

A fascinating unexplored connection exists between TMEM11's dual roles in mitophagy inhibition and cardiac regeneration suppression . Potential mechanisms linking these functions include: