Recombinant Macaca mulatta NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is MT-ND4L and what is its role in cellular metabolism?

MT-ND4L (mitochondrially encoded NADH-ubiquinone oxidoreductase chain 4L) is a critical subunit of complex I in the mitochondrial respiratory chain. This protein functions as part of the electron transport chain, specifically in the first step of oxidative phosphorylation. MT-ND4L participates in the transfer of electrons from NADH to ubiquinone, which creates an electrochemical gradient across the inner mitochondrial membrane that drives ATP production. This process is fundamental to cellular energy metabolism, converting the energy from food into a usable form for cellular functions . In most eukaryotes, the MT-ND4L gene is encoded in mitochondrial DNA, although exceptions exist in some species where it has been transferred to the nuclear genome .

How does the structure of Macaca mulatta MT-ND4L compare to human MT-ND4L?

Macaca mulatta (Rhesus macaque) MT-ND4L shares high sequence homology with human MT-ND4L, making it an excellent model for human studies. The amino acid sequence of Macaca mulatta MT-ND4L consists of 98 amino acids (MTLTYMNIMLFAFAISLLGMLTYRSHLVASLLCLEGMMMSLFIMATLIASNTHTFPLINIMPIILLVFAACETAVGLALLISISNTYGLDYVHNLNLLQC) . This protein maintains similar structural features to human MT-ND4L, including multiple transmembrane domains that anchor it within the inner mitochondrial membrane. The high conservation of sequence and structure between species reflects the evolutionary importance of this protein in maintaining proper mitochondrial function. Comparative sequence analysis reveals specific regions of higher conservation, particularly in transmembrane domains and functional motifs involved in electron transport.

What are the key differences between mitochondrially encoded versus nuclear-encoded complex I subunits?

While MT-ND4L is typically encoded in mitochondrial DNA in mammals including Macaca mulatta, there are fundamental differences between mitochondrially encoded and nuclear-encoded complex I subunits:

In organisms like Chlamydomonas reinhardtii, genes for subunits like ND3 and ND4L have been transferred to the nuclear genome, requiring special features to facilitate their expression and proper import of the proteins into mitochondria . This evolutionary process demonstrates the dynamic nature of mitochondrial and nuclear genome interactions throughout eukaryotic evolution .

What are the optimal conditions for handling and storing recombinant MT-ND4L protein?

Optimal handling and storage of recombinant Macaca mulatta MT-ND4L requires careful attention to protein stability conditions:

• Storage temperature: Store at -20°C for regular use, or -80°C for extended storage to prevent degradation .

• Buffer composition: Tris-based buffers with 50% glycerol provide optimal stability for MT-ND4L proteins. The addition of glycerol prevents freeze-thaw damage and maintains protein structure .

• Aliquoting strategy: Prepare small working aliquots to avoid repeated freeze-thaw cycles, as these significantly decrease protein activity. Working aliquots can be stored at 4°C for up to one week .

• Reconstitution: When using lyophilized preparations, reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL, then add glycerol to a final concentration of 30-50% for long-term storage .

• Handling precautions: Centrifuge vials briefly before opening to collect contents at the bottom, and minimize exposure to light and oxidizing conditions during experiments .

These conditions optimize protein stability while maintaining functional integrity for experimental applications, which is particularly important given the hydrophobic nature of this transmembrane protein.

How can researchers verify the functional activity of recombinant MT-ND4L in experimental systems?

Verifying the functional activity of recombinant MT-ND4L requires multiple complementary approaches:

• Complex I activity assays: Measure NADH:ubiquinone oxidoreductase activity using spectrophotometric methods that monitor NADH oxidation at 340 nm in the presence of ubiquinone analogs like coenzyme Q1 or decylubiquinone. Inhibitor sensitivity (rotenone sensitivity) provides confirmation of specific complex I activity .

• Membrane integration verification: Confirm proper incorporation of MT-ND4L into membrane structures using techniques such as:

  • Carbonate extraction followed by ultracentrifugation

  • Protease protection assays to assess membrane topology

  • Blue native PAGE combined with immunoblotting to verify incorporation into the complete complex I structure

• Electron microscopy: Negative staining and immunogold labeling can visualize the integration of recombinant MT-ND4L into reconstituted membrane systems or isolated mitochondria.

• Complementation studies: In systems where endogenous MT-ND4L is depleted or mutated, introduction of functional recombinant protein should restore complex I assembly and activity. Absence of ND4L prevents assembly of the 950-kDa whole complex I and eliminates enzyme activity .

• Protein-protein interaction studies: Crosslinking experiments or co-immunoprecipitation with other complex I subunits can confirm proper interactions with partner proteins.

The combination of these approaches provides robust verification of functional integrity beyond simple expression confirmation.

What are the key methodological considerations when using recombinant MT-ND4L in mitochondrial complex I reconstitution experiments?

Reconstitution of functional mitochondrial complex I using recombinant components requires addressing several methodological challenges:

• Protein expression system selection:

  • Bacterial expression systems (E. coli) offer high yield but may lack proper folding for membrane proteins

  • Eukaryotic systems (insect cells, yeast) provide better folding but lower yields

  • Cell-free systems allow incorporation of unnatural amino acids for mechanistic studies

• Membrane mimetic environment:

  • Detergent selection is critical (typically mild detergents like DDM, digitonin)

  • Lipid composition affects complex I stability and activity

  • Nanodiscs or liposomes may provide more native-like membrane environments

• Assembly process:

  • Sequential addition of subunits or subcomplexes improves success rates

  • Temperature, pH, and ionic strength must be carefully controlled

  • Chaperones may be required to facilitate proper assembly

• Verification of complete assembly:

  • Blue native PAGE combined with in-gel activity assays

  • Cryo-EM to verify structural integrity

  • Mass spectrometry to confirm subunit stoichiometry

• Activity measurements:

  • NADH oxidation (340 nm) coupled to artificial electron acceptors

  • Membrane potential measurements using voltage-sensitive dyes

  • Proton pumping assays using pH-sensitive probes

Assembly is particularly challenging because absence of ND4L prevents the formation of the entire 950-kDa complex I and eliminates enzyme activity . Researchers must verify both structural assembly and functional activity to confirm successful reconstitution.

How can researchers use recombinant MT-ND4L to investigate disease-associated mutations?

Investigating disease-associated mutations in MT-ND4L requires sophisticated experimental approaches:

• Site-directed mutagenesis strategy:

  • Create precisely defined mutations corresponding to disease variants (e.g., T10663C/Val65Ala mutation associated with Leber hereditary optic neuropathy)

  • Include appropriate controls (wild-type, known pathogenic, and benign variants)

  • Design experiments to include species-specific variations when using Macaca mulatta MT-ND4L as a model

• Functional characterization methods:

  • Electron transport chain kinetic analysis (Vmax, Km) for NADH oxidation

  • ROS production measurements using fluorescent probes (MitoSOX, DCF-DA)

  • Oxygen consumption rate analysis using respirometry

  • Membrane potential measurements using potentiometric dyes (TMRM, JC-1)

  • ATP synthesis capacity in reconstituted systems or cell models

• Structural impact assessment:

  • Hydrogen-deuterium exchange mass spectrometry to detect conformational changes

  • Thermal shift assays to evaluate protein stability changes

  • Molecular dynamics simulations informed by experimental data

  • Potential cryo-EM analysis of mutant complex structures

• Cellular models for validation:

  • Cybrids (cells with mitochondria containing the mutation of interest)

  • CRISPR-engineered cell lines with precise mutations

  • Primary cells from disease models or patient samples

  • iPSC-derived cell types relevant to affected tissues

These approaches allow researchers to establish clear mechanistic links between specific MT-ND4L mutations and disease phenotypes, providing insights into pathophysiology and potential therapeutic approaches for mitochondrial disorders like Leber hereditary optic neuropathy .

What experimental approaches can resolve contradictory findings regarding MT-ND4L's role in supercomplex formation?

Resolving contradictory findings about MT-ND4L's role in respiratory supercomplex formation requires multifaceted experimental strategies:

• Improved isolation techniques:

  • Use digitonin solubilization for gentler extraction preserving supercomplexes

  • Apply gradient purification to separate distinct supercomplex populations

  • Develop MT-ND4L-specific affinity tags that don't disrupt interactions

• Advanced structural biology approaches:

  • Cryo-EM analysis of supercomplexes with wild-type versus mutant/absent MT-ND4L

  • Crosslinking mass spectrometry to map protein-protein interaction interfaces

  • FRET-based proximity analysis between fluorescently labeled subunits

• Dynamic assembly monitoring:

  • Pulse-chase labeling to track temporal assembly of complexes

  • Inducible expression systems to control timing of MT-ND4L availability

  • Live-cell imaging with tagged components to visualize assembly process

• Functional supercomplex analysis:

  • Substrate channeling efficiency measurements

  • ROS production in intact versus disrupted supercomplexes

  • Electron flux kinetics through different respiratory chain segments

• Comparison across model systems:

  • Parallel studies in different species (human, Macaca mulatta, mouse)

  • Analysis in different tissues with varying supercomplex composition

  • Evaluation under different metabolic conditions (glycolytic vs. oxidative)

These approaches can help distinguish direct versus indirect effects of MT-ND4L on supercomplex formation and stability, resolving apparent contradictions in the literature. The finding that absence of ND4L prevents assembly of the 950-kDa whole complex I suggests its critical role in the structural foundation necessary for supercomplex formation.

How does post-translational modification affect MT-ND4L function and complex I assembly?

Post-translational modifications (PTMs) of MT-ND4L represent an emerging area of research with significant implications for complex I function:

To investigate these modifications:

• Site-directed mutagenesis to create non-modifiable variants (e.g., phosphomimetic or phosphodeficient)

• Mass spectrometry-based quantification of modification stoichiometry under different conditions

• In vitro enzyme assays with reconstituted complex I containing modified or unmodified MT-ND4L

• Identification of the responsible enzymes (kinases, acetyltransferases, etc.) through screening approaches

• Development of modification-specific antibodies or biosensors to track dynamic changes

PTM research on MT-ND4L is complicated by its membrane-embedded nature and relatively small size, requiring specialized techniques for enrichment and analysis. Understanding these modifications is critical as they may represent an important regulatory layer affecting complex I assembly, activity, and response to cellular stress conditions.

How has MT-ND4L evolved across species and what implications does this have for using Macaca mulatta as a model system?

The evolutionary conservation and divergence of MT-ND4L across species has important implications for research using Macaca mulatta as a model:

• Evolutionary pattern analysis:

  • MT-ND4L is among the most conserved mitochondrial proteins, reflecting its essential role in electron transport

  • Sequence conservation is highest in functional domains directly involved in electron transfer

  • Transmembrane domains show higher conservation than loop regions

  • Selection pressure analysis reveals negative/purifying selection predominates

• Comparative sequence alignment data:

• Genome location implications:

  • Unlike most mammals where MT-ND4L is mitochondrially encoded, in some species like Chlamydomonas reinhardtii, the gene has transferred to the nuclear genome

  • This transfer required adaptation of the protein for import into mitochondria

  • Such evolutionary events demonstrate the dynamic nature of mitochondrial genetics

• Macaca mulatta advantages and limitations:

  • High sequence similarity to humans makes it an excellent mammalian model

  • Conserved complex I structure and assembly process

  • Similar disease-associated mutations and phenotypes

  • Ability to perform controlled studies not possible in human subjects

  • Differences in regulatory elements and nuclear-encoded partners must be considered

The evolutionary conservation of MT-ND4L underscores its fundamental importance in mitochondrial function while providing valuable insights for translational research using non-human primates as models for human diseases.

What can researchers learn from comparing mitochondrially-encoded versus nuclear-encoded MT-ND4L in different species?

The comparison between mitochondrially-encoded and nuclear-encoded MT-ND4L across different species provides valuable insights into mitochondrial evolution and adaptation:

• Genomic transfer mechanisms:

  • In organisms like Chlamydomonas reinhardtii, MT-ND4L and ND3 genes have transferred to the nuclear genome

  • This transfer required acquisition of:
    a) Mitochondrial targeting sequences
    b) Adaptation to the standard genetic code (versus mitochondrial genetic code)
    c) Regulatory elements for nuclear gene expression

  • Molecular mechanisms for these transfers include RNA intermediates and genomic DNA fragments

• Functional adaptations:

  • Nuclear-encoded MT-ND4L typically requires post-translational import into mitochondria

  • Protein modifications may differ between nuclear and mitochondrially encoded versions

  • Nuclear control may allow more sophisticated regulation of expression

  • Co-evolution with import machinery components

• Experimental advantages:

  • Nuclear-encoded versions are more amenable to genetic manipulation

  • Expression systems can be optimized for higher yield

  • Fusion proteins and tags are more easily incorporated

  • Synthetic biology approaches benefit from nuclear encoding

• Comparative analysis findings:

  • Despite genomic relocation, the core function remains conserved

  • Complex I assembly depends on MT-ND4L regardless of genetic origin

  • Nuclear transfer may confer advantages in certain environmental conditions

  • Transfer events demonstrate ongoing evolutionary adaptation of mitochondria

These comparisons reveal fundamental principles about the co-evolution of mitochondrial and nuclear genomes, the plasticity of gene localization, and the essential constraints on respiratory chain components. The observation that "NUO3 and NUO11 [nuclear genes for ND3 and ND4L] display features that facilitate their expression and allow the proper import of the corresponding proteins into mitochondria" demonstrates the sophisticated adaptations required for functional maintenance after gene transfer.

How do mutations in MT-ND4L contribute to Leber hereditary optic neuropathy and other mitochondrial disorders?

Mutations in MT-ND4L have been implicated in several mitochondrial disorders, most notably Leber hereditary optic neuropathy (LHON):

• LHON-associated mutations and mechanisms:

  • The T10663C (Val65Ala) mutation in MT-ND4L has been identified in several families with LHON

  • This mutation changes valine to alanine at position 65 in the protein

  • Mechanistic consequences include:
    a) Altered complex I assembly efficiency
    b) Decreased NADH:ubiquinone oxidoreductase activity
    c) Increased reactive oxygen species (ROS) production
    d) Compromised ATP synthesis in affected tissues
    e) Specific vulnerability of retinal ganglion cells

• Pathophysiological cascade:

  • Primary biochemical defects:

    • Decreased complex I activity (typically 30-50% reduction)

    • Increased electron leakage leading to superoxide formation

    • Impaired proton pumping across inner mitochondrial membrane

  • Cellular consequences:

    • Energy deficit in high-energy demanding cells

    • Oxidative damage to proteins, lipids, and mtDNA

    • Impaired calcium homeostasis

    • Activation of apoptotic pathways

  • Tissue manifestations:

    • Selective degeneration of retinal ganglion cells

    • Optic nerve atrophy

    • Visual field defects and central vision loss

• Heteroplasmy and threshold effects:

  • MT-ND4L mutations typically show variable disease penetrance

  • Heteroplasmy levels (percentage of mutated mtDNA) correlate with disease severity

  • Tissue-specific thresholds determine clinical manifestations

  • Gender bias exists with higher male penetrance through mechanisms not fully understood

• Research approaches using recombinant MT-ND4L:

  • Creation of mutation-specific variants to study functional impacts

  • Cybrid cell models incorporating patient-derived mitochondria

  • Transgenic animal models expressing mutated MT-ND4L

  • High-throughput screening for compounds that rescue mutant phenotypes

Understanding these mechanisms provides a foundation for developing targeted therapies for LHON and related mitochondrial disorders caused by complex I dysfunction.

What therapeutic strategies targeting MT-ND4L are being developed for mitochondrial diseases?

Emerging therapeutic strategies targeting MT-ND4L and complex I-related mitochondrial diseases include:

• Gene therapy approaches:

  • Allotopic expression: Nuclear delivery of modified MT-ND4L genes with mitochondrial targeting sequences

  • RNA import: Engineered RNA molecules that can enter mitochondria and compensate for mutated mtDNA

  • Mitochondrially targeted nucleases to shift heteroplasmy (e.g., TALENs, zinc-finger nucleases)

  • Challenges include efficient mitochondrial targeting and ensuring proper incorporation into complex I

• Small molecule therapeutics:

  • Bypass therapies: Alternative electron carriers that can bypass complex I (e.g., idebenone)

  • Enhancers of mitochondrial biogenesis (e.g., AMPK activators, PGC-1α inducers)

  • ROS scavengers and mitochondrially targeted antioxidants (e.g., MitoQ, SS-31)

  • Compounds that stabilize MT-ND4L incorporation into complex I

• Mitochondrial replacement therapy:

  • Maternal spindle transfer or pronuclear transfer to prevent transmission

  • Mitochondrial augmentation therapy using healthy donor mitochondria

  • Ethical and regulatory considerations remain significant barriers

• Metabolic manipulation strategies:

  • Ketogenic diets to provide alternative energy substrates

  • Supplementation with nutrients that support mitochondrial function

  • Modulation of NAD+ levels to optimize remaining complex I function

  • Shifting metabolism to favor glycolysis in affected tissues

• Research tools enabling therapy development:

  • High-throughput screening platforms using MT-ND4L mutations

  • Patient-derived iPSCs differentiated to affected cell types

  • Organoid models that recapitulate tissue-specific pathology

  • CRISPR-based approaches for precise genetic manipulation

These therapeutic strategies are at various stages of development, from preclinical testing to early clinical trials. The complex nature of mitochondrial diseases suggests that combination therapies targeting multiple aspects of the disease process may ultimately prove most effective.

How can researchers use recombinant MT-ND4L to screen for compounds that may rescue complex I deficiency?

Screening for therapeutic compounds using recombinant MT-ND4L provides a powerful approach for identifying potential treatments for complex I deficiencies:

• In vitro screening platforms:

  • Reconstituted complex I systems with wild-type or mutant MT-ND4L

  • Membrane vesicles containing assembled complex I

  • Purified complex I preparations in various detergent or membrane mimetic systems

  • Measurements include NADH oxidation, ubiquinone reduction, and ROS production

• Cell-based screening approaches:

  • Patient-derived or CRISPR-engineered cells with MT-ND4L mutations

  • Cybrid cells containing patient-derived mitochondria

  • Conditional expression systems for controlled MT-ND4L variant testing

  • Readouts include ATP production, oxygen consumption, membrane potential, and cell viability

• Advanced screening methodologies:

• Compound categories for screening:

  • Complex I stabilizers/assembly enhancers

  • ROS modulators and antioxidants

  • Metabolic modifiers and alternative electron carriers

  • Mitochondrial biogenesis inducers

  • Inhibitors of mitophagy/mitochondrial degradation

• Translation to advanced models:

  • Validation in organoid systems

  • Testing in animal models of complex I deficiency

  • Structure-activity relationship studies for lead optimization

  • Assessment of compound effects on MT-ND4L stability and incorporation

These screening approaches enable researchers to identify compounds that specifically rescue the functional defects caused by MT-ND4L mutations or deficiencies, potentially leading to targeted therapies for complex I-related mitochondrial diseases.

What are the most promising techniques for studying the dynamic assembly of MT-ND4L into complex I?

Cutting-edge techniques for investigating MT-ND4L assembly into complex I offer new insights into this critical process:

• Time-resolved cryo-electron microscopy:

  • Capture assembly intermediates at different stages

  • Visualize MT-ND4L integration into the growing complex

  • Identify conformational changes during assembly

  • Challenges include sample heterogeneity and low abundance of intermediates

• Advanced fluorescence techniques:

  • Single-molecule FRET to measure distances between subunits during assembly

  • Fluorescence correlation spectroscopy to track diffusion coefficients as complexes grow

  • Super-resolution microscopy to visualize assembly sites within mitochondria

  • Fluorescence recovery after photobleaching (FRAP) to measure assembly kinetics

• Mass spectrometry-based approaches:

  • Cross-linking mass spectrometry to map interaction interfaces

  • Native mass spectrometry to determine composition of assembly intermediates

  • Pulse-SILAC to track newly synthesized components incorporating into complexes

  • Hydrogen-deuterium exchange to monitor structural changes during assembly

• Genetic approaches:

  • Inducible expression systems to trigger assembly in synchronized fashion

  • CRISPR interference for temporal control of assembly factor expression

  • Split-protein complementation assays to detect specific assembly steps

  • Ribosome profiling to measure translation coordination of complex components

• Computational and modeling approaches:

  • Molecular dynamics simulations of assembly processes

  • Kinetic modeling of assembly pathways

  • Network analysis of assembly factor interactions

  • Machine learning for predicting assembly defects from sequence variations

These techniques offer complementary information about the assembly process, which is particularly important since research shows that the absence of ND4L prevents assembly of the complete 950-kDa complex I and eliminates enzyme activity . Understanding the precise role of MT-ND4L in assembly will provide insights into both fundamental mitochondrial biology and disease mechanisms.

How might single-cell approaches reveal new insights about MT-ND4L function in heterogeneous tissues?

Single-cell approaches offer revolutionary potential for understanding MT-ND4L function in complex tissues:

• Single-cell transcriptomics applications:

  • Mapping nuclear-encoded assembly factors across cell types

  • Identifying compensatory responses to MT-ND4L dysfunction

  • Discovering cell type-specific vulnerability to complex I defects

  • Correlating MT-ND4L expression with mitochondrial content and function

• Single-cell proteomics approaches:

  • Quantifying MT-ND4L abundance across individual cells

  • Measuring heteroplasmy levels in individual cells

  • Detecting cell-specific post-translational modifications

  • Correlating protein levels with functional parameters

• Functional single-cell analysis:

  • Microfluidic platforms for measuring oxygen consumption in single cells

  • Single-cell imaging of membrane potential, ROS, and ATP levels

  • Patch-clamp techniques for mitochondrial electrophysiology

  • Metabolic flux analysis at single-cell resolution

• Spatial techniques for tissue context:

  • Spatial transcriptomics to map nuclear responses to MT-ND4L dysfunction

  • Highly multiplexed imaging to correlate MT-ND4L with multiple functional markers

  • Serial block-face electron microscopy for 3D ultrastructure of affected mitochondria

  • MERFISH or seqFISH for spatial mapping of transcriptional responses

• Integration approaches:

  • Combined single-cell multi-omics (e.g., RNA + protein + metabolites)

  • Trajectory analysis to map progression of dysfunction

  • Computational integration of spatial and functional data

  • Network analysis to identify cell type-specific vulnerabilities

These approaches are particularly valuable for understanding diseases like Leber hereditary optic neuropathy, where specific cell types (retinal ganglion cells) show selective vulnerability despite the MT-ND4L mutation being present in all cells. Single-cell technologies can reveal the basis for this differential susceptibility and identify potential protective mechanisms in resistant cell populations.

What are the implications of MT-ND4L research for developing mitochondrial medicine approaches?

The expanding understanding of MT-ND4L has significant implications for the emerging field of mitochondrial medicine:

• Precision medicine applications:

  • Mutation-specific therapies based on structural understanding

  • Patient stratification based on complex I assembly defects

  • Biomarker development to track disease progression and treatment response

  • Pharmacogenomic approaches for personalized treatment selection

• Drug development frameworks:

  • Structure-based drug design targeting MT-ND4L interaction surfaces

  • Allosteric modulators to stabilize mutant proteins

  • Compounds that enhance incorporation of recombinant MT-ND4L into complex I

  • Screening cascades that progress from biochemical to advanced cellular models

• Advanced therapeutic approaches:

  • Mitochondrially-targeted protein replacement strategies

  • Engineered complex I variants with improved stability or function

  • RNA-based therapeutics to modulate MT-ND4L expression or processing

  • Nanoparticle delivery systems for mitochondrial targeting

• Bioengineering approaches:

  • Synthetic biology platforms to produce optimized MT-ND4L variants

  • Cell-free systems for rapid testing of therapeutic molecules

  • Engineered mitochondrial delivery vectors

  • Organelle transplantation technologies

• Translational research priorities:

  • Development of clinically relevant biomarkers for complex I function

  • Establishment of model systems that accurately reflect human pathophysiology

  • Optimization of tissue-specific delivery systems for mitochondrially-targeted therapies

  • Clinical trial designs appropriate for mitochondrial disorders

These research directions collectively advance the field toward effective treatments for previously untreatable mitochondrial disorders. Understanding the critical role of MT-ND4L in complex I assembly and function provides a foundation for rational therapeutic design, while studies across species offer insights into evolutionary adaptations that might be leveraged for therapeutic purposes . As research progresses, mitochondrial medicine is moving from symptomatic management toward disease-modifying treatments that address the fundamental molecular defects.