Recombinant Oncorhynchus masou NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is the functional role of MT-ND4L in mitochondrial energy production?

The MT-ND4L gene provides instructions for making the NADH dehydrogenase 4L protein, a critical component of Complex I in the mitochondrial electron transport chain. This protein functions within a larger enzyme complex that initiates oxidative phosphorylation, the process by which mitochondria generate cellular energy . Within Complex I, MT-ND4L contributes to the transfer of electrons from NADH to ubiquinone, which represents the first step in the electron transport process .

The electron transfer facilitated by MT-ND4L and other Complex I components creates an electrochemical gradient across the inner mitochondrial membrane. This unequal distribution of electrical charge drives ATP synthesis, producing the primary energy currency of cells . The immediate electron acceptor for the enzyme is believed to be ubiquinone, making this interaction crucial for proper energy metabolism .

Research methodologies to investigate MT-ND4L function include:

• Spectrophotometric assays measuring NADH oxidation rates

• Membrane potential assessments using fluorescent probes

• Oxygen consumption measurements in isolated mitochondria

• Electron microscopy to analyze Complex I structural organization

How is MT-ND4L implicated in metabolic regulation and disease processes?

Changes in MT-ND4L gene expression have long-term consequences on energy metabolism and may represent a major predisposition factor for various conditions . Genome-wide association studies have revealed that a significant percentage (approximately 15%) of mitochondrial single nucleotide variants associated with metabolic alterations are located in the MT-ND4L gene . These variants are predominantly associated with metabolites from the glycerophospholipid class, suggesting an important interconnection between mitochondrial function and cellular metabolism .

In human medicine, mutations in MT-ND4L have been identified in several families with Leber hereditary optic neuropathy. One well-characterized mutation (T10663C or Val65Ala) changes a single amino acid in the NADH dehydrogenase 4L protein, although the precise mechanism linking this change to vision loss remains under investigation .

For experimental investigation of MT-ND4L variants:

• Use metabolomics approaches to measure changes in phosphatidylcholine ratios

• Apply CRISPR-based mitochondrial gene editing where feasible

• Develop cellular models with different MT-ND4L variants

• Employ transmission electron microscopy to assess mitochondrial ultrastructure

What sequencing approaches are recommended for studying MT-ND4L variants in Oncorhynchus masou?

For comprehensive analysis of MT-ND4L variants in Oncorhynchus masou, researchers should implement a multi-faceted sequencing strategy:

• Complete mitochondrial genome sequencing: This provides the broader genomic context for MT-ND4L, enabling identification of potential recombination events and evolutionary patterns. Next-generation sequencing technologies or approaches similar to the 454 method used in other mitochondrial studies are recommended .

• Population-level sampling: Collect samples across the geographic range of Oncorhynchus masou, ensuring representation of different ecological habitats to capture environmental adaptations.

• Bioinformatic analysis pipeline:

  • Sequence alignment using MUSCLE or MAFFT v.7 software

  • Polymorphism and divergence analysis using DnaSP v.6, PROSEQ v.2.9, and MEGA v.7

  • Phylogenetic reconstruction using maximum-likelihood methods in IQ-TREE v.2

  • Model selection based on Akaike Information Criterion (AIC) and Bayesian information criterion (BIC)

• Validation strategies:

  • Sanger sequencing to confirm key variants

  • Long-read sequencing to resolve complex structural variations

  • Digital PCR for quantitative assessment of heteroplasmy

This comprehensive approach enables researchers to identify both common and rare variants while understanding their population distribution and potential functional significance.

What expression systems are optimal for producing functional recombinant MT-ND4L protein?

Expressing functional recombinant MT-ND4L presents significant challenges due to its hydrophobic nature and involvement in a multi-protein complex. Successful expression requires careful consideration of the following methodological approaches:

• Codon optimization: Adapt the MT-ND4L coding sequence for expression in the chosen host system, considering the divergent codon usage between fish mitochondrial genomes and standard expression hosts.

• Expression system selection:

• Purification strategy:

  • Detergent screening to identify optimal solubilization conditions

  • Affinity chromatography using fusion tags

  • Size exclusion chromatography to separate aggregates

  • Reconstitution into nanodiscs or liposomes for functional studies

• Validation approaches:

  • Western blotting with validated antibodies tested on tissues known to express MT-ND4L positively and negatively

  • Mass spectrometry to confirm protein identity

  • Circular dichroism to assess secondary structure

  • Activity assays measuring electron transfer

The choice of expression system should be guided by the specific research questions, with bacterial systems suitable for structural studies and eukaryotic systems preferred for functional analyses.

How can researchers effectively analyze evolutionary selection patterns in MT-ND4L across salmonid species?

Analysis of selection patterns in MT-ND4L requires robust computational and statistical approaches. Based on studies of related mitochondrial genes in Atlantic salmon, researchers should implement:

• Multiple detection methods for selection:

  • MEME (Mixed Effects Model of Evolution) for detecting episodic positive selection

  • FUBAR (Fast Unconstrained Bayesian Approximation) for identifying sites under both positive and purifying selection

  • TreeSAAP for detecting selection based on physicochemical properties of amino acids

• Comparative analysis workflow:

  • Sequence MT-ND4L from Oncorhynchus masou and other salmonid species

  • Create multi-species alignments to identify variable sites

  • Apply at least two independent selection detection methods

  • Analyze the chemical properties of amino acid substitutions

  • Map selected sites onto protein structural models

• Environmental correlation analysis:

  • Examine geographical distribution of selected mutations

  • Test for associations with environmental variables (temperature, salinity, altitude)

  • Focus on adaptive patterns similar to those observed in Atlantic salmon, where some mutations were private to arctic populations

Evidence from Atlantic salmon indicates positive selection at multiple mitochondrial genes involved in the electron transport chain, including ND1, ND3, and ND4 genes , suggesting similar patterns may be present in the related MT-ND4L gene of Oncorhynchus masou.

What evidence exists for mitochondrial recombination involving MT-ND4L and how should researchers investigate this in Oncorhynchus masou?

Mitochondrial recombination, once considered rare, has been documented in several fish species, with the ND4L-ND4 gene region identified as a potential recombination hotspot . Investigating recombination in Oncorhynchus masou requires:

• Detection methodology:

  • Sliding window analysis to reveal non-uniform distribution of intraspecific differences

  • Pairwise homoplasy index (PHI) test to identify statistically significant recombination signals

  • Multiple recombination detection algorithms implemented in RDP4 software

• Sequence analysis approach:

  • Complete mitochondrial genome sequencing from multiple individuals

  • Comparison with closely related species to identify potential donor sequences

  • Focus on regions showing pronounced peaks of divergence, which often center at gene boundaries like ND4L-ND4

• Characterization of recombinant patterns:

  • Map precise recombination breakpoints

  • Determine if recombinant fragments are fixed in populations

  • Assess whether different mitochondrial genomes show mosaic patterns with varying numbers of recombinant events

Recent studies have shown that recombinant fragments in fish mitochondrial genomes can show high similarity (99-100%) to related species, indicating recent interspecific hybridization events . This finding suggests researchers should carefully examine Oncorhynchus masou populations for evidence of hybridization with other salmonid species, particularly in regions where ranges overlap.

How can metabolomic approaches enhance understanding of MT-ND4L function in Oncorhynchus masou?

Metabolomics provides powerful insights into the functional consequences of MT-ND4L variants and expression changes. Research has revealed significant associations between MT-ND4L variants and specific metabolite profiles:

• Key metabolomic associations:

  • MT-ND4L variants show strong associations with glycerophospholipid class metabolites

  • A large number of significant metabolite ratios involve phosphatidylcholine (PC) species in relation to MT-ND4L variants

  • These associations indicate an important interconnection between mitochondrial function and broader cellular metabolism

• Recommended analytical approach:

  • Targeted lipidomics focusing on phosphatidylcholine species

  • Untargeted metabolomics to discover novel metabolic signatures

  • Isotope tracing to track metabolic flux through pathways linked to mitochondrial function

  • Integration with transcriptomic and proteomic data

• Experimental design considerations:

  • Compare tissues from individuals with different MT-ND4L variants

  • Include environmental variables (temperature, oxygen levels) in experimental design

  • Establish metabolite ratio normalization protocols to enhance sensitivity

  • Develop tissue-specific metabolomic profiles relevant to energy demands

Understanding these metabolic signatures may have implications beyond basic research, as some of the significant metabolites associated with mitochondrial variants have been previously related to complex diseases, including neurological disorders and metabolic conditions .

What strategies should researchers employ to study the impact of temperature adaptation on MT-ND4L function in cold-water species like Oncorhynchus masou?

Cold-water species like Oncorhynchus masou likely exhibit specialized adaptations in mitochondrial function to maintain energy production efficiency at lower temperatures. Research strategies should include:

• Comparative functional analysis:

  • Measure MT-ND4L-containing Complex I activity across a temperature range

  • Compare enzymatic parameters (Km, Vmax) between populations from different thermal environments

  • Assess whether specific MT-ND4L variants correlate with thermal habitat

• Molecular evolution approach:

  • Identify amino acid substitutions in MT-ND4L that correlate with thermal habitat

  • Pay special attention to MT-ND4L variants that show geographical patterns similar to those observed in Atlantic salmon, where some mutations were private to arctic populations

  • Apply selection analysis methods to identify thermal adaptation signatures

• Experimental protocols:

  • Temperature-controlled enzyme assays of isolated mitochondria

  • Oxygen consumption measurements at different temperatures

  • Membrane fluidity assessments using fluorescence anisotropy

  • Expression of recombinant variants at different temperatures to assess stability

• Biophysical characterization:

  • Circular dichroism spectroscopy to assess protein stability across temperatures

  • Differential scanning calorimetry to determine thermal transition points

  • Molecular dynamics simulations to predict conformational changes at different temperatures

This multifaceted approach will help determine whether MT-ND4L variants in Oncorhynchus masou represent adaptations to specific thermal environments, providing insights into how this species might respond to changing water temperatures in different habitats.

How might research on Oncorhynchus masou MT-ND4L contribute to conservation genetics and fisheries management?

Understanding the genetic diversity and functional significance of MT-ND4L in Oncorhynchus masou has important implications for conservation and management:

• Population structure assessment:

  • MT-ND4L variants can serve as genetic markers for population differentiation

  • Mitochondrial genome analysis can help identify evolutionarily significant units

  • Recombination patterns may indicate hybridization events with implications for species integrity

• Adaptive potential evaluation:

  • Functional variants in MT-ND4L may represent adaptations to specific environmental conditions

  • Population-specific variants could indicate local adaptation that should be preserved

  • Diversity in energy metabolism genes may contribute to resilience against environmental change

• Methodological approach:

  • Develop population screening tools targeting key MT-ND4L variants

  • Combine genetic data with physiological assessments of metabolic function

  • Integrate findings into species management plans through stakeholder engagement

• Conservation applications:

  • Design stocking programs that maintain mitochondrial genetic diversity

  • Identify populations with unique adaptive variants for prioritized protection

  • Predict vulnerability to climate change based on metabolic adaptations

This research should be helpful to better understand the evolutionary status and population genetic diversity of Oncorhynchus masou, contributing to evidence-based management strategies .

What are the most promising future research directions for understanding MT-ND4L function in salmonid mitochondria?

Emerging technologies and integrative approaches offer exciting opportunities for advancing MT-ND4L research:

• Cutting-edge methodological approaches:

  • CRISPR-based mitochondrial genome editing for precise functional studies

  • Single-cell transcriptomics to examine cell-type specific expression patterns

  • Cryo-electron microscopy for high-resolution structural analysis of Complex I

  • Metabolic flux analysis to quantify the impact of variants on energy production

• Integrative research frameworks:

  • Combine genomics, transcriptomics, proteomics, and metabolomics

  • Link molecular findings to whole-organism physiology

  • Develop computational models of mitochondrial function incorporating MT-ND4L variants

  • Explore environmental interactions through controlled exposure studies

• Comparative evolutionary studies:

  • Expand analyses across diverse salmonid species and populations

  • Investigate convergent evolution in mitochondrial genes across cold-adapted species

  • Study the co-evolution of nuclear and mitochondrial genes encoding Complex I components

• Applied research directions:

  • Develop biomarkers based on MT-ND4L variants for monitoring population health

  • Investigate connections between mitochondrial function and climate resilience

  • Explore potential links between MT-ND4L variants and production traits in aquaculture

Understanding the connections between MT-ND4L variants, metabolic phenotypes, and environmental adaptation will contribute significantly to both fundamental knowledge of salmonid biology and practical applications in conservation and aquaculture.