Recombinant Sarcophyton glaucum NADH-ubiquinone oxidoreductase chain 4L (ND4L)

What is Sarcophyton glaucum NADH-ubiquinone oxidoreductase chain 4L (ND4L)?

Sarcophyton glaucum NADH-ubiquinone oxidoreductase chain 4L (ND4L) is a mitochondrial protein encoded by the mitochondrial genome of Sarcophyton glaucum, commonly known as toadstool umbrella leather coral. It functions as a subunit of NADH dehydrogenase (ubiquinone), also known as Complex I, which is a crucial component of the electron transport chain located in the mitochondrial inner membrane . ND4L is characterized as one of the most hydrophobic subunits forming the core of the transmembrane region of Complex I . In octocorals like Sarcophyton glaucum, the mitochondrial genome contains several unique features compared to other metazoans, including specific gene arrangements and additional genes involved in DNA repair mechanisms .

How does the structure of Sarcophyton glaucum ND4L compare to its human counterpart?

While the human MT-ND4L gene produces an 11 kDa protein composed of 98 amino acids and is located in human mitochondrial DNA from base pair 10,469 to 10,765 , the Sarcophyton glaucum ND4L differs in several significant aspects. The octocoral mitochondrial genome has unique structural characteristics that distinguish it from other metazoans . Both proteins serve similar functions as components of Complex I in the respiratory chain but have evolved differently, reflecting their divergent evolutionary histories. The octocoral mitochondrial genome contains the standard anthozoan complement of oxidative metabolic genes but also includes additional genetic elements not found in human mitochondria, such as the mtMutS gene involved in DNA repair . This structural difference suggests potential functional adaptations specific to the octocoral lineage.

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

ND4L serves as a critical subunit in the respiratory chain Complex I, which is essential for cellular energy production . As part of the NADH dehydrogenase complex, it participates in the transfer of electrons from NADH to ubiquinone, contributing to the generation of a proton gradient across the mitochondrial inner membrane. This electrochemical gradient drives ATP synthesis through oxidative phosphorylation. In octocorals like Sarcophyton glaucum, ND4L contributes to the core assembly of proteins required for the functional integrity of Complex I . The hydrophobic nature of ND4L allows it to be embedded within the membrane domain of the complex, facilitating electron transport and proton translocation processes essential for energy conversion in coral mitochondria.

What unique genetic features characterize the mitochondrial genome of Sarcophyton glaucum?

The mitochondrial genome of Sarcophyton glaucum, like other octocorals, possesses several distinctive characteristics that set it apart from typical metazoan mitochondrial DNA. Most notably, it contains not only the standard complement of anthozoan oxidative metabolic genes (13 protein-coding genes, two rRNAs, and a single tRNA) but also includes an unexpected open reading frame (ORF) that occupies almost 16% of the mtDNA (2949/18453 bp) . This novel gene, termed mtMutS, has not been found in any mitochondrial genome outside of the Octocorallia and appears to function in DNA mismatch repair, showing similarities to the yeast nuclear Mutation Suppressor Homolog 1 (MSH1) gene . The presence of this unique gene suggests specialized DNA repair mechanisms in octocoral mitochondria that may contribute to the group's evolutionary success and genetic stability.

How does the evolutionary history of ND4L in octocorals provide insights into horizontal gene transfer?

While ND4L itself is a conserved mitochondrial gene, the mitochondrial genome of octocorals like Sarcophyton glaucum contains unique features that offer compelling evidence of horizontal gene transfer (HGT). Phylogenetic analyses strongly support that the mtMutS gene found in octocoral mitochondria was acquired via HGT from either viral or bacterial origins . Research demonstrates that the closest relatives to octocoral mtMutS are MutS sequences from nucleocytoplasmic large DNA viruses (NCLDVs) and epsilonproteobacteria . Bayesian phylogenetic analysis showed that all octocoral mtMutS sequences form a monophyletic group with 100% posterior probability support, with the Heterocapsa circularisquama virus MutS sequence as the most probable sister group (though with only 62% support) . This evolutionary relationship provides a fascinating window into ancient genetic exchange events that have shaped the mitochondrial genome architecture in octocorals.

What does the overlapping gene structure between ND4L and ND4 suggest about mitochondrial genome evolution?

An intriguing feature observed in the human MT-ND4L gene—and potentially conserved in other species—is the 7-nucleotide overlap of its last three codons with the first three codons of the MT-ND4 gene . In humans, this overlap involves the sequence 5'-CAA TGC TAA-3' (coding for Gln, Cys, and Stop in ND4L) and 5'-ATG CTA AAA-3' (coding for Met-Leu-Lys in ND4) . This gene arrangement represents a remarkable instance of genomic economy and may reflect evolutionary pressure to minimize mitochondrial genome size while maintaining all necessary genetic information. While the search results don't explicitly confirm this same overlap in Sarcophyton glaucum, similar overlapping gene structures could exist, suggesting convergent evolutionary solutions to space constraints in compact mitochondrial genomes across diverse lineages.

What techniques are used to confirm the expression of mitochondrial genes like ND4L in octocorals?

Researchers employ several molecular techniques to verify that mitochondrial genes like ND4L are actively expressed in octocorals. PCR amplification of both genomic DNA (gDNA) and complementary DNA (cDNA) derived from RNA transcripts provides evidence of gene transcription. For instance, studies on octocoral mtMutS used PCR primers specific to different domains of the gene to amplify 687 bp DNA products from both gDNA and cDNA sources . Controls to eliminate genomic DNA contamination (cDNA preparations without reverse transcriptase) and foreign DNA contamination (no template added) are crucial to ensure results reflect genuine gene expression . Sequencing of PCR products followed by BLAST confirmation establishes the identity of the amplified fragments. These techniques collectively demonstrate that mitochondrial genes are transcribed and their mRNA is available for translation into potentially functional proteins in octocorals.

How can recombinant ND4L protein be properly prepared and stored for experimental use?

Recombinant Sarcophyton glaucum ND4L protein requires specific handling procedures to maintain its structural integrity and functional properties. The protein should be stored in a Tris-based buffer with 50% glycerol, optimized for protein stability . For short-term storage, -20°C is adequate, while extended storage requires either -20°C or -80°C to prevent degradation . When working with this recombinant protein, researchers should minimize freeze-thaw cycles, as these can compromise protein structure and activity. The production process typically determines the appropriate tag type for the recombinant protein , which should be considered when designing experiments to ensure the tag doesn't interfere with protein function or experimental readouts. Proper protein quantification methods, such as Bradford or BCA assays, should be employed to accurately determine protein concentration before use in experimental procedures.

What alignment and phylogenetic analysis methods are appropriate for studying ND4L evolutionary relationships?

For studying evolutionary relationships of ND4L and related mitochondrial genes, researchers employ sophisticated sequence alignment and phylogenetic analysis methods. Multiple sequence alignment can be performed using the E-INS-I method in MAFFT with appropriate scoring matrices such as BLOSUM45 . Manual adjustment in software like MacClade using pfam results ensures homologous domains are correctly matched . Conserved functional residues or motifs serve as anchors for alignment refinement.

For phylogenetic analysis, both Bayesian and maximum parsimony approaches have been successfully applied to mitochondrial genes . Trimming ambiguously aligned regions from amino acid alignments improves the reliability of phylogenetic inference. When analyzing domains with different evolutionary histories (like the HNH endonuclease domain in mtMutS), selective inclusion or exclusion of these regions may be necessary . These methodological approaches enable researchers to reconstruct evolutionary relationships and identify potential instances of horizontal gene transfer or other evolutionary phenomena affecting mitochondrial genes like ND4L.

What role might ND4L play in coral bleaching mechanisms and climate change resilience?

The function of ND4L in mitochondrial energy production suggests it could be a critical factor in coral resilience to environmental stressors associated with climate change. Coral bleaching—the loss of symbiotic algae under stress conditions—involves significant metabolic disruption and potentially altered mitochondrial function. Research could explore whether variants in ND4L sequence or expression correlate with bleaching susceptibility across different coral species or populations. Comparative studies between Sarcophyton glaucum and other coral species with different environmental tolerances could reveal whether specific ND4L characteristics contribute to stress resistance. Additionally, investigating how ND4L expression and activity change during bleaching events might provide insights into the cellular mechanisms underlying this phenomenon and potentially identify therapeutic targets for enhancing coral resilience to climate change impacts.

How can structural analysis of octocoral ND4L inform understanding of human mitochondrial diseases?

Variants in human MT-ND4L have been associated with conditions like increased BMI in adults and Leber's Hereditary Optic Neuropathy (LHON) . Comparative structural analysis between human and octocoral ND4L could provide valuable insights into the functional consequences of these mutations. While octocorals and humans are evolutionarily distant, the fundamental role of ND4L in Complex I suggests potential conservation of critical functional domains. Advanced structural biology techniques, including cryo-electron microscopy and computational modeling, could identify how specific residues contribute to protein stability and function. This cross-species comparison approach might reveal previously unrecognized functional regions in ND4L that could be targets for therapeutic interventions in mitochondrial disorders. Furthermore, the unique evolutionary history of octocoral mitochondrial genes might offer insights into alternative mechanisms for maintaining mitochondrial genome integrity that could inform human mitochondrial disease research.

What are the optimal expression systems for producing functional recombinant Sarcophyton glaucum ND4L?

The expression of functional recombinant Sarcophyton glaucum ND4L presents unique challenges due to its hydrophobic nature and mitochondrial origin. Bacterial expression systems like E. coli may struggle with proper folding of this membrane protein, potentially leading to inclusion body formation. Eukaryotic expression systems, including yeast (Saccharomyces cerevisiae or Pichia pastoris), insect cells (using baculovirus expression vectors), or mammalian cell lines, may provide better environments for correct folding and potential post-translational modifications. Cell-free protein synthesis represents another viable approach, particularly for hydrophobic membrane proteins, as it allows the incorporation of detergents or lipids during synthesis. For each expression system, optimization of codon usage for the host organism, selection of appropriate fusion tags (His, GST, MBP) to enhance solubility, and careful design of purification strategies are essential considerations. Validation of protein functionality through activity assays specific to NADH dehydrogenase would be necessary to confirm that the recombinant protein retains its native properties.

How can researchers effectively analyze the interaction between ND4L and other Complex I subunits?

Understanding the structural and functional interactions between ND4L and other subunits of Complex I requires sophisticated biochemical and biophysical approaches. Co-immunoprecipitation using antibodies against ND4L or other Complex I components can identify direct protein-protein interactions. Cross-linking mass spectrometry (XL-MS) offers higher resolution data on specific interaction sites between proteins. For detailed structural information, cryo-electron microscopy of purified Complex I can reveal the position and interactions of ND4L within the larger complex. Functional interaction studies might employ site-directed mutagenesis of key residues in ND4L followed by activity assays to determine their impact on Complex I function. Comparative analysis between octocoral and mammalian Complex I could highlight conserved and divergent interaction patterns. Blue native polyacrylamide gel electrophoresis (BN-PAGE) provides information on the assembly state of Complex I and can assess whether mutations or conditions affect the incorporation of ND4L into the complete complex.

What bioinformatic tools are most effective for analyzing ND4L sequence conservation across species?

Several specialized bioinformatic approaches can effectively analyze ND4L sequence conservation across evolutionary diverse species. Multiple sequence alignment tools like MAFFT (using the E-INS-I method) with appropriate scoring matrices (BLOSUM45) are well-suited for aligning hydrophobic membrane proteins like ND4L . Visualization and manual adjustment of alignments in programs such as MacClade, Jalview, or AliView allow refinement based on structural features and functional domains. For conservation analysis, tools like ConSurf can map evolutionary conservation onto protein structures, while PAML or HyPhy can detect sites under positive or negative selection pressure. Transmembrane topology prediction tools (TMHMM, Phobius) help identify conserved membrane-spanning regions critical for function. Protein structure prediction using AlphaFold2 can generate models for comparing structural conservation beyond primary sequence. Database resources including specialized mitochondrial genome databases and general repositories like NCBI GenBank provide comprehensive sequence data for cross-species comparison. Together, these tools enable researchers to identify key conserved features that underlie ND4L function across diverse taxonomic groups.