Recombinant Marchantia polymorpha ATP synthase protein MI25 (YMF39)

What is Marchantia polymorpha ATP synthase protein MI25 (YMF39) and what is its biological function?

Marchantia polymorpha ATP synthase protein MI25 (YMF39) is a mitochondrial protein that functions as subunit b of the F0 component in the F0F1-ATP synthase complex. Initially classified as a "conserved hypothetical protein-coding gene" in mitochondrial genomes of land plants and certain protists, YMF39 has been definitively identified as an essential component of the ATP synthase machinery . The protein plays a critical role in maintaining the structural integrity of the F0 domain and facilitating proton translocation across the mitochondrial membrane, which drives ATP synthesis. Within the complete ATP synthase complex (α3:β3:γ:δ:ε:a:b:b′:c9), the b subunit forms part of the peripheral stalk that connects the F1 catalytic domain to the membrane-embedded F0 domain . This structural arrangement is essential for the rotational mechanism that couples proton movement to ATP synthesis.

What is the relationship between YMF39 and atp4 gene designations?

The relationship between YMF39 and atp4 represents a nomenclature evolution based on functional characterization. YMF39 was the original designation for this "hypothetical mitochondrial open reading frame" before its function was confirmed. Through sequence analysis and biochemical studies, researchers demonstrated that YMF39 is homologous to the bacterial atpF gene, which codes for ATP synthase subunit b . Based on this functional identification, researchers proposed redesignating ymf39 as atp4 to better reflect its biological role . This nomenclature change is significant for researchers as both designations appear in scientific literature and databases, and understanding their equivalence is crucial for comprehensive literature searches and data interpretation. Gene name databases now often list both "YMF39" and "atp4" as synonyms for the same genetic locus in Marchantia polymorpha .

How was YMF39's function as ATP synthase subunit b initially confirmed?

The confirmation of YMF39's function involved multiple complementary experimental approaches that demonstrate the power of comparative biochemistry in assigning function to hypothetical genes. Initially, researchers noticed weak sequence similarity between YMF39 from a green alga and the atpF gene product from Bradyrhizobium, suggesting a potential role in ATP synthesis . To test this hypothesis, researchers:

• Sequenced ymf39 from five protists (jakobids) with minimally derived mitochondrial genomes

• Isolated the mitochondrial ATP synthase complex from the jakobid Seculamonas ecuadoriensis

• Determined the partial protein sequence of a 19-kDa subunit, which matched the expected size for YMF39

• Confirmed that the obtained peptide sequence perfectly matched a region in the ymf39 gene sequence

This biochemical evidence, combined with statistical tests assessing sequence similarity between YMF39 proteins and known ATP synthase subunits from various organisms, provided conclusive evidence that YMF39 functions as ATP synthase subunit b. This methodological approach illustrates how protein purification, sequencing, and comparative analysis can be used to assign functions to previously uncharacterized genes .

What expression systems are available for producing recombinant YMF39, and what are their relative advantages?

Multiple expression systems have been developed for producing recombinant Marchantia polymorpha ATP synthase protein MI25 (YMF39), each offering specific advantages depending on research objectives:

The choice of expression system should align with specific experimental requirements. For structural studies where large quantities of protein are needed, E. coli systems may be preferable. For functional studies investigating protein-protein interactions or requiring native folding, mammalian or yeast expression systems might yield more biologically relevant results. The cell-free expression system offers advantages for proteins that might be toxic when expressed in living cells .

What purification strategies are most effective for recombinant YMF39?

Effective purification of recombinant YMF39 typically employs a multi-step approach designed to achieve high purity (≥85% as determined by SDS-PAGE) while maintaining protein structure and function:

• Initial Capture: Affinity chromatography based on the specific tag attached to the recombinant protein (His-tag, Avi-tag, etc.) provides efficient initial capture. For biotinylated YMF39 variants, streptavidin-based affinity columns yield excellent specificity .

• Intermediate Purification: Ion exchange chromatography (IEX) exploiting YMF39's charge properties at specific pH values helps remove contaminants with different charge characteristics.

• Polishing Step: Size exclusion chromatography (SEC) separates the target protein from aggregates and smaller contaminants based on molecular size.

• Quality Control: Final purity assessment via SDS-PAGE, with accepted batches typically showing ≥85% purity .

What tagging strategies are recommended for recombinant YMF39 in different experimental applications?

The choice of protein tagging strategy for recombinant YMF39 depends critically on the intended experimental application. Several validated approaches include:

• Standard Affinity Tags: His-tags (6x or 10x) and GST-tags facilitate purification and can be positioned at either N- or C-terminus, though C-terminal tagging is often preferred to avoid interfering with the protein's mitochondrial targeting sequence .

• Biotinylation Tags: The Avi-tag system, which allows for in vivo biotinylation using E. coli biotin ligase (BirA), is particularly valuable for protein-protein interaction studies and immobilization applications. This approach creates a highly specific covalent attachment of biotin to the 15 amino acid AviTag peptide .

• Fluorescent Protein Fusions: For localization studies in living cells, YMF39 has been successfully fused with fluorescent proteins like CITRINE or GFP. The successful use of such constructs is demonstrated in promoter studies examining MpANT gene expression, suggesting similar approaches could work for YMF39 .

• Custom Tag Selection: Some suppliers determine appropriate tag types during the manufacturing process based on protein properties, offering flexibility to meet specific research requirements .

When designing tagged constructs, researchers should consider potential interference with protein folding, oligomerization, or functional interactions. For projects where tag interference is a concern, tag removal using specific proteases (e.g., TEV or PreScission) followed by a second purification step can yield untagged protein for functional studies .

How can recombinant YMF39 be used in structural biology studies of mitochondrial ATP synthase?

Recombinant YMF39 offers several important applications in structural biology studies of mitochondrial ATP synthase:

• Cryo-EM Structure Determination: Purified recombinant YMF39 can be reconstituted with other ATP synthase subunits to form subcomplexes suitable for cryo-electron microscopy. This approach has been successfully used with other ATP synthase components to determine high-resolution structures, as demonstrated with the mycobacterial F-ATP synthase complex .

• Protein-Protein Interaction Mapping: Using techniques such as chemical cross-linking coupled with mass spectrometry (XL-MS), researchers can identify contact points between YMF39 and other subunits of the ATP synthase complex. This is particularly valuable for understanding how the peripheral stalk components (including subunit b/YMF39) interact with both the F1 catalytic domain and the membrane-embedded F0 domain .

• Conformational Dynamics Studies: Site-specific labeling of recombinant YMF39 with fluorescent probes or spin labels enables investigation of protein dynamics during ATP synthesis and hydrolysis cycles using techniques such as FRET or electron paramagnetic resonance (EPR) spectroscopy.

• Comparative Structural Analysis: The availability of purified recombinant YMF39 facilitates structural comparisons with its functionally equivalent counterparts (ATP4/ATP5F) from fungi and animals, providing insights into evolutionary adaptations of ATP synthase components .

When designing structural biology experiments, researchers should consider the potential impact of expression tags on protein structure and function. Where possible, tags should be removed prior to structural analysis, or their potential impact on structure should be carefully assessed and controlled for .

What role does YMF39/atp4 play in the evolution of mitochondrial genomes across species?

YMF39/atp4 serves as a fascinating case study in mitochondrial genome evolution and gene transfer between organelles and the nucleus:

• Retention in Plant Mitochondrial Genomes: In land plants including Marchantia polymorpha, YMF39/atp4 is encoded in the mitochondrial genome, representing a conserved feature of plant mitochondrial genetics .

• Nuclear Migration in Fungi and Animals: Comparative analysis reveals that the functional counterparts of YMF39 in fungi and animals (ATP4/ATP5F) are encoded in the nuclear genome rather than the mitochondrial genome. This indicates a historical gene transfer event from the mitochondrion to the nucleus during evolution .

• Sequence Divergence Following Transfer: Statistical analyses show that the nuclear-encoded ATP4/ATP5F proteins in fungi and animals are highly diverged forms of mitochondrial YMF39. This divergence likely occurred following the gene transfer event, potentially reflecting adaptation to nuclear expression and cytosolic translation followed by mitochondrial import .

• Protist Model Systems: The investigation of YMF39 in jakobids (protists with minimally derived mitochondrial genomes) provides a window into the ancestral state of mitochondrial gene content. These organisms retain many mitochondrial genes that have been transferred to the nucleus in other lineages, making them valuable models for studying mitochondrial genome evolution .

This evolutionary context is important for researchers working with YMF39, as it explains patterns of sequence conservation and divergence across taxa and provides insights into the functional constraints operating on ATP synthase components. Understanding these evolutionary patterns can inform comparative studies and guide experimental design when working with YMF39 from different species .

What methodological approaches can be used to study YMF39's role in ATP synthase assembly and function?

Several complementary methodological approaches can be employed to investigate YMF39's role in ATP synthase assembly and function:

• Gene Editing via CRISPR-Cas9: The CRISPR-Cas9 system has been successfully applied to Marchantia polymorpha genes, as demonstrated with MpANT . Similar approaches can be used to create targeted mutations or knockouts of YMF39/atp4 to study its function. For example, guide RNAs targeting specific regions of the YMF39 gene can be designed and cloned under appropriate promoters (such as the MpU6 promoter) into CRISPR-Cas9 expression vectors for transformation into wild-type Marchantia polymorpha .

• Blue Native PAGE and Complex Isolation: Mitochondrial complexes can be isolated using gentle detergents followed by blue native polyacrylamide gel electrophoresis (BN-PAGE) to separate intact ATP synthase complexes. This technique was successfully used with the jakobid Seculamonas ecuadoriensis to isolate ATP synthase and identify YMF39 as a component .

• In vitro Reconstitution Assays: Purified recombinant YMF39 can be combined with other ATP synthase subunits in vitro to study complex assembly. This approach can identify which subunits directly interact with YMF39 and determine the sequential steps in complex formation.

• ATP Synthesis/Hydrolysis Assays: Functional assays measuring ATP synthesis or hydrolysis rates can be performed using reconstituted complexes containing wild-type or mutant YMF39 to determine how specific regions of the protein contribute to enzymatic activity. Similar approaches have been applied to mycobacterial F-ATP synthase to study the regulatory role of specific subunits .

• Immunolocalization Studies: Antibodies against YMF39 or tagged versions of the protein can be used to track its subcellular localization and assembly into the ATP synthase complex in vivo. Cross-reactivity considerations must be evaluated when using antibodies, as demonstrated in the approach used for MpPIN1 detection using AtPIN1 antibodies .

These methodological approaches provide a toolkit for researchers investigating different aspects of YMF39 biology, from basic questions about subcellular localization to complex inquiries about structure-function relationships and evolutionary adaptations .

How does YMF39 function compare to its homologs in other organisms?

YMF39 exists within a broader evolutionary context of ATP synthase subunit b proteins across diverse taxa, exhibiting both conserved and divergent features:

Understanding these comparative aspects provides important context for researchers working with YMF39, particularly when interpreting experimental results or designing heterologous expression systems .

What insights from YMF39 research might apply to studies of other mitochondrial membrane proteins?

Research on YMF39 offers valuable methodological and conceptual insights applicable to studies of other mitochondrial membrane proteins:

• Gene Identification Approaches: The confirmation of YMF39's function as ATP synthase subunit b demonstrates an effective methodological pipeline for assigning functions to hypothetical mitochondrial genes. This approach—combining weak sequence similarity detection, targeted sequencing, protein complex isolation, and partial protein sequencing—provides a template for functional annotation of other hypothetical mitochondrial proteins .

• Expression System Selection: The challenges and solutions developed for recombinant expression of YMF39 in various systems (E. coli, yeast, baculovirus, mammalian cells) provide practical guidance for expressing other mitochondrial membrane proteins. The comparative advantages of different expression systems outlined for YMF39 can inform strategic decisions for other challenging membrane proteins .

• Evolutionary Insights: The observation that YMF39 remains mitochondrially encoded in plants while its homolog has transferred to the nucleus in animals and fungi raises broader questions about the selective pressures governing mitochondrial gene retention. This evolutionary perspective can guide investigations into why certain genes remain in organellar genomes while others migrate to the nucleus .

• Complex Assembly Studies: Methodologies developed to study YMF39's integration into the ATP synthase complex can be adapted for investigating assembly pathways of other multi-subunit mitochondrial complexes, particularly those spanning the inner mitochondrial membrane .

• Structural Biology Approaches: Techniques applied to determine how YMF39 interacts with other ATP synthase subunits, such as cross-linking and mass spectrometry, provide templates for structural characterization of other mitochondrial membrane protein complexes .

These transferable insights make YMF39 research valuable beyond its specific role in ATP synthase, contributing to broader understanding of mitochondrial biology and membrane protein biochemistry .

What are the major challenges in expressing and purifying recombinant YMF39, and how can they be overcome?

Expression and purification of recombinant YMF39 present several technical challenges common to mitochondrial membrane proteins, along with effective solutions:

• Challenge: Membrane Protein Solubility

  • Solution: Use of detergents appropriate for mitochondrial proteins (e.g., n-dodecyl β-D-maltoside, digitonin) during extraction and purification. Selection of suitable detergents is critical for maintaining protein structure while ensuring solubilization .

• Challenge: Maintaining Native Conformation

  • Solution: Expression in eukaryotic systems (yeast, insect, or mammalian cells) can help maintain native folding and post-translational modifications. For E. coli expression, specialized strains designed for membrane proteins can improve folding .

• Challenge: Low Expression Yields

  • Solution: Optimization of expression conditions including temperature (often lower than standard), induction parameters, and growth media composition. Codon optimization of the YMF39 sequence for the chosen expression host can significantly improve yields .

• Challenge: Protein Aggregation During Purification

  • Solution: Addition of stabilizing agents such as glycerol (5-50%) to purification buffers helps maintain protein solubility. Performing purification steps at reduced temperatures (4°C) and minimizing freeze-thaw cycles are also recommended .

• Challenge: Assessing Functional Integrity

  • Solution: Development of functional assays specific to YMF39's role in ATP synthase, such as reconstitution with partner proteins or binding assays with interacting subunits. These functional tests should complement standard purity assessments by SDS-PAGE .

• Challenge: Protein Degradation

  • Solution: Inclusion of protease inhibitors throughout purification and minimizing exposure to room temperature. For long-term storage, lyophilization is recommended, with reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL, and addition of 5-50% glycerol for stability .

Addressing these challenges requires a systematic approach, often involving iteration and optimization of multiple parameters simultaneously. The specific solutions may need to be tailored to the particular variant of YMF39 being studied and the research questions being addressed .

How can researchers validate the functional integrity of recombinant YMF39?

Validating the functional integrity of recombinant YMF39 requires multiple complementary approaches that assess both structural and functional properties:

• Structural Validation:

  • Circular Dichroism (CD) Spectroscopy: Provides information about secondary structure content (α-helices, β-sheets) and can confirm proper folding compared to native protein standards.

  • Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): Assesses the oligomeric state of purified YMF39, confirming whether it maintains expected quaternary structure.

  • Limited Proteolysis: Properly folded proteins often show characteristic proteolytic fragmentation patterns different from misfolded variants.

• Functional Validation:

  • Binding Assays with Partner Proteins: Using techniques such as surface plasmon resonance (SPR) or microscale thermophoresis (MST) to quantify binding to known interaction partners within the ATP synthase complex.

  • Reconstitution Studies: Combining purified YMF39 with other ATP synthase components to assess complex formation, which can be monitored by native PAGE or analytical ultracentrifugation.

  • Activity Assays: While YMF39 itself lacks enzymatic activity, its ability to support ATP synthase function can be assessed by reconstituting it with other subunits and measuring ATP synthesis or hydrolysis activity of the assembled complex.

• Cellular Validation:

  • Complementation Studies: Testing whether recombinant YMF39 can restore function in systems where the endogenous protein has been depleted or inactivated.

  • Subcellular Localization: Confirming proper mitochondrial targeting of tagged versions using fluorescence microscopy, similar to approaches used with other mitochondrial proteins .

• Mass Spectrometry Validation:

  • Peptide Mapping: Confirming complete sequence coverage and identifying any post-translational modifications that might be important for function.

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Assessing protein dynamics and conformational properties, which can be compared between recombinant and native proteins.

These validation approaches should be combined to provide comprehensive assessment of recombinant YMF39's integrity, as different methods address complementary aspects of protein structure and function .

What are the most promising future research directions involving YMF39 and ATP synthase?

Several promising research directions involving YMF39 and ATP synthase warrant further investigation:

• Structural Biology Advances: Integration of high-resolution structural techniques (cryo-EM, X-ray crystallography) with functional studies to determine how YMF39 contributes to ATP synthase architecture and mechanism. Particular focus should be placed on how YMF39 interacts with other subunits during the catalytic cycle .

• Regulatory Mechanisms: Investigation of potential regulatory roles for YMF39 in controlling ATP synthase activity under different physiological conditions. By analogy with the regulatory role of the α C-terminus in mycobacterial ATP synthase, YMF39 might participate in activity regulation in plants .

• Evolutionary Studies: Comprehensive phylogenetic analysis across diverse taxa to better understand the selective pressures that have maintained YMF39 in the mitochondrial genome of plants while facilitating its transfer to the nucleus in animals and fungi .

• Protein-Protein Interaction Network: Systematic mapping of YMF39's interactions with other proteins, both within and outside the ATP synthase complex, to understand its potential roles in mitochondrial organization and bioenergetics .

• Stress Response and Adaptation: Investigation of how YMF39 expression and function change under various stress conditions (e.g., oxidative stress, temperature stress) to understand its role in cellular adaptation and energy homeostasis.

• Development of Specific Inhibitors: Design of small molecules that specifically target the interaction between YMF39 and other ATP synthase subunits, providing new tools for studying ATP synthase function in vivo .

• Gene Editing Applications: Utilizing CRISPR-Cas9 and other gene editing tools to create specific mutations in YMF39/atp4 and assess their phenotypic consequences, similar to approaches used with other Marchantia polymorpha genes .

These research directions build upon current knowledge while addressing important gaps in our understanding of YMF39 biology and ATP synthase function in different organisms .

How might studies of YMF39 contribute to our understanding of mitochondrial diseases?

Although YMF39/atp4 has not been directly implicated in human mitochondrial diseases, research on this protein contributes valuable insights to the broader field of mitochondrial medicine:

• Comparative Functional Analysis: Studies of YMF39 in plants and protists provide comparative context for understanding the function of its homolog ATP5F1/ATP4 in humans. ATP5F1 mutations have been associated with mitochondrial disorders characterized by ATP synthase dysfunction, making mechanistic insights from YMF39 research potentially relevant to human disease .

• Model Systems Development: Marchantia polymorpha, with its relatively simple genetic architecture and amenability to gene editing techniques like CRISPR-Cas9, provides a valuable model system for studying fundamental aspects of mitochondrial biology that might be obscured in more complex organisms .

• Evolutionary Medicine Perspectives: The evolutionary history of YMF39/atp4, including its nuclear transfer in animals, provides context for understanding the unique vulnerabilities of human mitochondrial genetics. The retention of certain genes in mitochondrial DNA versus nuclear transfer has significant implications for inheritance patterns and tissue-specific manifestations of mitochondrial diseases .

• Therapeutic Target Identification: Understanding the structure-function relationships in ATP synthase components like YMF39 could inform the development of targeted therapeutics aimed at modulating ATP synthase activity in mitochondrial disorders. The development of pharmacophore models for interacting with specific ATP synthase subunits, as demonstrated with the α C-terminus in mycobacterial ATP synthase, provides a template for similar approaches in human mitochondrial medicine .

• Diagnostic Biomarker Development: Insights into how YMF39 and its homologs contribute to ATP synthase assembly and function could lead to improved biochemical assays for diagnosing mitochondrial disorders affecting this critical enzyme complex.

While direct clinical applications may not be immediate, these contributions highlight how basic research on evolutionarily conserved mitochondrial components like YMF39 enriches our conceptual framework for understanding and eventually treating mitochondrial diseases .