Recombinant Ustilago maydis ATP synthase subunit 9, mitochondrial (ATP9)

What is ATP synthase subunit 9 in Ustilago maydis and what is its function?

ATP synthase subunit 9 (ATP9) in Ustilago maydis is a highly hydrophobic protein component of the F₁F₀ ATP synthase complex located in the inner mitochondrial membrane. The mature protein is 73 amino acids in length with the sequence: MLAAAKYIGSGVAALGLIGAGIGVGIVFAALIQGVSRNPSLRGQLFTYAILGFALSEATGLFALMVSFLLLYS .

This subunit is classified as a proteolipid because it can be easily extracted from mitochondria using organic solvents. It functions as part of the proton-translocating domain (F₀) of ATP synthase . During ATP synthesis, the subunit 9 forms a ring structure (containing approximately ten copies in yeast) that rotates as protons flow through the complex, causing conformational changes in the catalytic head (F₁) that facilitate ATP production and release into the mitochondrial matrix .

How does the genetic organization of ATP9 vary across fungal species?

The ATP9 gene shows remarkable diversity in its genomic location across fungal species. A phylogenetic analysis of 26 fungal species revealed five different ATP9 gene distributions :

The phylogenetic distribution suggests that ATP9 has undergone at least two independent transfers from mitochondria to nucleus during fungal evolution, followed by several independent episodes of loss of either the mitochondrial or nuclear gene copy .

How can researchers effectively store and handle recombinant U. maydis ATP9 protein?

Recombinant U. maydis ATP9 protein requires specific storage and handling conditions due to its hydrophobic nature:

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

• Formulation: The protein is typically provided as:

  • Lyophilized powder for maximum stability

  • In Tris/PBS-based buffer with 6% trehalose at pH 8.0 or

  • In Tris-based buffer with 50% glycerol

• Reconstitution protocol:

  • Centrifuge the vial briefly before opening

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to 5-50% final concentration (recommended 50%)

  • Aliquot for long-term storage

• Stability considerations:

  • Avoid repeated freeze-thaw cycles

  • Working aliquots can be stored at 4°C for up to one week

  • Aliquoting is necessary for multiple use

What experimental approaches are used to express and purify recombinant U. maydis ATP9?

Expression and purification of recombinant U. maydis ATP9 involves several specialized approaches:

Expression Systems:

Escherichia coli is the predominant expression system for recombinant U. maydis ATP9 . The challenging nature of expressing this highly hydrophobic membrane protein requires specific strategies:

• Vector design: Full-length ATP9 (1-73 amino acids) is typically expressed with an N-terminal His-tag to facilitate purification .

• Codon optimization: The native U. maydis ATP9 sequence must be codon-optimized for efficient expression in E. coli .

• Expression conditions: Lower temperatures (16-25°C) and reduced inducer concentrations may improve proper folding of this hydrophobic protein.

Purification Protocol:

• Cell lysis: Mechanical disruption in the presence of detergents to solubilize membrane proteins

• Affinity chromatography: Ni-NTA purification utilizing the His-tag

• Detergent considerations: Selection of appropriate detergents (e.g., DDM, LDAO) critical for maintaining protein solubility

• Final formulation: Either lyophilized powder or in solution with stabilizing agents like trehalose or glycerol

The typical purity achieved is greater than 90% as determined by SDS-PAGE .

How do the properties of nuclear-encoded ATP9 differ from mitochondrially-encoded versions?

The relocation of ATP9 from the mitochondrial to nuclear genome requires several adaptations that affect protein properties:

Key Differences:

Functional Implications:

Experimental relocation of ATP9 from mitochondria to nucleus in Saccharomyces cerevisiae using P. anserina nuclear ATP9 genes demonstrated:

• Respiratory capacity was restored in Δatp9 yeast but not to wild-type levels

• Oxygen consumption rates with NADH were:

  • 80% of wild-type with PaAtp9-5 (multicopy)

  • 40% of wild-type with PaAtp9-7 (multicopy)

• Mitochondrial function parameters showed significant differences:

  • ATP synthesis rates were reduced

  • Membrane potential was affected

  • Assembly of ATP synthase complex was altered

This suggests that while nuclear expression of ATP9 is possible, it may not achieve the same functional efficiency as the native mitochondrially-encoded version.

How have ATP9 genes been used to study mitochondrial genome evolution in fungi?

ATP9 genes serve as excellent models for studying mitochondrial genome evolution due to their variable genomic location across fungi:

Evolutionary Insights from ATP9 Distribution:

• Multiple independent gene transfers: Phylogenetic analysis suggests at least two independent transfers of ATP9 from mitochondria to nucleus in fungi, rather than gene duplication events .

• Evidence for distinct events: The mitochondrial targeting sequences (MTS) of ATP9-5 homologs are well conserved in sequence and length, while those of ATP9-7 homologs are more divergent, supporting separate transfer events .

• Complex evolutionary history: The distribution pattern of ATP9 genes is generally consistent with established fungal phylogeny (Assembling the Fungal Tree Of Life classification) .

Experimental Approaches:

Researchers utilize several methodologies to study ATP9 evolution:

• Comparative genomics: Analyzing ATP9 gene presence/absence across species

• Phylogenetic analysis: Constructing evolutionary trees based on ATP9 sequences

• Functional complementation: Testing whether nuclear ATP9 genes from one species can function in another species lacking its own ATP9

• Sequence divergence analysis: Examining the rate and pattern of sequence changes in ATP9 genes from different genomic compartments

The study of ATP9 gene transfer provides valuable insights into the ongoing process of mitochondrial genome reduction in eukaryotes.

What specific adaptations are required for successful expression of ATP9 from nuclear DNA?

Successful expression of ATP9 from nuclear DNA requires several critical adaptations:

Experimental Success Rates:

In experimental relocation of ATP9 to the nucleus in yeast, success varied with different constructs:

This demonstrates that even with appropriate modifications, nuclear expression of ATP9 achieves only partial functionality compared to native mitochondrial expression .

How is the expression of ATP9 regulated during the fungal life cycle?

The expression of ATP9 shows sophisticated regulation patterns during the fungal life cycle, particularly in species with nuclear-encoded ATP9 genes:

Regulation in Podospora anserina:

P. anserina contains two nuclear ATP9 genes with distinct expression profiles :

Functional Significance:

• Energy Demand Adaptation:

  • PaAtp9-5 is strongly expressed during germination, a process requiring substantial biomass production with high energy demands

  • PaAtp9-7 is predominantly expressed during sexual reproduction

• Phenotypic Effects:

  • Deletion of PaAtp9-5 is lethal

  • Deletion of PaAtp9-7 strongly impairs ascospore production

  • Swapping regulatory regions between genes demonstrated that the coding sequences are functionally interchangeable

• Control Mechanism:

  • Preliminary microarray data suggests that other ATP synthase subunits maintain consistent expression

  • This indicates that ATP9 expression may be the limiting factor controlling ATP synthase assembly

This sophisticated regulation of ATP9 parallels observations in mammals, where nuclear ATP9 isogenes show tissue-specific expression patterns, suggesting convergent evolution of regulatory mechanisms despite independent gene transfer events .

How can recombinant U. maydis ATP9 be used in structural and functional studies?

Recombinant U. maydis ATP9 offers valuable opportunities for structural and functional studies of mitochondrial ATP synthase:

Structural Applications:

• Cryo-EM Structure Determination:

  • Purified recombinant ATP9 can be reconstituted into liposomes or nanodiscs

  • Assembly of complete or partial ATP synthase for high-resolution structural analysis

  • Investigation of the c-ring structure and proton-binding sites

• Cross-linking Studies:

  • Identification of interaction partners within the ATP synthase complex

  • Mapping of protein-protein interactions using modified ATP9 with crosslinking agents

Functional Applications:

• Site-Directed Mutagenesis:

  • Systematic mutation of key residues (particularly in transmembrane domains) to identify those essential for proton translocation

  • Structure-function relationship studies of the c-ring rotation mechanism

• Reconstitution Experiments:

  • Incorporation of recombinant ATP9 into liposomes to measure proton transport

  • Assembly with other ATP synthase components to assess complex formation efficiency

• Comparative Studies:

  • Analysis of U. maydis ATP9 alongside ATP9 from other fungi with different genomic locations (nuclear vs. mitochondrial)

  • Investigation of adaptations specific to smut fungi

Specialized Techniques:

• Hydrogen/Deuterium Exchange Mass Spectrometry:

  • Analysis of protein dynamics and conformational changes

  • Identification of solvent-accessible regions

• Solid-State NMR:

  • Investigation of ATP9 structure in membrane environments

  • Analysis of proton-binding site properties

The hydrophobic nature of ATP9 makes it challenging to study but provides important insights into the function of membrane-embedded components of the ATP synthase complex.

What are the implications of ATP9 research for understanding mitochondrial diseases?

Research on ATP9 has significant implications for understanding and potentially treating mitochondrial diseases:

Disease Relevance:

• ATP Synthase Deficiencies:

  • Mutations in ATP synthase components, including subunit c (ATP9), are associated with mitochondrial diseases

  • Understanding the assembly and function of ATP9 provides insights into disease mechanisms

• Allotopic Expression Potential:

  • Successful nuclear expression of mitochondrial genes like ATP9 demonstrates the feasibility of allotopic expression

  • This approach has therapeutic implications for diseases caused by mitochondrial DNA mutations

Research Applications:

• Model System:

  • U. maydis and other fungi serve as models for studying the consequences of ATP synthase dysfunction

  • The ability to express and manipulate ATP9 in these systems facilitates mechanistic studies

• Therapeutic Strategy Development:

  • Experiments relocating ATP9 from mitochondria to nucleus provide proof-of-concept for gene therapy approaches

  • Identification of factors that enhance nuclear expression efficiency could improve therapeutic strategies

• Comparative Studies:

  • Analysis of different regulatory mechanisms across species reveals fundamental aspects of ATP synthase biogenesis

  • Identification of conserved pathways that might be targeted therapeutically

The successful relocation of ATP9 from mitochondria to nucleus in fungi, though not without functional compromises, suggests that similar approaches might eventually be applicable to human mitochondrial genes implicated in disease.