Recombinant Chondrus crispus ATP synthase subunit 9, mitochondrial (ATP9)

How does Chondrus crispus ATP9 differ from similar proteins in other organisms?

Comparative analysis reveals both conservation and divergence in ATP9 across species:

Despite sequence variations, functional domains are conserved across species, particularly the hydrophobic transmembrane regions that form the proton-conducting channel . This conservation highlights the essential role of ATP9 in mitochondrial function while species-specific variations may reflect evolutionary adaptations to different environmental conditions .

What is known about the transcription and expression of ATP9 in Chondrus crispus mitochondria?

The mitochondrial DNA of C. crispus is transcribed into two large RNA molecules as primary transcripts. These are then processed through multiple maturation events to produce either mono- or poly-cistronic RNAs .

Transcription initiation for both transcription units has been mapped to inverse repeated sequences near the "north pole" of the mitochondrial genome. Notably, C. crispus mitochondrial promoter motifs share significant similarities with chicken and Xenopus mitochondrial promoters, suggesting conservation of transcription mechanisms across diverse eukaryotic lineages .

The expression of ATP9, like other mitochondrial genes in C. crispus, is regulated through this transcription machinery. Interestingly, while many mitochondrial genes have been transferred to the nuclear genome during evolution in various lineages, genes involved in ATP synthesis, including atp6, atp8, and atp9, are consistently found in red algal mitochondrial DNA, with only a few exceptions .

What are the optimal conditions for expressing recombinant C. crispus ATP9 protein?

For high-yield expression of recombinant C. crispus ATP9, Escherichia coli is the preferred heterologous expression system. The protocol typically involves:

• Cloning the ATP9 coding sequence (1-76aa) into an expression vector with an N-terminal His-tag

• Transforming the construct into an E. coli expression strain optimized for membrane proteins

• Inducing expression at lower temperatures (16-20°C) to minimize protein aggregation

• Using specialized media supplements like betaine or sorbitol to enhance proper folding of this hydrophobic protein

The hydrophobic nature of ATP9 presents challenges for expression and purification. Adding fusion partners like thioredoxin or GST may enhance solubility, but these should be removed post-purification if they interfere with functional studies .

How should researchers purify and store recombinant C. crispus ATP9 protein?

Purification of recombinant C. crispus ATP9 requires specialized approaches due to its hydrophobic properties:

Purification protocol:

• Cell lysis using detergent-based buffers (e.g., Triton X-100, n-dodecyl β-D-maltoside)

• Immobilized metal affinity chromatography (IMAC) using the His-tag

• Size exclusion chromatography for further purification if needed

• Quality assessment by SDS-PAGE (>90% purity is standard for research applications)

Storage and stability:

• Lyophilize the purified protein in a Tris/PBS-based buffer with 6% trehalose at pH 8.0

• Store the lyophilized powder at -20°C/-80°C

• Upon reconstitution, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

• Add glycerol (5-50% final concentration) for long-term storage at -20°C/-80°C

• For working solutions, maintain aliquots at 4°C for up to one week

• Avoid repeated freeze-thaw cycles

What methods are most effective for studying ATP9 interactions with other ATP synthase subunits?

Investigating ATP9 interactions requires specialized techniques for membrane protein complexes:

• Co-immunoprecipitation (Co-IP): Using antibodies against the His-tag or ATP9 protein to pull down interacting partners, followed by mass spectrometry identification.

• Blue Native-PAGE: This technique preserves protein-protein interactions and can resolve the entire ATP synthase complex with ATP9 in its native state. Label-free LC-MS/MS analysis following BN-PAGE can identify all interacting proteins, as demonstrated in studies of C. crispus protein complexes .

• Proximity labeling: Methods such as BioID or APEX can capture transient interactions within the mitochondrial membrane.

• Structural studies: Cryo-electron microscopy (cryo-EM) has emerged as the method of choice for resolving ATP synthase structure and subunit interactions at near-atomic resolution.

Research on C. crispus ATP9 has shown that isolation of the mitochondrial ATP synthase complex followed by determination of partial protein sequences through mass spectrometry is an effective approach for confirming ATP9's role in the complex .

How can C. crispus ATP9 be used as a model for studying mitochondrial genome evolution?

Chondrus crispus ATP9 provides an excellent model for studying mitochondrial genome evolution for several reasons:

• Evolutionary conservation: The presence of ATP9 in the mitochondrial genome of C. crispus, while it has been transferred to the nuclear genome in some lineages, makes it valuable for studying gene transfer events during evolution .

• Comparative genomics: Analysis of ATP9 across diverse species reveals patterns of conservation and divergence. Statistical tests have been employed to assess the significance of sequence similarity between mitochondrially-encoded Ymf39 (now known as ATP9) proteins and their nucleus-encoded counterparts (ATP4/ATP5F) from fungi and animals .

• Ancient origins: The similarity between C. crispus ATP9 and α-proteobacterial ATP synthase b-subunits provides evidence for the endosymbiotic origin of mitochondria .

• Transcriptional mechanisms: C. crispus mitochondrial DNA transcription shares surprising similarities with animal mitochondrial transcription rather than with other algae, suggesting convergent evolution of transcription mechanisms based on genome organization rather than phylogeny .

These characteristics make C. crispus ATP9 an important marker for investigating the evolution of mitochondrial genomes across diverse eukaryotic lineages.

What are the current approaches for studying ATP9 function in reconstituted systems?

Advanced researchers are employing several sophisticated approaches to study ATP9 function:

• Liposome reconstitution: Purified recombinant ATP9 can be incorporated into liposomes to study proton translocation activity. This requires:

  • Preparation of liposomes with defined lipid composition

  • Incorporation of purified ATP9 protein

  • Measurement of proton flux using pH-sensitive fluorescent dyes

• Nanodiscs technology: ATP9 can be incorporated into nanodiscs (disc-shaped lipid bilayers stabilized by scaffold proteins) for structural and functional studies in a native-like membrane environment.

• Complementation studies: Expression of C. crispus ATP9 in ATP9-deficient systems can assess functional conservation. Researchers have found that despite sequence divergence, ATP9 function is often conserved across species .

• Site-directed mutagenesis: Systematic mutation of conserved residues followed by functional assays can identify critical amino acids for ATP9 function. Key targets include the conserved residues in the transmembrane regions that form the proton-conducting channel.

How do mutations in ATP9 affect ATP synthase assembly and function?

Mutations in ATP9 can have profound effects on ATP synthase assembly and function, providing insights into structure-function relationships:

• Oligomerization defects: Mutations in the highly conserved transmembrane regions can disrupt the formation of the c-ring structure (composed of multiple ATP9 subunits), compromising proton translocation and ATP synthesis.

• Proton conductance: Alterations in key residues involved in proton binding and release can affect the efficiency of proton translocation, impacting the proton motive force that drives ATP synthesis.

• Assembly defects: Some mutations may allow c-ring formation but disrupt interactions with other ATP synthase subunits, leading to improper assembly of the complete F0F1 complex.

• Stability issues: Certain mutations can affect protein stability, leading to rapid degradation and reduced steady-state levels of ATP9 in the mitochondrial membrane.

The evolutionary conservation of specific amino acid residues in ATP9 across diverse species, including C. crispus, indicates their critical importance for function . Comparative analysis of ATP9 sequences can guide targeted mutagenesis to understand the role of specific residues.

How does C. crispus ATP9 relate to ATP synthase components in other organisms?

Comparative analysis reveals that C. crispus ATP9 is homologous to:

• The ATP synthase c-subunit (subunit 9) in other eukaryotes

• The AtpE subunit in bacteria

• The previously unknown Ymf39 protein, now recognized as ATP4/ATP9

This evolutionary relationship is supported by:

• Sequence similarity analysis showing conservation of key functional domains

• Statistical tests confirming significant sequence similarity between Ymf39 proteins and α-proteobacterial ATP synthase b-subunits

• Functional studies demonstrating similar roles in ATP synthesis

Interestingly, while C. crispus retains ATP9 in its mitochondrial genome, some lineages have transferred this gene to the nuclear genome. The nuclear-encoded ATP4/ATP5F proteins in fungi and animals appear to be highly diverged forms of mitochondrial ATP9/Ymf39 that have migrated to the nucleus during evolution .

What insights does studying red algal ATP9 provide about mitochondrial genome organization?

Red algal mitochondrial genomes, including that of C. crispus, offer unique insights into mitochondrial genome organization:

• Genome structure: The mitochondrial DNA of red algae like C. crispus is transcribed into two large RNA molecules that undergo extensive processing, similar to animal mitochondrial genomes rather than other algae .

• Gene content conservation: In red algae, genes involved in ATP synthesis, including atp6, atp8, and atp9, are consistently found in mitochondrial DNA, with only rare exceptions (like the absence of atp8 in some species) .

• Densely packed genomes: C. crispus has a densely packed mitochondrial genome with genes encoded on both positive and negative strands, with considerable sequence synteny across other red algal species .

• tRNA complement: Like other red algae, C. crispus mitochondrial DNA does not contain a full set of tRNAs needed for translation, indicating the import of some tRNAs from the cytosol .

These characteristics demonstrate that mitochondrial genome organization in red algae represents an intermediate state in the reduction of the ancestral α-proteobacterial genome to the minimal mitochondrial genomes seen in some lineages.

How can phylogenetic analysis of ATP9 inform our understanding of eukaryotic evolution?

Phylogenetic analysis of ATP9 provides valuable insights into eukaryotic evolution:

• Endosymbiotic origin: The similarity between C. crispus ATP9 and α-proteobacterial ATP synthase subunits reinforces the endosymbiotic origin of mitochondria .

• Gene transfer events: By comparing mitochondrially-encoded ATP9 with nuclear-encoded homologs across diverse eukaryotes, researchers can trace the history of gene transfer events from mitochondria to the nucleus .

• Convergent evolution: The surprising similarity in transcription mechanisms between C. crispus and animal mitochondrial genomes suggests convergent evolution based on similar genome organization rather than phylogenetic relatedness .

• Rate of sequence evolution: Studies incorporating ATP9 sequences from multiple species have revealed varying rates of sequence evolution, with some lineages showing accelerated rates compared to others .

Phylogenetic trees constructed using mitochondrial protein-coding genes, including ATP9, have helped resolve evolutionary relationships among green algae, red algae, and land plants, contributing to our understanding of the diversification of photosynthetic eukaryotes .

What are common challenges in working with recombinant C. crispus ATP9 and how can they be addressed?

Researchers face several challenges when working with this hydrophobic membrane protein:

• Low expression yields:

  • Solution: Optimize codon usage for the expression host

  • Solution: Use specialized E. coli strains designed for membrane protein expression

  • Solution: Express at lower temperatures (16-20°C) to reduce aggregation

• Protein aggregation:

  • Solution: Include mild detergents during extraction and purification

  • Solution: Use fusion partners to enhance solubility

  • Solution: Add stabilizing agents like glycerol or trehalose

• Degradation during purification:

  • Solution: Include protease inhibitors in all buffers

  • Solution: Perform purification steps at 4°C

  • Solution: Minimize purification time by optimizing protocols

• Loss of activity after reconstitution:

  • Solution: Reconstitute protein in buffers containing lipids

  • Solution: Avoid repeated freeze-thaw cycles

  • Solution: Store working aliquots at 4°C for up to one week

How can researchers verify the functional integrity of purified C. crispus ATP9?

Verifying functional integrity of purified ATP9 requires specialized assays:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm proper secondary structure

  • Fluorescence spectroscopy to assess tertiary structure

  • Size exclusion chromatography to verify oligomeric state

• Functional assays:

  • Proton translocation assays in reconstituted liposomes

  • ATP synthesis/hydrolysis assays in reconstituted systems

  • Binding studies with other ATP synthase subunits

• Interaction studies:

  • Co-immunoprecipitation with known interacting partners

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters of interactions

These approaches provide complementary information about the structural and functional integrity of purified ATP9 protein.

What considerations are important for designing experiments using C. crispus ATP9 in comparative studies?

When designing comparative studies using C. crispus ATP9:

• Sequence alignment considerations:

  • Use multiple sequence alignment tools that handle membrane proteins well

  • Pay special attention to conserved functional residues in transmembrane domains

  • Consider structural predictions alongside sequence conservation

• Experimental design for comparative studies:

  • Include positive and negative controls from well-characterized systems

  • Use consistent experimental conditions across all proteins being compared

  • Account for differences in protein stability and solubility

• Functional comparisons:

  • Develop standardized assays that can be applied to ATP9 from multiple species

  • Normalize activity measurements to account for differences in protein purity and stability

  • Consider chimeric proteins to identify domains responsible for functional differences

• Data interpretation:

  • Be cautious about attributing differences to species-specific adaptations versus experimental artifacts

  • Consider the effects of expression systems on post-translational modifications

  • Account for differences in lipid environments that may affect protein function

By carefully addressing these considerations, researchers can maximize the reliability and significance of comparative studies using C. crispus ATP9.