Recombinant Ashbya gossypii ATP synthase subunit 9, mitochondrial (ATP9)

What is the genomic location of ATP9 in Ashbya gossypii, and how does this compare to other fungi?

ATP9 genes in fungi show remarkable diversity in genomic location, existing in either the mitochondrial genome, nuclear genome, or both depending on the species. Research across 26 fungal species has identified five distinct ATP9 gene distribution patterns, suggesting at least two independent transfers from mitochondria to the nucleus during fungal evolution, followed by multiple independent gene losses . To determine the location in A. gossypii specifically, researchers should conduct both mitochondrial and nuclear genome analyses using comparative genomic approaches. The complete sequencing of the A. gossypii genome facilitates such investigations . Researchers should also consider that, like in Podospora anserina, A. gossypii might possess multiple ATP9 genes with distinct regulatory patterns and functional roles.

How can CRISPR/Cas9 technology be utilized to study ATP9 function in A. gossypii?

A one-vector CRISPR/Cas9 system has been specifically adapted for A. gossypii, enabling marker-free genetic engineering of this organism . For ATP9 studies, researchers should:

• Identify suitable target sites containing the required PAM sequence (5'-NGG-3')

• Design guide RNAs complementary to these target regions

• Construct mutagenic donor DNA containing the desired modification flanked by homology arms

• Generate the complete CRISPR vector containing the Cas9 nuclease, guide RNA expression cassette, and donor DNA

• Transform A. gossypii using established protocols

• Screen for successful editing through PCR amplification and sequencing

This approach enables precise modifications to ATP9, including point mutations, deletions, or replacements, without introducing selection markers that might interfere with downstream analyses. For studying essential genes like ATP9, consider using inducible or tissue-specific promoters to control expression timing or creating heterozygous mutations if possible .

What is the relationship between ATP9 expression and mitochondrial function during different life cycle stages?

Based on studies in related fungi like P. anserina, ATP9 expression likely varies significantly throughout the A. gossypii life cycle. In P. anserina, two nuclear ATP9 genes (PaAtp9-5 and PaAtp9-7) show distinct expression patterns: PaAtp9-5 is strongly expressed during ascospore germination, while PaAtp9-7 predominates during sexual reproduction . These patterns correspond to stage-specific energy demands, with germination requiring substantial ATP for rapid biomass production.

To investigate this relationship in A. gossypii, researchers should:

• Collect samples from multiple developmental stages (spores, germinating spores, active mycelium, mature mycelium)

• Quantify ATP9 transcript levels using RT-qPCR with stage-specific normalization controls

• Measure corresponding mitochondrial function parameters (oxygen consumption, membrane potential, ATP production)

• Correlate ATP9 expression with morphological and physiological changes

Understanding these relationships would provide insights into how A. gossypii modulates energy metabolism throughout its life cycle and potentially inform strategies for optimizing biotechnological applications .

What experimental approaches can identify transcriptional regulators of ATP9 in A. gossypii?

To identify the transcriptional regulators controlling ATP9 expression in A. gossypii, a multi-faceted approach is recommended:

• Promoter analysis and reporter assays:

  • Clone the putative ATP9 promoter region upstream of a reporter gene (e.g., GFP)

  • Create a series of promoter truncations to map regulatory regions

  • Analyze the promoter sequence for known transcription factor binding motifs

  • Test reporter activity under various conditions reflecting different life cycle stages

• Chromatin immunoprecipitation (ChIP) approaches:

  • Perform ChIP-seq to identify proteins binding to the ATP9 promoter

  • Use antibodies against known transcription factors or tagged versions of candidate regulators

  • Validate binding through electrophoretic mobility shift assays (EMSA)

• Genetic screens:

  • Create an ATP9 promoter-reporter strain

  • Perform random mutagenesis and screen for altered reporter expression

  • Alternatively, systematically test deletion/overexpression of candidate transcription factors

• Comparative genomics:

  • Analyze ATP9 promoter regions across related fungi for conserved elements

  • Compare expression patterns with P. anserina PaAtp9-5 and PaAtp9-7 regulators

Through these approaches, researchers can map the regulatory network controlling ATP9 expression during different developmental stages and environmental conditions.

How do post-translational modifications affect ATP9 function in mitochondrial ATP synthase assembly?

While specific data on ATP9 post-translational modifications (PTMs) in A. gossypii is limited, research in related organisms suggests several critical modifications:

• N-terminal processing:

  • If nuclear-encoded, ATP9 requires removal of the mitochondrial targeting sequence after import

  • The timing and efficiency of this cleavage may affect incorporation into the ATP synthase complex

• Phosphorylation sites:

  • Potential phosphorylation of serine/threonine residues could regulate:

    • Protein-protein interactions within the ATP synthase complex

    • Proton channel formation and function

    • Assembly kinetics or stability

• Methods to study ATP9 PTMs:

  • Mass spectrometry analysis of purified ATP9 under different conditions

  • Site-directed mutagenesis of putative modification sites

  • In vitro kinase/phosphatase assays

  • Antibodies specific to modified forms

• Functional implications:

  • PTMs may coordinate ATP synthase assembly with cellular energy demands

  • Modifications could fine-tune proton conductance properties

  • PTMs might mediate responses to oxidative stress or other cellular signals

Understanding these modifications would provide insights into how A. gossypii regulates ATP synthase activity at the post-translational level and potentially reveal novel regulatory mechanisms.

What are the structural differences between mitochondrial-encoded and nuclear-encoded ATP9 proteins?

Nuclear-encoded and mitochondrial-encoded ATP9 proteins exhibit several key structural differences with important functional implications:

• Mitochondrial targeting sequence (MTS):

  • Nuclear-encoded ATP9 contains an N-terminal MTS absent in mitochondrial-encoded variants

  • In fungi with dual ATP9 genes, MTS sequences show different patterns of conservation (ATP9-5-like MTS are well conserved while ATP9-7-like MTS show greater divergence)

• Transmembrane domains:

  • Core functional transmembrane helices are generally conserved

  • Nuclear-encoded variants may show adaptations in hydrophobic residues that facilitate import

• C-terminal regions:

  • Significant divergence often occurs in C-terminal regions

  • These differences may affect interactions with other ATP synthase subunits

• Codon usage:

  • Nuclear-encoded variants adapt to nuclear codon usage patterns

  • This transition requires coordinated evolution of the gene after nuclear transfer

The dual ATP9 proteins in P. anserina (PaAtp9-5 and PaAtp9-7) share only 44% sequence identity yet are functionally interchangeable in complementation studies . This suggests significant structural plasticity in ATP9, allowing considerable sequence variation while maintaining essential functions. Comparative structural analysis between mitochondrial and nuclear variants could reveal evolutionary constraints on ATP synthase function.

What protocols are most effective for isolating functional mitochondria from A. gossypii for ATP9 studies?

Isolating functional mitochondria from filamentous fungi like A. gossypii requires specialized protocols due to their robust cell walls. An optimized procedure would include:

• Sample preparation:

  • Grow A. gossypii in liquid medium for 24-48 hours

  • Harvest mycelium by filtration and wash with ice-cold isolation buffer

  • Consider using different growth conditions to study life-cycle specific variations

• Cell disruption:

  • Enzymatic digestion of cell wall using lysing enzymes from Trichoderma harzianum

  • Gentle mechanical disruption using a Dounce homogenizer

  • Maintain samples at 4°C throughout processing

• Mitochondrial isolation:

  • Differential centrifugation (1,500g to remove cell debris, 12,000g to pellet mitochondria)

  • Further purification using Percoll gradient centrifugation

  • Resuspend in appropriate buffer containing ATP and substrate

• Quality assessment:

  • Oxygen consumption measurements using a Clark-type electrode

  • Membrane potential assessment using fluorescent dyes (JC-1, TMRM)

  • Citrate synthase activity as a marker enzyme

• ATP synthase activity measurement:

  • Spectrophotometric assays coupling ATP production to NADPH generation

  • Direct measurement of proton pumping using pH-sensitive fluorescent probes

  • Blue native PAGE to assess complex integrity

This protocol can be adapted for different developmental stages, allowing researchers to correlate ATP9 expression patterns with mitochondrial function throughout the A. gossypii life cycle .

How can researchers distinguish between the functions of multiple ATP9 isoforms if present in A. gossypii?

Based on the findings in P. anserina, A. gossypii might possess multiple ATP9 isoforms with distinct expression patterns and potentially specialized functions . To distinguish between their roles:

• Isoform-specific gene deletion:

  • Generate strains with individual ATP9 isoform deletions using CRISPR/Cas9

  • Analyze phenotypic effects at different developmental stages

  • Create double/multiple deletions with conditional complementation if essential

• Isoform swapping experiments:

  • Replace the coding sequence of one isoform with another

  • Maintain native regulatory elements to test functional equivalence

  • P. anserina studies showed that despite 56% sequence divergence, ATP9-5 and ATP9-7 proteins were functionally interchangeable when properly expressed

• Domain swapping analysis:

  • Create chimeric proteins with domains from different isoforms

  • Identify regions responsible for any functional differences

• Expression pattern analysis:

  • Use isoform-specific quantitative PCR primers

  • Employ reporter constructs with isoform-specific promoters

  • In P. anserina, PaAtp9-5 dominates during germination while PaAtp9-7 predominates during sexual reproduction

• Biochemical characterization:

  • Purify individual isoforms and reconstitute in liposomes

  • Compare proton conductance, oligomerization properties, and stability

These approaches would determine whether ATP9 isoforms in A. gossypii are functionally redundant or have evolved specialized roles for different developmental stages or environmental conditions.

What are the most informative phenotypic assays for characterizing ATP9 mutants in A. gossypii?

Comprehensive phenotypic characterization of ATP9 mutants should include:

• Growth and morphology analysis:

  • Growth rate on different carbon sources (fermentable vs. non-fermentable)

  • Colony morphology and hyphal extension rate

  • Microscopic examination of mitochondrial distribution using MitoTracker stains

  • Electron microscopy to assess mitochondrial ultrastructure and cristae formation

• Developmental phenotypes:

  • Sporulation efficiency and germination rates

  • Time-course analysis through complete life cycle

  • In P. anserina, different ATP9 isoforms are critical for specific developmental stages

• Bioenergetic parameters:

  • Oxygen consumption rates using respirometry

  • ATP/ADP ratios via luciferase-based assays or HPLC

  • Membrane potential measurements with potentiometric dyes

  • Reactive oxygen species (ROS) production using fluorescent indicators

• Stress response:

  • Sensitivity to oxidative stress (H₂O₂, menadione)

  • Response to mitochondrial inhibitors (oligomycin, CCCP)

  • Temperature sensitivity

  • Osmotic stress tolerance

• Metabolic profiling:

  • Riboflavin production capacity (A. gossypii is used industrially for vitamin B2 production)

  • Central carbon metabolism intermediates

  • Amino acid and lipid profiles

This multi-parameter assessment would provide insights into how ATP9 variants affect both mitochondrial function specifically and cellular physiology broadly, potentially revealing unexpected roles beyond ATP synthesis.

How can the evolutionary history of ATP9 gene transfer from mitochondria to nucleus inform experimental design?

The complex evolutionary history of ATP9 genes in fungi provides valuable context for experimental design:

• Phylogenetic analysis as a guide:

  • Five distinct patterns of ATP9 gene distribution exist across 26 fungal species

  • At least two independent mitochondria-to-nucleus transfers occurred

  • Multiple independent losses of either mitochondrial or nuclear genes followed these transfers

• Mitochondrial targeting sequence (MTS) considerations:

  • If generating recombinant ATP9, researchers must consider appropriate MTS design

  • ATP9-5-like and ATP9-7-like sequences have different conservation patterns

  • Optimal MTS may differ between developmental stages or growth conditions

• Evolutionary rate analysis:

  • Compare substitution rates between mitochondrial and nuclear ATP9 genes

  • Identify conserved residues likely critical for function

  • Target mutational analysis to evolutionarily variable regions first

• Horizontal comparison approach:

  • Test functional complementation between ATP9 genes from different fungi

  • In P. anserina, despite only 44% sequence identity, ATP9 isoforms were functionally interchangeable

  • Cross-species complementation can reveal fundamental functional constraints

• Experimental evolution strategies:

  • Subject A. gossypii to conditions that might select for altered ATP9 function

  • Monitor for compensatory mutations when ATP9 is modified

  • Compare with natural evolutionary patterns

This evolutionary perspective helps researchers focus on the most informative experimental approaches and interpret results within a broader context of ATP9 functional constraints and adaptability.

How do regulatory mechanisms for ATP9 expression differ between A. gossypii and other model fungi?

While specific information about A. gossypii ATP9 regulation is limited, comparative analysis with other fungi reveals diverse regulatory strategies:

This comparative approach would reveal both conserved and species-specific aspects of ATP9 regulation, providing insights into how energy metabolism is tailored to specific ecological niches and life histories.

What insights from Podospora anserina ATP9 studies can be applied to A. gossypii research?

The extensive studies of ATP9 in P. anserina provide valuable insights applicable to A. gossypii research:

• Dual nuclear genes with specialized functions:

  • P. anserina has two nuclear ATP9 genes (PaAtp9-5 and PaAtp9-7) with distinct expression patterns

  • PaAtp9-5 is critical during germination while PaAtp9-7 is essential for sexual reproduction

  • A. gossypii researchers should search for multiple ATP9 genes and assess their expression across life stages

• Functional interchangeability despite sequence divergence:

  • Despite only 44% sequence identity, P. anserina ATP9 proteins are functionally interchangeable when properly expressed

  • This suggests experimental flexibility when designing complementation studies

  • The critical factor may be expression pattern rather than precise sequence

• Promoter swapping strategy:

  • In P. anserina, swapping promoters between ATP9 genes revealed the importance of proper regulation

  • Similar approaches in A. gossypii could:

    • Determine if expression timing is more important than protein sequence

    • Identify critical regulatory elements

    • Establish minimal expression requirements for different developmental stages

• Correlation with energy demands:

  • High ATP9 expression in P. anserina correlates with high-energy-demand processes

  • In A. gossypii, researchers should examine ATP9 expression during riboflavin production

  • Engineering ATP9 expression might optimize bioenergetics for biotechnological applications

• Experimental design considerations:

  • Use stage-specific sampling as in P. anserina (germination, active growth, reproduction)

  • Consider the possibility that deletion of one ATP9 isoform may be compensated by others

  • Design complementation constructs with appropriate regulatory elements

These insights provide a valuable framework for investigating ATP9 biology in A. gossypii, potentially accelerating research by building on established patterns from P. anserina.

How can recombinant ATP9 be used to enhance riboflavin production in A. gossypii?

A. gossypii is industrially significant for riboflavin (vitamin B2) production . Strategic manipulation of ATP9 could enhance this capability:

• Energy optimization strategies:

  • Riboflavin biosynthesis requires significant ATP

  • Modulating ATP9 expression to coordinate with production phases could:

    • Increase ATP availability during peak biosynthetic activity

    • Reduce energy expenditure during preparatory phases

    • Balance growth and production requirements

• Expression timing engineering:

  • Based on P. anserina findings, ATP9 genes show stage-specific expression

  • Creating modified promoters with production-phase specific expression could:

    • Enhance energy availability during riboflavin synthesis

    • Reduce metabolic burden during growth phases

• Redox balance considerations:

  • ATP synthase function affects cellular redox state

  • Riboflavin production involves multiple redox reactions

  • Strategic ATP9 variants could optimize NADH/NAD+ and FADH₂/FAD ratios

• Experimental approaches:

  • Generate strains with modified ATP9 expression patterns

  • Test inducible promoters to synchronize ATP production with riboflavin synthesis

  • Employ metabolic flux analysis to identify energy bottlenecks

  • Use adaptive laboratory evolution to select for enhanced ATP9 variants

• Process integration:

  • Coordinate ATP9 regulation with other genetic modifications

  • Consider two-stage fermentation processes with different ATP9 expression patterns

  • Optimize feed strategies based on ATP9-mediated energy production capacity

This targeted bioenergetic engineering could significantly enhance the industrial utility of A. gossypii for riboflavin production and potentially other high-value compounds.

What methodological approaches can resolve contradictory data about ATP9 localization and function?

When faced with contradictory data regarding ATP9 localization or function, researchers should employ multiple complementary approaches:

• Genomic location verification:

  • Southern blotting with mitochondrial and nuclear DNA fractions

  • Long-read sequencing to resolve complex genomic regions

  • PCR with primers specific to mitochondrial vs. nuclear contexts

  • In situ hybridization to visualize genomic loci

• Protein localization approaches:

  • Immunogold electron microscopy with ATP9-specific antibodies

  • Fractionation studies with western blot validation

  • Fluorescent protein tagging with appropriate controls

  • Import assays with isolated mitochondria

• Functional validation:

  • Complementation of ATP9-deficient strains with:

    • Native gene versions

    • Tagged constructs

    • Genes expressed from different promoters

  • Measurement of ATP synthase activity in isolated mitochondria

  • Respiratory chain complex assembly analysis by blue native PAGE

• Temporal and spatial resolution:

  • Time-course experiments throughout development

  • Cell-type specific analysis if appropriate

  • In P. anserina, different ATP9 genes function at different life cycle stages

• Integration of approaches:

  • Combine genetic, biochemical, and imaging techniques

  • Use quantitative rather than qualitative assessments when possible

  • Consider dynamic rather than static models of ATP9 function

How can synthetic biology approaches be used to engineer novel ATP9 variants with enhanced properties?

Synthetic biology offers powerful tools for engineering novel ATP9 variants with enhanced or altered properties:

• Rational design strategies:

  • Structure-guided mutations targeting:

    • Proton-binding sites to modify conductance properties

    • Oligomerization interfaces to affect complex stability

    • Interaction sites with other ATP synthase subunits

  • Codon optimization for improved expression

  • Modified regulatory elements for precise expression control

• Directed evolution approaches:

  • Create ATP9 variant libraries using error-prone PCR

  • Design selection systems based on:

    • Growth under energy-limiting conditions

    • Resistance to oxidative stress

    • Enhanced riboflavin production

  • Employ continuous evolution systems with iterative selection

• Domain swapping and chimeric proteins:

  • Create fusion proteins with parts from different fungal ATP9 variants

  • Test interchangeability of transmembrane domains and termini

  • P. anserina studies showed functional interchangeability despite 56% sequence divergence

• Orthogonal ATP synthase engineering:

  • Introduce modified ATP9 variants that function with specific partner subunits

  • Create parallel energy production systems within the same organism

  • Design synthetic regulatory circuits controlling different ATP9 variants

• Experimental validation approaches:

  • In vitro reconstitution in liposomes

  • Biophysical characterization of proton conductance

  • In vivo growth and development analysis

  • Metabolic output measurements (ATP/ADP ratio, riboflavin production)

These synthetic biology approaches could yield ATP9 variants with precisely tuned properties for specific research applications or industrial processes, potentially enhancing riboflavin production or enabling new biotechnological capabilities in A. gossypii.