Recombinant Neurospora crassa ATP synthase subunit 9, mitochondrial (atp-9)

How is ATP synthase subunit 9 synthesized and processed in Neurospora crassa?

In Neurospora crassa, ATP synthase subunit 9 is synthesized as a precursor protein with an N-terminal extension that serves as a mitochondrial targeting sequence (MTS). The biosynthetic pathway involves:

• Initial Synthesis: The precursor is synthesized on free cytosolic ribosomes (not membrane-bound) as directed by nuclear mRNA associated with free polysomes .

• Post-translational Processing: After synthesis, the precursor forms a high molecular weight aggregate in the cytosol before being imported into mitochondria. The import process involves recognition by a receptor on the mitochondrial surface and is dependent on the electrical potential across the inner mitochondrial membrane .

• Proteolytic Cleavage: During import, the N-terminal presequence is cleaved by mitochondrial processing peptidases, converting the 16,400 Da precursor to the 10,500 Da mature form .

• Assembly: Following import and processing, the mature protein integrates into the inner mitochondrial membrane, where it acquires specific properties including protease resistance, solubility in chloroform/methanol, and the ability to be immunoprecipitated with antibodies to F₁-ATPase .

This processing is inhibited by carbonylcyanide m-chlorophenylhydrazone (CCCP), which disrupts the mitochondrial membrane potential required for protein import .

Why is the location of the ATP9 gene different across fungal species?

The ATP9 gene exhibits variable genomic localization across fungal species, representing a fascinating example of gene transfer during evolution:

This variable localization reflects evolutionary gene transfer events from the mitochondrial to the nuclear genome. In P. anserina, the gene appears to be exclusively nuclear-encoded, with no sequence similarity detected in the mitochondrial genome. The nuclear-encoded version contains a 63-amino acid N-terminal presequence that directs mitochondrial import of the precursor protein .

The successful evolutionary transfer required several adaptations, particularly in the protein's structure to reduce hydrophobicity, which facilitates proper import into mitochondria from the cytosol .

What specific challenges exist in relocating ATP9 from mitochondrial to nuclear genomes experimentally?

The experimental relocation of ATP9 from mitochondrial to nuclear genomes presents significant challenges due to:

• Extreme Hydrophobicity: ATP9 encodes one of the most hydrophobic proteins in the mitochondrial proteome, making cytosolic synthesis and subsequent import particularly difficult. The hydrophobic segments can cause protein aggregation or misfolding in the cytosol .

• Import Barriers: For successful relocation, the nuclear-encoded protein must include an effective mitochondrial targeting sequence (MTS) and have reduced hydrophobicity in its first transmembrane segment to facilitate membrane passage during import .

• Cellular Adaptations: Experiments with P. anserina ATP9 genes expressed in S. cerevisiae demonstrate that even when functional import occurs, it perturbs cellular properties including morphology and activates stress responses like the heat shock response .

• Assembly Efficiency: Nuclear-expressed ATP9 (even when successfully imported) shows reduced efficiency in assembly into the ATP synthase complex. In experimental relocations, oxygen consumption rates of strains with nuclear ATP9 reach only 40-80% of wild-type levels depending on the specific construct used .

Successful experimental relocation has been achieved using naturally nuclear versions from filamentous fungi expressed in yeast, with the P. anserina ATP9-5 gene providing better functionality than ATP9-7 when expressed in S. cerevisiae .

What methods are used for recombinant expression and purification of Neurospora crassa ATP synthase subunit 9?

Recombinant expression and purification of N. crassa ATP synthase subunit 9 involves several specialized techniques:

• Expression Systems Selection:

  • Bacterial systems (E. coli) can be used for N. crassa ATP9, though expression of highly hydrophobic membrane proteins often requires optimization

  • Codon optimization based on the host organism is essential for efficient expression

• Vector Design Considerations:

  • Addition of appropriate mitochondrial targeting sequence (MTS) if mitochondrial localization is desired

  • Selection of appropriate promoters (e.g., Tet-off system for controlled expression)

  • Choice between centromeric (low-copy) or multicopy plasmids based on required expression levels

• Purification Strategy:

  • Detergent-based extraction from membranes using mild detergents to preserve native structure

  • Organic solvent extraction (chloroform/methanol) exploiting the proteolipid properties

  • Affinity chromatography using tags added to the recombinant protein

  • Buffer optimization to maintain protein stability during purification

• Storage Considerations:

  • Optimal storage at -20°C in Tris-based buffer with 50% glycerol

  • Avoiding repeated freeze-thaw cycles

  • Preparing working aliquots for short-term storage at 4°C

The particularly hydrophobic nature of this protein requires special attention to solubilization methods and buffer components throughout the purification process.

How can researchers verify proper expression and assembly of recombinant ATP9 in experimental systems?

Verification of proper expression and assembly of recombinant ATP9 requires a multi-faceted approach:

• Western Blot Analysis:

  • Detection of both precursor and mature forms using specific antibodies

  • Monitoring processing efficiency by comparing precursor/mature protein ratios

  • Assessment of expression levels compared to native controls

• Functional Assays:

  • Oxygen consumption measurements to evaluate oxidative phosphorylation capacity

  • ATP synthesis rates in isolated mitochondria or reconstituted systems

  • Membrane potential measurements to assess proton-pumping activity

• Structural Integration Assessment:

  • Blue native PAGE to analyze incorporation into ATP synthase complexes

  • Protease protection assays (properly assembled subunit 9 is resistant to added proteases)

  • Solubility in organic solvents (chloroform/methanol) to confirm proteolipid properties

  • Immunoprecipitation with antibodies to F₁-ATPase to verify association with the ATP synthase complex

• Subcellular Localization:

  • Fractionation studies to confirm mitochondrial localization

  • Immunofluorescence microscopy to visualize mitochondrial targeting

  • In vitro import assays using isolated mitochondria to assess import efficiency

For example, in experiments expressing P. anserina ATP9 in S. cerevisiae, respiratory growth on glycerol medium provides an initial screen, followed by detailed bioenergetic characterization showing 40-80% of wild-type oxygen consumption rates depending on the construct used .

How does ATP synthase subunit 9 contribute to studies of mitochondrial permeability transition?

ATP synthase subunit 9 is instrumental in understanding the mitochondrial permeability transition (mPT), a process linked to cell death mechanisms:

• Channel Formation Properties:

  • Reconstituted ATP synthase complexes containing subunit 9 can form large Ca²⁺-dependent channels that resemble the mitochondrial permeability transition pore

  • These channels exhibit conductances up to 600 pS and voltage-dependent gating properties

  • The channel formation capacity provides direct evidence for the role of ATP synthase components in mitochondrial permeability regulation

• Regulatory Interactions:

  • The channels formed by subunit 9-containing complexes respond to classical mPT modulators:

    • Inhibition by ADP and bongkrekate

    • Modulation by cyclophilin

    • Sensitivity to cyclosporin A (which abolishes cyclophilin effects)

    • Response to oxidative stress (e.g., tert-butyl hydroperoxide reversibly suppresses voltage gating)

• Experimental Approaches:

  • Patch-clamp experiments with reconstituted recombinant ADP/ATP carrier and ATP synthase components

  • Electrophysiological characterization of channel properties under various conditions

  • Pharmacological manipulations to identify regulatory mechanisms

These studies with recombinant components provide mechanistic insights into how ATP synthase components may contribute to the formation of the permeability transition pore, a key event in mitochondrial-mediated cell death pathways .

What can be learned from comparing different nuclear-encoded ATP9 variants from filamentous fungi?

Comparative analysis of nuclear-encoded ATP9 variants from filamentous fungi provides valuable insights into protein evolution and mitochondrial bioenergetics:

• Functional Differences:

  • The P. anserina nuclear genome contains two ATP9 genes (ATP9-5 and ATP9-7) that are differentially expressed during the organism's life cycle

  • When expressed in S. cerevisiae, ATP9-5 provides greater respiratory capacity than ATP9-7 (80% vs. 40% of wild-type oxygen consumption)

  • These functional differences suggest distinct roles or expression regulation during development

• Structural Determinants of Import Efficiency:

  • Comparison of N-terminal sequences reveals differences in mitochondrial targeting sequences

  • Analysis of transmembrane domains shows variations in hydrophobicity profiles that correlate with import efficiency

  • Chimeric constructs demonstrate that reduced hydrophobicity in the first transmembrane segment is critical for successful mitochondrial import

• Evolutionary Adaptations:

  • Sequence comparison between species shows extensive conservation in the mature protein region but significant variation in the N-terminal targeting sequences

  • These differences reflect adaptations to species-specific import machinery

  • P. anserina ATP9 shows sequence identity with corresponding proteins from N. crassa, Aspergillus nidulans, and A. niger, indicating evolutionary relationships

These comparative studies provide a framework for understanding the requirements for successful gene transfer from mitochondria to nucleus during evolution .

What methodological approaches are most effective for studying the assembly of ATP synthase subunit 9 into functional complexes?

Investigating the assembly of ATP synthase subunit 9 into functional complexes requires specialized methodological approaches:

• Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE):

  • Allows visualization of intact ATP synthase complexes and assembly intermediates

  • Can be combined with second-dimension SDS-PAGE to identify specific components

  • Western blotting with subunit-specific antibodies confirms the presence of subunit 9 in complexes

  • Enables quantitative assessment of assembly efficiency under different conditions

• Pulse-Chase Experiments:

  • Monitor the kinetics of subunit 9 synthesis, import, and assembly

  • Radioactive labeling allows tracking of newly synthesized proteins

  • Immunoprecipitation at different time points captures assembly intermediates

  • Particularly useful for comparing efficiency between wild-type and mutant or recombinant variants

• Crosslinking Studies:

  • Identify interaction partners during assembly process

  • Chemical crosslinkers of various lengths can probe spatial relationships

  • Mass spectrometry analysis of crosslinked products identifies specific contacts

  • Helps establish the sequence of assembly events

• In Organello Assembly Systems:

  • Isolated mitochondria can import labeled precursor proteins

  • Assembly can be monitored in a controlled environment

  • Effects of specific inhibitors or conditions can be systematically tested

  • Allows direct comparison between different protein variants or mutants

• Cryo-Electron Microscopy:

  • Provides structural insights into the arrangement of subunit 9 in the c-ring

  • Allows visualization of assembly intermediates at near-atomic resolution

  • Can reveal structural differences between variants from different species

  • Helps correlate functional properties with structural features

Applying these complementary approaches provides a comprehensive understanding of the complex process by which subunit 9 is incorporated into the ATP synthase complex and identifies the factors that influence assembly efficiency in different experimental systems .

What are the major technical difficulties in working with recombinant ATP synthase subunit 9 and how can they be overcome?

Working with recombinant ATP synthase subunit 9 presents several technical challenges:

• Extreme Hydrophobicity:

  • Challenge: The highly hydrophobic nature causes aggregation during expression and purification

  • Solution: Use specialized detergents (e.g., digitonin, DDM) for extraction; include stabilizing agents like glycerol; optimize buffer conditions; consider fusion partners that enhance solubility

• Proper Mitochondrial Targeting:

  • Challenge: Efficient import into mitochondria requires specific N-terminal sequences and reduced hydrophobicity

  • Solution: Design chimeric constructs with proven mitochondrial targeting sequences; select naturally nuclear versions from filamentous fungi that have evolved efficient import mechanisms; test multiple presequence variants

• Functional Reconstitution:

  • Challenge: Maintaining native conformation and activity after purification

  • Solution: Use gentle extraction methods; reconstitute into liposomes with lipid compositions mimicking the mitochondrial membrane; validate function through proton conductance or ATP synthesis assays

• Expression Level Optimization:

  • Challenge: Balancing expression levels to avoid toxicity while ensuring sufficient protein production

  • Solution: Use regulatable promoters (e.g., Tet-off system); compare centromeric (low-copy) versus multicopy plasmids; optimize codon usage for expression host

• Proper Assembly Assessment:

  • Challenge: Confirming correct integration into ATP synthase complexes

  • Solution: Combine functional assays (oxygen consumption, ATP synthesis) with structural analysis (BN-PAGE, crosslinking studies); use protease protection assays to confirm proper membrane insertion

Researchers have successfully addressed these challenges by using naturally nuclear versions from filamentous fungi as templates, which have already evolved solutions to these problems during the evolutionary transfer of the gene from mitochondria to nucleus .

How might research on ATP synthase subunit 9 contribute to understanding mitochondrial disease mechanisms?

Research on ATP synthase subunit 9 provides valuable insights into mitochondrial disease mechanisms:

• Bioenergetic Dysfunction Models:

  • Manipulating subunit 9 expression or structure creates models with defined defects in oxidative phosphorylation

  • These models allow systematic investigation of how ATP synthase deficiencies affect cellular metabolism

  • Comparative studies between nuclear and mitochondrial expression systems help understand tissue-specific impacts of mitochondrial gene mutations

• Mitochondrial Permeability Transition Insights:

  • Subunit 9's role in channel formation relates to mitochondrial permeability transition, a process implicated in neurodegenerative diseases and ischemia-reperfusion injury

  • Studying how subunit 9 contributes to channel formation and regulation provides mechanistic understanding of cell death pathways

  • The Ca²⁺-dependent channels formed by recombinant systems offer controllable models to test therapeutic interventions

• Protein Import and Processing Defects:

  • Studies of subunit 9 import illuminate general principles of mitochondrial protein import

  • Defects in import machinery components affect highly hydrophobic proteins like subunit 9 first

  • Understanding these mechanisms helps explain pathologies resulting from mutations in mitochondrial import machinery

• Mitochondrial-Nuclear Communication:

  • The natural existence of both mitochondrial and nuclear-encoded versions provides insights into retrograde signaling

  • Expression of nuclear ATP9 in systems with mitochondrial ATP9 deletions demonstrates how cells adapt to changes in mitochondrial function

  • These adaptations include altered cellular morphology and activation of stress responses like the heat shock pathway

• Therapeutic Strategy Development:

  • Successful expression of nuclear-encoded ATP9 provides proof-of-concept for allotopic expression as a therapeutic approach

  • This strategy could potentially address mitochondrial DNA mutations by providing functional nuclear-encoded alternatives

  • The techniques developed may apply to other mitochondrially-encoded proteins involved in human diseases

By continuing to investigate the complex biology of ATP synthase subunit 9, researchers can develop more effective approaches to diagnosing and treating mitochondrial disorders that affect ATP production and mitochondrial membrane integrity .