Recombinant Neosartorya fumigata Mitochondrial inner membrane protease atp23 (atp23)

What is the primary function of ATP23 in fungal mitochondria?

ATP23 serves dual functions in mitochondria: it acts as a metalloprotease that processes the N-terminal presequence of F₀-ATPase subunit 6, and it functions as a chaperone that facilitates the assembly of the F₀ complex by mediating the association of subunit 6 with the subunit 9 ring . The proteolytic activity of ATP23 depends on its HEXXH motif, which is characteristic of metalloproteases. This motif contains two histidine residues and a glutamic acid that participate in zinc binding and catalytic mechanism, respectively . Interestingly, research in Saccharomyces cerevisiae has shown that the chaperone function can operate independently of the protease function, as mutants with inactive protease domains can still assemble functional ATPase complexes despite accumulating the subunit 6 precursor .

How is ATP23 localized within fungal mitochondria?

ATP23 is an inner mitochondrial membrane protein with its C-terminus facing the intermembrane space . This localization has been determined through protease protection assays and subcellular fractionation studies. For example, in S. cerevisiae, ATP23 tagged with HA epitope shows sensitivity to proteinase K in mitoplasts (mitochondria with disrupted outer membranes) but not in intact mitochondria, similar to known intermembrane space proteins like Sco1p . This orientation is consistent with its role in processing the N-terminus of subunit 6, which faces the intermembrane space. For recombinant expression studies, this localization must be considered when designing constructs and purification strategies.

How conserved is ATP23 across fungal species?

ATP23 is highly conserved across eukaryotes from yeast to humans . The conservation is particularly strong in the metalloprotease domain containing the HEXXH motif. When working with Neosartorya fumigata (Aspergillus fumigatus) ATP23, researchers should note that while core functions may be conserved, species-specific differences in protein interactions or regulatory mechanisms might exist. Comparative sequence analysis between S. cerevisiae and A. fumigatus ATP23 can help identify conserved domains that are likely crucial for function versus regions that may have evolved differently in these distantly related fungi.

What are the optimal expression systems for producing recombinant N. fumigata ATP23?

For expressing recombinant N. fumigata ATP23, researchers should consider several expression systems based on experimental goals. For structural studies requiring high protein yields, bacterial expression in E. coli with appropriate modifications may work, though eukaryotic post-translational modifications will be absent. Yeast expression systems (S. cerevisiae or Pichia pastoris) offer advantages for a mitochondrial protein like ATP23, as they provide a eukaryotic environment with appropriate folding machinery.

For functional studies, consider expressing ATP23 in S. cerevisiae atp23 null mutants (W303ΔATP23) to assess complementation . This approach would determine whether N. fumigata ATP23 can functionally replace the S. cerevisiae ortholog. When designing expression constructs, include appropriate tags (His, GST, or HA) for purification and detection, but place tags at positions that don't interfere with the HEXXH catalytic domain or membrane association.

What methods are most effective for assessing ATP23 proteolytic activity in vitro?

To evaluate the proteolytic activity of recombinant N. fumigata ATP23, researchers should design assays using purified recombinant ATP23 and suitable substrates. Based on research with S. cerevisiae ATP23, the natural substrate is the N-terminal presequence of ATPase subunit 6 .

A typical in vitro assay would include:

• Purified recombinant ATP23 protein

• Synthetic peptides corresponding to the N-terminal region of N. fumigata subunit 6

• Appropriate buffer conditions (pH, salt concentration)

• Metal ions (particularly zinc, given ATP23's metalloprotease nature)

Activity can be monitored by:

• SDS-PAGE to observe substrate cleavage

• Mass spectrometry to identify precise cleavage sites

• Fluorescence-based assays with quenched fluorescent peptide substrates

Controls should include ATP23 with mutations in the HEXXH motif (particularly E→Q mutation) that eliminate proteolytic activity while maintaining protein folding . The effect of metal chelators (EDTA) should also be tested to confirm the metal-dependent nature of the proteolytic activity.

How can protein-protein interactions between ATP23 and other mitochondrial proteins be studied?

To investigate interactions between N. fumigata ATP23 and other mitochondrial proteins, particularly subunit 6 and potential homologs of ATP10, several complementary approaches can be employed:

• Co-immunoprecipitation: Express epitope-tagged ATP23 in Aspergillus fumigatus or in a heterologous system, isolate mitochondria, solubilize with mild detergents, and perform immunoprecipitation followed by mass spectrometry to identify interacting partners.

• Yeast two-hybrid assays: While limited for membrane proteins, modified membrane yeast two-hybrid systems can be used for specific domains of ATP23.

• Bimolecular Fluorescence Complementation (BiFC): Split fluorescent proteins fused to ATP23 and potential interacting partners can reveal interactions in vivo.

• Cross-linking studies: Chemical cross-linking coupled with mass spectrometry can identify transient or weak interactions.

• Suppressor studies: Following the approach used with S. cerevisiae, test whether N. fumigata ATP23 overexpression can suppress phenotypes of mutations in other assembly factors (like ATP10 homologs) . The research with S. cerevisiae demonstrated that an extra copy of ATP23 was an effective suppressor of an atp10 null mutant, suggesting functional overlap between these proteins .

How do mutations in the HEXXH motif affect ATP23 function in mitochondrial ATPase assembly?

Mutations in the HEXXH motif of ATP23, particularly the glutamic acid residue essential for catalytic activity, have distinct effects on its dual functions. Based on studies in S. cerevisiae, substituting the catalytic glutamic acid with glutamine (E→Q) eliminates the proteolytic activity of ATP23, resulting in accumulation of the unprocessed subunit 6 precursor .

Interestingly, despite this proteolytic deficiency, the E→Q mutant can still restore respiratory growth in atp23 null mutants and facilitate assembly of a functional ATPase complex. This indicates that the chaperone function of ATP23 remains intact and can operate independently of its protease activity . To study this phenomenon in N. fumigata ATP23:

• Generate recombinant ATP23 with the E→Q mutation in the HEXXH motif

• Assess its ability to process subunit 6 using in vitro assays

• Test for ATPase assembly by complementation studies

• Measure ATPase activity and oligomycin sensitivity (as shown in Table 2 from the S. cerevisiae studies, where wild-type ATPase shows ~80% inhibition by oligomycin, whereas mutants show much lower inhibition)

What is the relationship between ATP23 and mitochondrial respiratory function in N. fumigata?

Given the role of ATP23 in ATPase assembly and the importance of mitochondrial function for fungal growth, particularly under hypoxic conditions, this question addresses a critical aspect of N. fumigata physiology. In Aspergillus fumigatus, adaptation to hypoxia is an important component of growth during pulmonary infections .

To investigate this relationship:

• Generate ATP23 deletion or conditional mutants in A. fumigatus

• Assess growth and mitochondrial respiration under normoxic and hypoxic conditions

• Measure oxygen consumption rates and ATPase activity

• Analyze mitochondrial membrane potential and ROS production

• Evaluate the expression of alternative respiratory pathways (like alternative oxidase) that might compensate for defects in the main respiratory chain

From studies in A. fumigatus, we know that the mitochondrial electron transport chain plays important roles in hypoxia adaptation and virulence . Specifically, cytochrome C is required for A. fumigatus germination and growth in normoxia and hypoxia, while the alternative oxidase shows different roles in stress responses . The relationship between ATP23 and these respiratory components would be an important area of investigation.

How does ATP23 activity change in response to antifungal stressors?

This question explores how ATP23 function might be modulated during antifungal stress responses. Given that azole drugs are primary treatments for A. fumigatus infections and mitochondrial function can influence stress responses, understanding ATP23's role under these conditions is valuable.

Research approaches should include:

• Exposing A. fumigatus to sub-lethal concentrations of different antifungals (azoles, polyenes, echinocandins)

• Measuring ATP23 expression levels, protein abundance, and localization

• Assessing mitochondrial morphology and function

• Analyzing ATPase assembly and activity

• Comparing wild-type and ATP23 mutant strains for antifungal susceptibility

Of particular interest would be whether ATP23 is involved in adaptive responses that contribute to antifungal resistance. Some transcription factors, like AtrR in A. fumigatus, are central players in azole resistance , and mitochondrial function may interact with these resistance mechanisms.

What are the structural determinants of ATP23's dual protease and chaperone functions?

Understanding how a single protein performs distinct protease and chaperone functions represents an advanced research question. For N. fumigata ATP23, structural biology approaches would be valuable:

• Determine the crystal or cryo-EM structure of ATP23, both wild-type and proteolytically inactive mutants

• Identify domains responsible for membrane association, substrate binding, and chaperone function

• Perform molecular dynamics simulations to understand conformational changes

• Use structure-guided mutagenesis to create separation-of-function mutants (defective in only one function)

• Characterize protein dynamics using hydrogen-deuterium exchange mass spectrometry

The HEXXH motif is clearly critical for the proteolytic function , but the structural elements required for the chaperone function remain poorly defined. Identifying these elements would provide insights into how ATP23 facilitates the association of subunit 6 with the subunit 9 ring during F₀ assembly .

How does ATP23 coordinate with other assembly factors in the biogenesis of fungal F₁F₀-ATPase?

To address this question:

• Identify all putative ATPase assembly factors in N. fumigata through homology searches

• Generate an interaction map using proteomics approaches

• Perform genetic interaction studies (synthetic lethality, suppressor screens)

• Analyze assembly intermediates that accumulate in various mutant backgrounds

• Use live-cell imaging to track the temporal sequence of assembly events

In S. cerevisiae, the interaction between ATP23 and ATP10 appears to be functional rather than physical, as they do not cosediment in sucrose gradients . The model suggests a cooperative interaction where both proteins interact with subunit 6 to stabilize it and facilitate its assembly into the F₀ complex .

What is the role of ATP23 in virulence and pathogenicity of N. fumigata in pulmonary infections?

This question connects fundamental mitochondrial biology to fungal pathogenesis. Given that hypoxia is an important component of host microenvironments during pulmonary fungal infections , and mitochondrial function is critical for adaptation to these conditions, ATP23's role in virulence merits investigation.

Research approaches should include:

• Generating conditional ATP23 mutants in A. fumigatus

• Assessing virulence in murine models of invasive pulmonary aspergillosis

• Measuring fungal burden, inflammatory responses, and survival rates

• Analyzing transcriptional and metabolic adaptations of wild-type versus ATP23 mutants during infection

• Evaluating ATP23 as a potential antifungal target

Research on the A. fumigatus electron transport chain has shown that components like cytochrome C play important roles in virulence, as evidenced by the attenuated virulence of cytochrome C null mutants in murine models of invasive pulmonary aspergillosis . Whether ATP23, through its role in ATPase assembly, similarly contributes to virulence remains to be determined.

What purification strategies yield the highest activity for recombinant N. fumigata ATP23?

Purifying membrane-associated proteases like ATP23 while maintaining their activity presents significant challenges. Optimal strategies include:

• Membrane protein extraction using mild detergents (digitonin, DDM, or CHAPS)

• Affinity chromatography using tags that don't interfere with function

• Size exclusion chromatography to separate monomeric from aggregated protein

• Ion exchange chromatography for further purification

Critical considerations include:

• Detergent concentration must be sufficient for solubilization without denaturing the protein

• Including zinc or other divalent cations in buffers to maintain metalloprotease activity

• Using protease inhibitors that don't target metalloproteases during early purification steps

• Avoiding freeze-thaw cycles that can reduce activity

Activity assays should be performed at each purification step to track retention of both proteolytic and chaperone functions.

How can ATP23 activity be measured in whole cells versus isolated mitochondria?

This question addresses the methodological challenges of measuring ATP23 activity in different experimental contexts.

For whole-cell assays:

• Use reporter constructs with ATP23 target sequences fused to detectable markers

• Monitor ATPase assembly through respiration measurements

• Assess subunit 6 processing using Western blotting from cell extracts

For isolated mitochondria:

• Prepare mitochondria using standard isolation techniques

• Measure subunit 6 processing directly by incubating precursor proteins with mitochondrial extracts

• Assess ATPase assembly by blue native PAGE

• Measure oligomycin-sensitive ATPase activity (as shown in Table 2 from the S. cerevisiae studies)

Comparative analysis between these approaches can reveal important differences between in vivo and in vitro activity, potentially identifying additional regulatory factors present in the cellular environment.

What assays can distinguish between the protease and chaperone functions of ATP23?

Separating and specifically measuring the dual functions of ATP23 requires specialized assays:

For protease activity:

• In vitro cleavage assays using synthetic peptides corresponding to the subunit 6 N-terminus

• Western blotting to detect precursor versus mature forms of subunit 6

• Mass spectrometry to identify specific cleavage sites

For chaperone activity:

• Complementation of atp23 null mutants with proteolytically inactive ATP23 (E→Q mutant)

• Monitoring ATPase assembly by blue native PAGE

• Measuring oligomycin-sensitive ATPase activity (in S. cerevisiae, the E→Q mutant showed substantial oligomycin sensitivity of 80-92%, indicating formation of properly assembled F₀)

• Protein aggregation prevention assays using model substrates

• Co-immunoprecipitation to detect interaction with subunit 6

The ability to distinguish these functions is critical for understanding ATP23's complete role in mitochondrial biogenesis. The table below, based on data from S. cerevisiae, illustrates how ATPase activity and oligomycin sensitivity can be used to assess F₀ assembly even when proteolytic processing is defective: