Recombinant Debaryomyces hansenii Mitochondrial inner membrane protease ATP23 (ATP23)

What is ATP23 and what are its primary functions in yeast mitochondria?

ATP23 is a metalloprotease encoded by the nuclear genome that performs dual critical functions in mitochondria:

• It processes the N-terminal presequence (10 residues) from the precursor form of subunit 6 (Atp6p) of the mitochondrial ATP synthase (F₁-F₀ complex)

• It functions as a chaperone that mediates the assembly of the F₀ sector of ATP synthase, particularly facilitating the association of subunit 6 with the subunit 9 ring

ATP23 is associated with the mitochondrial inner membrane and is localized to the intermembrane space . The protein is conserved from yeast to humans, suggesting evolutionary importance . The human ATP23 gene is located on chromosome 12 and has been associated with diseases including osteogenesis imperfecta type IV .

How can we distinguish between the proteolytic and chaperone functions of ATP23?

Experimental evidence with mutant studies has definitively separated ATP23's dual functions:

• The HEXXH motif (specifically residue E168 in S. cerevisiae) is critical for metalloprotease activity

• Mutation of this residue (E168Q) eliminates protease activity while maintaining chaperone function

• Cells expressing only the E168Q mutant accumulate the subunit 6 precursor but can still assemble a functional F₁-F₀ complex

This indicates that ATP23's chaperone function is sufficient for ATP synthase assembly even when its proteolytic activity is absent. The table below shows ATPase activity measurements demonstrating this phenomenon:

Table shows ATPase activity without/with oligomycin and percent inhibition by oligomycin, which indicates functional F₀ assembly

What are the optimal approaches for expressing recombinant ATP23 in D. hansenii?

D. hansenii has considerable biotechnological potential due to its osmotolerance and stress resistance. For recombinant expression of proteins like ATP23:

• PCR-based gene targeting with homologous recombination has proven highly efficient (>75% success rate)

• Transformants can be generated using PCR products with just 50 bp flanks identical to the target site

• Heterologous selectable markers conferring Hygromycin B or G418 resistance can be used

For expression vector construction:

• Promoters like TEF1 from Arxula adeninivorans have shown high expression levels

• In vivo DNA assembly is feasible in D. hansenii, allowing co-transformation of up to three different DNA fragments with 30-bp homologous overlapping overhangs

A recently developed CRISPR-Cas9 toolbox for D. hansenii provides additional genetic engineering options for precise manipulation of the ATP23 gene .

How can researchers assess the functionality of recombinant ATP23?

Several complementary approaches can be used to evaluate ATP23 function:

• Proteolytic activity assessment:

  • Monitoring the processing of subunit 6 precursor using in vivo labeling of mitochondrial translation products with [³⁵S]methionine

  • Western blot analysis to detect precursor vs. mature forms of subunit 6

• Chaperone function assessment:

  • Measuring oligomycin-sensitive ATPase activity (indicates proper F₀ assembly)

  • Analyzing ATP synthase complex integrity via blue native PAGE

  • Growth phenotype analysis on non-fermentable carbon sources (e.g., glycerol/ethanol)

• Protein interaction studies:

  • Co-immunoprecipitation (Co-IP) to detect interactions with ATP10 or subunit 6

  • Sucrose gradient sedimentation to assess complex formation

Which domains and motifs are essential for ATP23 activity?

ATP23 contains several crucial structural elements:

• Metalloprotease domain:

  • The HEXXH motif is characteristic of metalloproteases and essential for proteolytic activity

  • The two conserved histidine residues and the glutamic acid residue in this motif are critical for protease function

• Chaperone domain:

  • Residues 112-115 (LRDK) have been identified as required for the assembly function of ATP23

  • This chaperone function is independent of the proteolytic activity

• Mitochondrial targeting:

  • ATP23 is targeted to the mitochondrial intermembrane space

  • The protein associates with the inner membrane despite lacking membrane-spanning segments

How does Mia40 contribute to ATP23 biogenesis and function?

Mia40 plays a crucial role in ATP23 biogenesis through:

• Oxidative folding:

  • Mia40 directly interacts with ATP23 and introduces disulfide bonds

  • Under anaerobic conditions, Mia40 can completely oxidize all cysteine residues in ATP23

  • Both the substrate-binding region and the CPC motif of Mia40 are critical for its ability to oxidize ATP23

• Folding assistance:

  • Mia40 promotes proper folding of ATP23 into a protease-resistant conformation

  • Following translocation of reduced ATP23 precursor into the intermembrane space, Mia40 introduces five disulfide bonds

  • This oxidation converts ATP23 into its folded, functional state

The Mia40-dependent folding can be assessed using protease accessibility assays, where properly folded ATP23 shows resistance to trypsin digestion .

How do ATP23 and ATP10 coordinately regulate ATP synthase assembly?

ATP23 and ATP10 work together in a coordinated manner to facilitate ATP synthase assembly:

• Physical association:

  • Co-immunoprecipitation and blue native PAGE experiments demonstrate that ATP23 and ATP10 physically associate with each other

  • This interaction appears to be transient as they do not consistently cosediment in sucrose gradients

• Functional cooperation:

  • ATP10 forms a complex with newly synthesized subunit 6 and confers stability

  • ATP10 is required for association of subunit 6 with the subunit 9 ring

  • ATP23 can partially compensate for ATP10 deficiency when overexpressed

• Complementary roles:

  • In an ATP10 null mutant, overexpression of ATP23 increases the stability of subunit 6

  • Expression of ATP23 increases in ATP10 null mutants compared to wild type

  • After 72 hours of growth, ATP10 null mutants show leaky growth on respiratory substrates, presence of low levels of subunit 6, and partial recovery of oligomycin sensitivity

What is the mechanism behind ATP23 suppression of ATP10 deletion?

The suppression mechanism involves:

• Increased ATP23 expression:

  • ATP23 levels naturally increase in ATP10 null mutants after 24 hours of growth

  • This compensatory mechanism stabilizes some subunit 6 molecules

• Dual function requirement:

  • Both functions of ATP23 (proteolytic and chaperone) are required for the partial rescue

  • The residues 112-115 (LRDK) of ATP23 are essential for this suppression

• Stabilization effect:

  • Overexpression of ATP23 increases the stability of subunit 6 in ATP10 null strains

  • This allows a small percentage (5-15%) of F₁-F₀ ATPase to be assembled even in the absence of ATP10

The suppression is partial, suggesting that while ATP23 can compensate for some ATP10 functions, both proteins normally act cooperatively for optimal ATP synthase assembly.

How might salt stress affect ATP23 expression and function in D. hansenii?

D. hansenii is renowned for its halotolerance, and research considerations regarding ATP23 under salt stress should include:

• Expression regulation:

  • D. hansenii shows differential transcriptomic and proteomic responses to Na⁺ vs. K⁺

  • Studying ATP23 expression changes under different salt conditions could reveal regulatory mechanisms

• Energy metabolism implications:

  • High salt environments require additional energy for ion homeostasis

  • ATP synthase assembly and function become crucial under these conditions

  • ATP23's role in maintaining ATP synthase function may be particularly important during salt stress

• Potential interaction with salt-response mechanisms:

  • D. hansenii employs multiple transporters for salt tolerance, including H⁺-ATPases (DhPma1, DhVma2), Na⁺/H⁺-antiporter (DhNha1), and Na⁺-ATPase (DhEna1)

  • Potential functional interactions between ATP23 and these salt-responsive proteins warrant investigation

How can researchers address the challenges of studying ATP23 in different yeast species?

When investigating ATP23 across different yeast species, researchers should consider:

• Genetic manipulation strategies:

  • Species-specific transformation protocols (S. cerevisiae vs. D. hansenii)

  • Optimize homologous recombination efficiency (50 bp flanks sufficient for D. hansenii)

  • Selection marker compatibility across species

• Functional conservation assessment:

  • Complementation studies (can ATP23 from one species rescue defects in another?)

  • Cross-species protein interaction analyses

  • Comparative analysis of ATP23 processing activity on subunit 6 from different species

• Experimental design considerations:

  • Growth conditions optimization (D. hansenii thrives in salt-rich media)

  • Mitochondrial isolation protocols may need adjustment for different yeast species

  • Consider growth rate differences when designing time-course experiments

This cross-species approach can provide valuable insights into both conserved mechanisms and species-specific adaptations of ATP23 function.