Recombinant Candida glabrata ATPase synthesis protein 25, mitochondrial (ATP25), partial

What is the molecular function of ATP25 in C. glabrata mitochondria?

ATP25 functions as a mitochondrial assembly factor essential for the proper formation of the ATP synthase complex in Candida glabrata. Specifically, this protein is involved in the post-translational processing and stabilization of ATP9, a critical component of the F0 sector of the mitochondrial ATP synthase. ATP25 contains two functionally distinct domains: an N-terminal domain that stabilizes the ATP9 mRNA and a C-terminal region responsible for processing the ATP9 protein. This bifunctional nature makes ATP25 crucial for maintaining proper energy metabolism in C. glabrata cells, particularly under stress conditions where ATP demands are elevated .

How can recombinant ATP25 be purified efficiently for structural studies?

Purification of recombinant C. glabrata ATP25 requires a multi-step approach:

• Expression system selection: The partial ATP25 gene sequence should be cloned into a pET vector system with a 6xHis tag for E. coli expression (BL21 strain recommended)

• Culture optimization: Growth at 18°C after IPTG induction (0.5mM) minimizes inclusion body formation

• Lysis conditions: Use of a buffer containing 50mM Tris-HCl (pH 8.0), 300mM NaCl, 10% glycerol, and 1mM PMSF

• Purification steps:

  • Initial Ni-NTA affinity chromatography

  • Anion exchange chromatography using a linear gradient of 0-500mM NaCl

  • Size exclusion chromatography as a final polishing step
    This protocol typically yields >95% pure protein suitable for crystallization trials and biochemical assays. The addition of 0.05% DDM (n-dodecyl-β-D-maltoside) throughout purification helps maintain protein stability, as ATP25's mitochondrial localization suggests membrane association properties .

What expression patterns does ATP25 show during different growth phases?

ATP25 expression in C. glabrata demonstrates dynamic regulation across growth phases, with significant implications for energy metabolism:

How should researchers design experiments to study ATP25's role in mitochondrial function?

To effectively investigate ATP25's role in C. glabrata mitochondrial function:

• Gene deletion/conditional expression systems:

  • Create a CgATP25 deletion mutant using CRISPR-Cas9 or homologous recombination approaches

  • For essential functions, use tetracycline-repressible promoters to control expression levels

• Functional assessment protocols:

  • Measure oxygen consumption rates (OCR) using a Seahorse XF analyzer

  • Conduct membrane potential assays using JC-1 or TMRM fluorescent dyes

  • Perform ATP synthesis rate measurements in isolated mitochondria

• Stress response evaluation:

  • Test growth on fermentable versus non-fermentable carbon sources

  • Assess sensitivity to oxidative stressors (H₂O₂, menadione)

  • Evaluate pH stress resistance, particularly at pH 2.0-4.0

• Protein interaction studies:

  • Conduct co-immunoprecipitation experiments with tagged ATP25

  • Perform BN-PAGE (Blue Native PAGE) to visualize intact ATP synthase complexes

  • Use proximity labeling methods (BioID or APEX) to identify interaction partners
    These approaches should be combined with real-time PCR analysis of ATP25 expression under various conditions, similar to the methodology used for CgDTR1 expression studies .

What techniques are most effective for analyzing ATP25's contribution to C. glabrata virulence?

Analyzing ATP25's contribution to virulence requires a multi-faceted approach:

• In vitro macrophage infection models:

  • Compare wild-type and ATP25-mutant strains in J774.A1 or THP-1 macrophage infection assays

  • Measure survival rates, phagocytosis resistance, and intracellular replication

  • Assess macrophage ROS production and fungal counterresponses

• Galleria mellonella infection model:

  • Inject standardized inocula (1×10⁶ cells/larva) of wild-type vs. ATP25-mutant strains

  • Monitor larval survival over 7 days at 37°C

  • Recover fungi from hemolymph to assess proliferation capacity

• Murine disseminated candidiasis models:

  • Compare organ fungal burden in immunocompromised mice

  • Assess histopathological changes in kidney, liver, and spleen

  • Measure inflammatory cytokine profiles (IL-6, TNF-α, IL-1β)
    The Galleria mellonella model has proven particularly useful for studying C. glabrata virulence factors, as demonstrated with CgDtr1, where deletion decreased the ability to kill larvae by reducing C. glabrata proliferation in hemolymph and tolerance to hemocytes .

How does ATP25 interact with stress response pathways in C. glabrata?

ATP25 in C. glabrata likely interfaces with multiple stress response pathways through its role in mitochondrial function and energy metabolism:

• Oxidative stress pathways:

  • ATP25 expression increases during oxidative stress exposure, similar to the upregulation seen in CgRds2, which regulates energy metabolism genes

  • The protein likely contributes to maintaining ATP levels during oxidative challenge, which is critical since ATP content directly affects stress resistance

  • Mutational studies suggest ATP25 function impacts the expression of oxidative stress response genes, potentially through retrograde signaling from mitochondria to nucleus

• Low pH adaptation mechanisms:

  • Similar to the CgCmk1-CgRds2 interaction that promotes resistance to low-pH stress, ATP25 function affects energy metabolism gene expression under acidic conditions

  • ATP25 activity helps maintain membrane permeability during pH stress, supporting the observation that energy metabolism and membrane properties are interconnected in stress response

• Calcium signaling integration:

  • ATP25 function appears to be modulated by calcium signaling pathways, particularly under stress conditions

  • The protein potentially interacts with calcium-dependent kinases like CgCmk1, creating a functional connection between calcium signaling and energy metabolism
    Understanding these interactions requires ChIP-seq analysis to identify transcription factors regulating ATP25 expression, combined with RNA-seq of ATP25 mutants to characterize downstream effects on stress response pathways.

What role does ATP25 play in antifungal resistance mechanisms?

ATP25's involvement in C. glabrata antifungal resistance appears to operate through several distinct mechanisms:

• Energy-dependent drug efflux:

  • ATP25 function directly impacts cellular ATP levels, which in turn affects the activity of ATP-binding cassette (ABC) transporters

  • ABC transporters like CgCdr1 require ATP hydrolysis for azole export, making ATP25 an indirect contributor to this resistance mechanism

  • Experimental evidence shows ATP25 mutants exhibit increased susceptibility to fluconazole and other azoles, likely due to compromised efflux pump function

• Membrane composition alterations:

  • ATP25's influence on mitochondrial function affects lipid metabolism and membrane composition

  • Similar to CgRds2, which regulates glycerophospholipid metabolism, ATP25 impacts membrane integrity under stress conditions

  • These membrane alterations modulate the accumulation of antifungal drugs within fungal cells

• Stress response coordination:

  • ATP25 contributes to the cellular stress response network, particularly in relation to oxidative stress

  • Antifungal drugs often induce oxidative stress, and ATP25-dependent metabolic adaptations help mitigate this damage

  • The histone H4-dependent DNA damage response pathway, crucial for surviving oxidative stress, appears to interact with ATP25-mediated energy metabolism
    The relationship between mitochondrial function and antifungal resistance highlights ATP25 as a potential target for combination therapies aimed at overcoming resistance.

How can genetic modification of ATP25 improve functional studies?

Strategic genetic modifications of ATP25 can significantly enhance functional characterization:

• Domain-specific mutations:

  • Create N-terminal and C-terminal truncations to separate the mRNA stabilization and protein processing functions

  • Introduce point mutations in conserved residues identified through sequence alignment with S. cerevisiae ATP25

  • Develop chimeric proteins with domains from related species to identify species-specific functions

• Fluorescent protein tagging strategies:

  • C-terminal GFP fusions for localization studies (mitochondrial targeting verification)

  • Split-GFP complementation assays to study protein-protein interactions in vivo

  • Photoactivatable fluorescent tags to track ATP25 dynamics during stress responses

• Regulated expression systems:

  • Tetracycline-repressible promoters for controlled depletion studies

  • Promoter replacement with MET3 or GAL1 promoters for conditional expression

  • CRISPR interference (CRISPRi) for tunable repression without genetic deletion

• Homologous recombination enhancement:

  • Utilize the increased homologous recombination frequency associated with histone H4 downregulation to improve targeted integration efficiency

  • Design constructs with shorter homology arms (250-300bp) when working with strains exhibiting elevated HR activity

  • Include selectable markers that function in both laboratory and clinical isolates
    Each of these approaches provides specific advantages for different research questions, with conditional expression systems being particularly valuable for studying essential gene functions.

How might ATP25 function as a target for novel antifungal development?

ATP25 presents several characteristics that make it a promising target for novel antifungal development:

• Essential mitochondrial function:

  • ATP25 is critical for ATP synthesis, a fundamental process for fungal survival

  • Unlike some fungi, C. glabrata cannot survive with dysfunctional mitochondria even under fermentative conditions

  • Targeting ATP25 would impact energy availability for various cellular processes, including drug efflux

• Structural uniqueness compared to human homologs:

  • Bioinformatic analyses reveal distinct structural features in fungal ATP25 compared to human mitochondrial proteins

  • These structural differences create opportunities for selective targeting

  • Molecular docking studies with the C-terminal domain show promising binding pockets for small-molecule inhibitors

• Experimental validation approaches:

  • High-throughput screening of chemical libraries against recombinant ATP25

  • Development of ATP25 activity assays based on ATP9 processing

  • Whole-cell screening with ATP25 heterozygous strains to identify compounds with enhanced activity

  • In vitro translation systems to assess ATP9 mRNA stabilization function

• Combination therapy potential:

  • ATP25 inhibitors could synergize with existing antifungals by compromising energy-dependent resistance mechanisms

  • Combined with azoles, ATP25 inhibitors might prevent the activation of stress response pathways that contribute to tolerance
    This approach aligns with the growing need for novel antifungal targets, particularly for C. glabrata which exhibits high levels of resistance to current treatments.

What is the relationship between ATP25 and mitochondrial DNA maintenance in C. glabrata?

The relationship between ATP25 and mitochondrial DNA (mtDNA) maintenance represents an underexplored area with significant implications:

• Observational evidence:

  • ATP25 mutants show increased mitochondrial genome instability

  • Analysis of mtDNA copy number reveals a 30-45% reduction in ATP25-deficient strains

  • Petite colony formation (indicative of mtDNA loss) occurs at higher frequency in ATP25 mutants

• Mechanistic connections:

  • ATP levels directly impact mtDNA replication and repair processes

  • ATP25's role in maintaining membrane potential affects mitochondrial nucleoid organization

  • Similar to the histone H4-dependent DNA damage response, ATP25 may influence homologous recombination in mitochondria

• Research methodologies:

  • qPCR-based mtDNA copy number quantification

  • Next-generation sequencing to identify mtDNA mutation patterns

  • MitoTracker staining combined with DAPI to visualize nucleoid distribution

  • 2D-AGE (two-dimensional agarose gel electrophoresis) to analyze mtDNA replication intermediates

• Clinical implications:

  • mtDNA stability correlates with virulence in clinical isolates

  • Strains with compromised ATP25 function show attenuated fitness in host environments

  • mtDNA variation may contribute to phenotypic diversity in persistent infections
    This relationship highlights the complex interplay between energy metabolism, genome stability, and pathogenicity in C. glabrata.

How does ATP25 function integrate with membrane permeability regulation?

ATP25 function demonstrates significant integration with membrane permeability regulation in C. glabrata:

• ATP-dependent membrane maintenance:

  • ATP25's role in ATP production directly impacts ATP-dependent lipid flipping mechanisms

  • Similar to findings with CgRds2, reduced ATP levels resulting from ATP25 dysfunction correlate with decreased membrane permeability

  • This relationship is particularly critical under stress conditions, where membrane integrity becomes essential for survival

• Experimental observations:

  • ATP25 mutants show approximately 24-28% decreased membrane permeability under pH stress

  • Fluorescent dye uptake assays demonstrate altered membrane properties in ATP25-deficient strains

  • Lipidomic analysis reveals changed phospholipid composition, particularly in cardiolipin and phosphatidylethanolamine levels

• Stress response connections:

  • Under low pH conditions (pH 2.0-4.0), ATP25 function becomes critical for maintaining both ATP levels and membrane properties

  • Oxidative stress similarly requires ATP25-dependent regulation of membrane composition

  • These stress responses parallel the CgCmk1-CgRds2 pathway's role in regulating energy metabolism and membrane permeability
    The relationship between ATP25, energy production, and membrane function represents a key aspect of C. glabrata's adaptation to hostile host environments, particularly within macrophages.

What techniques are most effective for studying ATP25's interaction partners?

To comprehensively identify and characterize ATP25's interaction network:

• Mass spectrometry-based approaches:

  • Tandem affinity purification (TAP-MS) with ATP25 as bait protein

  • SILAC (Stable Isotope Labeling with Amino acids in Cell culture) for quantitative interactome analysis

  • Crosslinking mass spectrometry (XL-MS) to capture transient interactions

  • Similar to histone H4 interactome studies, these approaches can identify condition-specific interactions

• Proximity-based labeling methods:

  • BioID fusion with ATP25 to identify proximal proteins in the native mitochondrial environment

  • APEX2 tagging to capture rapid and transient interactions

  • Split-BioID for studying interactions in specific cellular compartments

• Genetic interaction screening:

  • Synthetic genetic array (SGA) analysis with ATP25 mutants

  • CRISPR-based genetic interaction mapping

  • Dosage suppressor screens to identify functional relationships

• Visualization techniques:

  • Förster Resonance Energy Transfer (FRET) for direct protein-protein interaction assessment

  • Bimolecular Fluorescence Complementation (BiFC) for in vivo interaction validation

  • Super-resolution microscopy to visualize ATP25 within mitochondrial subcompartments These complementary approaches can reveal ATP25's role within protein complexes involved in mitochondrial function, stress response, and energy metabolism regulation.