Recombinant Candida glabrata Mitochondrial import inner membrane translocase subunit TIM14 (PAM18)

What is C. glabrata PAM18 and what is its role in mitochondrial function?

C. glabrata PAM18 (also known as TIM14, protein identifier Q6FPU1) is a mitochondrial import inner membrane translocase subunit that plays an essential role in protein import into mitochondria. As a member of the DnaJ family of proteins, PAM18 contains a canonical tripeptide HPD (Histidine-Proline-Aspartate) motif that is crucial for stimulating the ATPase activity of Hsp70 . This stimulation drives the translocation of preproteins into the mitochondrial matrix, making PAM18 an essential component of the Presequence translocase-Associated Motor (PAM) complex in the mitochondrial inner membrane protein import machinery .

How does C. glabrata PAM18 compare to its orthologs in other species?

C. glabrata PAM18 (Q6FPU1) shares orthologous relationships with proteins from multiple species. According to ortholog group analyses, it shows similarity to mitochondrial import proteins in other fungi, including Aspergillus kawachii and Ustilago maydis . Unlike plant PAM18 orthologs, which can be dual-targeted to both mitochondria and plastids depending on tissue type, C. glabrata PAM18 is exclusively mitochondria-targeted . Understanding these evolutionary relationships helps contextualize functional conservation and divergence across species.

What is the relationship between PAM18 and PAM16 in the mitochondrial import machinery?

PAM18 functions in close association with PAM16, a DnaJ-like protein that lacks the HPD motif essential for Hsp70 ATPase stimulation. PAM16 forms a heterodimer with PAM18 and regulates its activity . This interaction is crucial for proper functioning of the mitochondrial protein import apparatus. While PAM18 directly stimulates the ATPase activity of Hsp70, PAM16 modulates this stimulation through its interaction with PAM18. Both proteins are essential for yeast viability, highlighting their critical roles in mitochondrial function .

What expression systems are optimal for producing recombinant C. glabrata PAM18?

For recombinant expression of C. glabrata PAM18, researchers typically use either homologous expression in C. glabrata itself or heterologous expression in Saccharomyces cerevisiae. For C. glabrata expression, copper-inducible promoters such as the MTI promoter have proven effective for controlled expression . The construction typically involves:

• PCR amplification of the PAM18 gene (CAGL0M06281g or similar designation)

• Creation of a recombinant plasmid using homologous recombination

• Replacement of standard promoters (like GAL1) with the copper-inducible MTI promoter

• Verification by DNA sequencing

For protein purification, a histidine tag is often added to facilitate affinity chromatography while maintaining protein function.

What are the key considerations for designing PAM18 gene deletion experiments in C. glabrata?

When designing PAM18 gene deletion experiments in C. glabrata, researchers should consider:

• Essential gene status: Since PAM18 is essential for viability in S. cerevisiae , conditional deletion strategies may be necessary

• Complementation system: Include a rescuing plasmid expressing PAM18 to confirm phenotype specificity

• Phenotypic analysis: Assess mitochondrial function through multiple parameters:

  • Respiratory capacity

  • Mitochondrial protein import efficiency

  • Cell growth under various stress conditions

  • Mitochondrial morphology

• Control comparisons: Include related deletions (e.g., other PAM complex components) to distinguish specific from general effects

• Strain background: Deletion phenotypes may vary between clinical and laboratory C. glabrata strains

How can researchers effectively detect and localize PAM18 in C. glabrata cells?

For detection and localization of PAM18 in C. glabrata, researchers can employ:

• Fluorescent fusion proteins: Creating GFP-tagged PAM18 under native or controlled promoters for in vivo localization

• Immunodetection: Developing specific antibodies against C. glabrata PAM18 or using epitope tags for Western blotting and immunofluorescence

• Subcellular fractionation: Isolating mitochondria and subfractionating to confirm inner membrane localization

• In vitro import assays: Using radiolabeled PAM18 precursors with isolated mitochondria to analyze import kinetics and requirements

• Co-localization studies: With known mitochondrial markers to confirm submitochondrial localization

How does PAM18 contribute to stress tolerance and virulence in C. glabrata?

While PAM18's direct role in C. glabrata virulence is not fully characterized, evidence from related mitochondrial systems suggests significant contributions:

• Mitochondrial function impact: Altered mitochondrial function (as in mip1Δ mutants) can increase C. glabrata's resistance to azole antifungals and enhance survival within phagocytes

• Stress response integration: Proper protein import into mitochondria is essential for cellular responses to various stresses encountered during host infection

• Energy metabolism: PAM18-dependent mitochondrial function affects cellular energy production necessary for virulence factor expression

• Potential connection to "petite" phenotype: Mitochondrial dysfunction creates a "petite" phenotype that can increase resistance to various stresses and improve survival in macrophages

To experimentally address this question, researchers should assess PAM18 conditional mutants in:

• Phagocyte survival assays

• Infection models like Galleria mellonella

• Stress response to oxidative agents, azoles, and acetic acid

What is the structural basis for PAM18's interaction with other components of the PAM complex?

The structural basis of PAM18's interactions within the PAM complex involves:

• J-domain interactions: The canonically conserved HPD motif in PAM18's J-domain is crucial for stimulating Hsp70's ATPase activity

• PAM16 heterodimer formation: Specific domains mediate the interaction between PAM18 and PAM16

• Membrane anchoring: PAM18 contains domains for anchoring to the inner mitochondrial membrane

• Tim44 interactions: PAM complex assembly involves interactions with the peripheral membrane protein Tim44

Research approaches to elucidate these interactions include:

• Site-directed mutagenesis of key residues

• Co-immunoprecipitation studies

• Crosslinking experiments

• Structural studies using X-ray crystallography or cryo-EM

• In vitro reconstitution of minimal functional complexes

How does C. glabrata PAM18 function compare with homologs in pathogenic versus non-pathogenic fungi?

Comparative analysis reveals both similarities and differences:

To investigate these differences, researchers should:

• Perform complementation studies across species

• Compare protein-protein interaction networks

• Assess functional conservation in heterologous expression systems

• Analyze expression patterns during infection processes

What techniques are most effective for assessing PAM18's role in mitochondrial protein import?

To evaluate PAM18's role in mitochondrial protein import:

• In vitro import assays:

  • Isolate mitochondria from wild-type and PAM18-depleted cells

  • Use radiolabeled precursor proteins to track import efficiency

  • Analyze import kinetics through time-course experiments

• Reconstitution systems:

  • Purify recombinant components of the import machinery

  • Reconstitute minimal systems with liposomes

  • Test the impact of PAM18 variants on import function

• Genetic approaches:

  • Create conditional mutants or protein depletion systems

  • Analyze synthetic genetic interactions with other import machinery components

  • Perform high-throughput screens for suppressors of PAM18 deficiency

• Structural analysis:

  • Use protein crosslinking to map interaction surfaces

  • Perform hydrogen-deuterium exchange mass spectrometry

  • Employ cryo-EM for complex visualization

How can researchers effectively study the PAM18-PAM16 interaction in C. glabrata?

The critical PAM18-PAM16 interaction can be studied through:

• Co-expression systems:

  • Express both proteins with different tags in C. glabrata

  • Perform reciprocal co-immunoprecipitation

  • Use proximity-based labeling techniques like BioID

• Mutagenesis approaches:

  • Create systematic mutations in predicted interaction surfaces

  • Assess impact on complex formation and function

  • Identify suppressor mutations that restore interaction

• Biochemical characterization:

  • Measure ATPase stimulation activity of PAM18 with and without PAM16

  • Determine binding constants through surface plasmon resonance or isothermal titration calorimetry

  • Analyze oligomeric states using size exclusion chromatography

• In vivo analyses:

  • Create fluorescent protein fusions to visualize co-localization

  • Use split-fluorescent protein complementation to detect interactions

  • Assess impact of stress conditions on complex formation

What are the best approaches for studying PAM18's role in C. glabrata pathogenesis?

To investigate PAM18's contribution to C. glabrata pathogenesis:

• Infection models:

  • Use Galleria mellonella larvae as an invertebrate model

  • Employ murine models of disseminated candidiasis

  • Develop conditional expression systems to bypass essentiality

• Phagocyte interaction studies:

  • Analyze survival within macrophages and neutrophils

  • Measure proliferation in hemolymph or after phagocytosis

  • Assess resistance to antimicrobial mechanisms

• Stress response analysis:

  • Test tolerance to oxidative stress

  • Evaluate resistance to acetic acid stress and other relevant stressors

  • Assess antifungal susceptibility patterns

• Transcriptomic and proteomic approaches:

  • Compare global expression profiles between wild-type and PAM18-altered strains

  • Analyze mitochondrial proteome changes

  • Identify potential regulatory networks connecting mitochondrial function to virulence traits

What are the major challenges in expressing and purifying functional recombinant PAM18?

Researchers face several challenges when working with recombinant PAM18:

• Membrane protein purification:

  • PAM18 is anchored to the inner mitochondrial membrane

  • Requires careful detergent selection for solubilization

  • May lose functionality when removed from native membrane environment

• Maintaining protein stability:

  • J-domain proteins often require specific conditions for stability

  • May require co-expression with interaction partners

  • Aggregation can occur during concentration steps

• Achieving proper folding:

  • Heterologous expression systems may lack specific chaperones

  • Mitochondrial targeting sequences can interfere with recombinant expression

  • Post-translational modifications may differ between expression systems

• Functional assessment:

  • Activity assays require reconstitution of complex interactions

  • Multiple protein components needed for meaningful functional tests

  • Difficult to distinguish direct from indirect effects

How can contradictory findings about PAM18 function across different fungal species be reconciled?

When faced with contradictory findings about PAM18 function:

• Methodological standardization:

  • Compare results using identical experimental conditions

  • Develop consensus protocols for functional assays

  • Ensure proper controls for species-specific factors

• Evolutionary context analysis:

  • Conduct phylogenetic analysis of PAM18 sequences

  • Identify lineage-specific adaptations

  • Consider selective pressures in different ecological niches

• Systems-level comparison:

  • Map protein interaction networks across species

  • Consider compensatory mechanisms in different genetic backgrounds

  • Analyze differences in regulatory mechanisms

• Cross-species complementation:

  • Test functional interchangeability through heterologous expression

  • Identify domains responsible for species-specific functions

  • Create chimeric proteins to pinpoint functional differences

What emerging technologies might advance our understanding of PAM18's role in mitochondrial biogenesis and fungal pathogenicity?

Several cutting-edge approaches show promise for PAM18 research:

• CRISPR-based technologies:

  • CRISPRi for conditional repression of essential genes

  • Base editors for precise mutagenesis without double-strand breaks

  • CRISPR screens to identify genetic interactions

• Advanced imaging techniques:

  • Super-resolution microscopy for submitochondrial localization

  • Live-cell imaging to track dynamic interactions

  • Correlative light and electron microscopy for structural context

• Single-cell approaches:

  • Single-cell RNA-seq to capture heterogeneity in pathogen populations

  • Microfluidics for tracking individual cell responses to stress

  • Single-molecule tracking to analyze protein dynamics

• Computational methods:

  • Molecular dynamics simulations of protein interactions

  • Systems biology models of mitochondrial import

  • Machine learning approaches to predict functional consequences of mutations