Recombinant Candida glabrata Mitochondrial import inner membrane translocase subunit TIM16 (PAM16)

What is the fundamental role of PAM16/TIM16 in Candida glabrata mitochondria?

PAM16 (also known as TIM16) in Candida glabrata functions as an essential component of the presequence translocase-associated motor (PAM) complex, which drives protein import into the mitochondrial matrix. The protein plays a critical regulatory role in the ATP-dependent translocation of proteins across the inner mitochondrial membrane by modulating the activity of mitochondrial heat shock proteins. In particular, PAM16 forms a complex with PAM18 (also known as TIM14) to regulate the ATPase activity of mortalin/mtHsp70, preventing premature ATP hydrolysis and ensuring efficient protein import .

What experimental approaches can verify the subcellular localization of recombinant PAM16 in C. glabrata?

To verify the subcellular localization of recombinant PAM16 in C. glabrata, researchers can employ several complementary approaches:

• Fluorescence microscopy with GFP fusion proteins: Express PAM16-GFP fusion proteins in C. glabrata cells using inducible promoters (such as the copper-inducible MTI promoter). Visualize the localization pattern at standardized cell density (OD600nm of 0.5 ± 0.05) after approximately 5 hours of induction .

• Subcellular fractionation: Isolate mitochondria using differential centrifugation, followed by Western blot analysis with antibodies specific to PAM16 and markers for different mitochondrial compartments.

• Immunogold electron microscopy: For precise localization within mitochondrial subcompartments, use immunogold labeling with anti-PAM16 antibodies.

• Controls: Include known mitochondrial membrane proteins (like CgDtr1, which shows predominantly cell periphery localization) as comparative controls for localization patterns .

What are the optimal cloning strategies for recombinant C. glabrata PAM16 expression?

For optimal cloning and expression of recombinant C. glabrata PAM16, consider the following methodological approach:

• Gene amplification: Design primers with appropriate restriction sites or overlapping sequences for the cloning method of choice. Include sequences for adding purification tags (His, GST, etc.) if required.

• Vector selection: For expression in yeast systems (recommended for proper folding), use vectors with selectable markers and inducible promoters like the copper-inducible MTI promoter, which has been successfully used for C. glabrata proteins .

• Promoter considerations: Replace standard promoters (like GAL1) with C. glabrata-specific promoters for expression in the native organism. This can be accomplished through PCR-based methods using primers containing:

  • Homology to the target promoter sequence (e.g., MTI promoter)

  • Homology to vector cloning site flanking regions

  • The resulting fragment can be co-transformed with the linearized vector for recombination-based cloning

• Verification: Confirm recombinant plasmids by DNA sequencing before transformation into expression hosts.

Which expression systems are most effective for producing functional recombinant C. glabrata PAM16?

The selection of an appropriate expression system is critical for obtaining functional recombinant C. glabrata PAM16. Consider these methodological options:

For mitochondrial proteins like PAM16, yeast expression systems (particularly C. glabrata itself or S. cerevisiae) typically provide the most biologically relevant environment for proper folding and function .

How can researchers assess the protein import function of recombinant PAM16 in vitro?

To assess the protein import function of recombinant PAM16, researchers can employ these methodological approaches:

• Reconstituted in vitro import assay:

  • Isolate mitochondria from PAM16-depleted C. glabrata cells

  • Add recombinant PAM16 protein in varying concentrations

  • Assess import of radiolabeled mitochondrial precursor proteins

  • Measure import efficiency through autoradiography and quantitative analysis

• ATPase activity modulation assay:

  • Purify recombinant mtHsp70 (HSPA9 homolog) and PAM18

  • Add recombinant PAM16 in various concentrations

  • Measure ATP hydrolysis rates using colorimetric phosphate detection assays

  • Plot the inhibitory effect of PAM16 on PAM18-stimulated ATPase activity

• Protein-protein interaction analysis:

  • Use pull-down assays with tagged recombinant PAM16

  • Identify interaction partners through mass spectrometry

  • Confirm specific interactions with PAM complex components through co-immunoprecipitation

  • Quantify binding affinities using surface plasmon resonance or microscale thermophoresis

• Complementation in PAM16-deficient strains:

  • Transform PAM16-depleted yeast with recombinant PAM16 variants

  • Assess restoration of mitochondrial protein import and cell viability

  • Compare with wild-type controls to determine functional efficacy

What experimental controls are essential when studying C. glabrata PAM16 interaction with other mitochondrial import components?

When studying C. glabrata PAM16 interactions with other mitochondrial import components, include these essential controls:

• Negative interaction controls:

  • Empty vector/tag-only controls to identify non-specific binding

  • Unrelated mitochondrial proteins of similar size/charge

  • Mutated PAM16 variants lacking key interaction domains

• Positive interaction controls:

  • Known interaction partners (PAM18, mitochondrial HSP70 homologs)

  • Conserved interactions demonstrated in related species

• Specificity controls:

  • Competition assays with unlabeled proteins

  • Dose-dependent interaction analyses

  • Cross-linking followed by mass spectrometry to confirm direct interactions

• System validation controls:

  • Expression level verification through Western blotting

  • Subcellular localization confirmation

  • Functional complementation in deletion strains

• Technical controls:

  • Non-denaturing conditions preservation during isolation

  • Multiple methodologies to confirm interactions (e.g., Y2H, BiFC, co-IP)

  • Reproducibility across independent biological replicates

What methods can distinguish between the specific functions of PAM16 and its interaction partner PAM18 in C. glabrata?

To distinguish between the specific functions of PAM16 and PAM18 in C. glabrata:

• Domain swap experiments:

  • Create chimeric proteins containing domains from PAM16 and PAM18

  • Express these in appropriate deletion backgrounds

  • Assess mitochondrial import function restoration

  • Identify which domains confer which specific functions

• Differential interactome analysis:

  • Perform immunoprecipitation with tagged PAM16 and PAM18 separately

  • Use mass spectrometry to identify unique and common interaction partners

  • Create network maps highlighting specific protein associations

  • Validate key differential interactions through targeted approaches

• Selective depletion studies:

  • Develop conditional mutants with independently controllable PAM16 and PAM18 expression

  • Monitor time-course effects on specific mitochondrial functions

  • Measure import of different classes of precursor proteins

  • Determine temporal requirements for each protein in the import process

• Structural biology approaches:

  • Obtain crystal or cryo-EM structures of PAM16, PAM18, and their complex

  • Identify key interaction residues through structural analysis

  • Create point mutations affecting specific functional residues

  • Test mutant proteins in functional assays to map structure-function relationships

How can researchers investigate the impact of PAM16 mutations on mitochondrial protein import efficiency in C. glabrata?

To investigate how PAM16 mutations affect mitochondrial protein import in C. glabrata:

• Mutation library creation:

  • Generate site-directed mutants targeting conserved residues

  • Create random mutagenesis libraries

  • Design mutations mimicking known pathogenic variants from homologous proteins

  • Express mutant proteins with appropriate tags for detection

• In vivo import assessment:

  • Transform PAM16 mutants into PAM16-depleted C. glabrata

  • Express reporter proteins with mitochondrial targeting sequences

  • Measure reporter protein accumulation in mitochondria versus cytosol

  • Quantify import efficiency through fractionation and Western blotting

• Real-time import kinetics:

  • Isolate mitochondria from strains expressing PAM16 variants

  • Add fluorescently labeled precursor proteins

  • Monitor import rates through fluorescence quenching assays

  • Calculate import rate constants for different mutations

• Structure-function correlation:

  • Map mutations to structural models of PAM16

  • Correlate functional defects with specific structural perturbations

  • Use molecular dynamics simulations to predict mutation effects

  • Validate predictions with targeted biochemical assays

How does C. glabrata PAM16 function compare with homologs in other pathogenic fungi and potential implications for antifungal targets?

Understanding the comparative biology of PAM16 across pathogenic fungi offers insights into potential antifungal targets:

• Homology analysis methodology:

  • Perform multiple sequence alignments of PAM16 proteins from C. glabrata, C. albicans, Aspergillus species, and Cryptococcus species

  • Identify conserved functional domains versus species-specific regions

  • Construct phylogenetic trees to visualize evolutionary relationships

  • Map conservation onto structural models to identify functional hotspots

• Functional conservation assessment:

  • Test cross-species complementation by expressing PAM16 homologs in C. glabrata PAM16 deletion strains

  • Measure mitochondrial import efficiency restoration

  • Assess growth under various stress conditions

  • Determine virulence phenotype restoration in model systems

• Target potential evaluation:

  • Identify fungal-specific regions absent in human homologs

  • Screen for small molecules disrupting PAM16 function in fungi but not humans

  • Develop assays to measure PAM16 complex formation as a screening platform

  • Test promising compounds in vitro and in infection models

• Resistance potential analysis:

  • Assess natural variation in PAM16 sequences across clinical isolates

  • Determine functional impacts of natural polymorphisms

  • Predict evolutionary constraints on PAM16 function

  • Evaluate barrier to resistance development through directed evolution experiments

What advanced techniques can be used to study the real-time dynamics of PAM16-containing complexes during mitochondrial protein import?

To study real-time dynamics of PAM16-containing complexes during mitochondrial protein import:

• Live-cell imaging methodologies:

  • Express PAM16 fused to photoactivatable fluorescent proteins

  • Use super-resolution microscopy techniques (STED, PALM, STORM)

  • Track protein complex assembly/disassembly during active import

  • Quantify localization changes using automated image analysis algorithms

• FRET-based interaction studies:

  • Create donor-acceptor pairs with PAM16 and interaction partners

  • Measure energy transfer during active protein import

  • Calculate interaction kinetics and binding/unbinding rates

  • Analyze conformational changes during functional cycles

• Single-molecule tracking:

  • Label PAM16 with quantum dots or other bright, stable fluorophores

  • Track individual molecules at the mitochondrial import site

  • Measure residency times and diffusion characteristics

  • Correlate molecular behavior with import events

• Cryo-electron tomography:

  • Capture mitochondria during active protein import

  • Visualize PAM16-containing complexes in near-native state

  • Reconstruct 3D architecture of the active import machinery

  • Identify structural rearrangements during function

• Mass spectrometry-based temporal interactomics:

  • Use pulse-SILAC or TMT labeling to capture dynamic interactions

  • Identify temporal assembly/disassembly of complexes

  • Quantify stoichiometric changes during functional cycles

  • Map post-translational modifications regulating complex activity

What are the most common technical challenges in producing active recombinant C. glabrata PAM16 and how can they be overcome?

Researchers frequently encounter several technical challenges when producing active recombinant C. glabrata PAM16:

• Protein solubility issues:

  • Challenge: Formation of inclusion bodies or aggregation

  • Solution: Express with solubility-enhancing tags (MBP, SUMO), optimize induction conditions (lower temperature, reduced inducer concentration), use specialized strains designed for membrane-associated proteins, add stabilizing agents during purification

• Proper folding:

  • Challenge: Obtaining correctly folded, functional protein

  • Solution: Express in eukaryotic systems (preferably yeast), include molecular chaperones during expression, use slow refolding protocols if purified from inclusion bodies, verify folding through circular dichroism spectroscopy

• Maintaining protein stability:

  • Challenge: Protein degradation during purification

  • Solution: Include protease inhibitors, maintain low temperature throughout purification, optimize buffer conditions (test various pH values, salt concentrations, and stabilizing additives), minimize freeze-thaw cycles

• Yield optimization:

  • Challenge: Low expression levels

  • Solution: Test multiple expression constructs with different promoters (MTI promoter has been successful for C. glabrata proteins ), optimize codon usage for the expression host, evaluate different induction times and conditions, scale up production using bioreactors

• Functional verification:

  • Challenge: Confirming protein activity

  • Solution: Develop sensitive activity assays, compare with native protein isolates, assess ability to complement deletion mutants, verify correct interactions with known partners through pull-down assays

How can researchers resolve discrepancies in PAM16 functional data between in vitro biochemical assays and in vivo phenotypic studies?

Resolving discrepancies between in vitro and in vivo PAM16 functional data requires systematic investigation:

• Methodological reconciliation approach:

  • Carefully compare experimental conditions between in vitro and in vivo systems

  • Identify key variables (pH, ion concentrations, protein concentrations, temperature)

  • Systematically modify in vitro conditions to better mimic the in vivo environment

  • Develop intermediate complexity systems (semi-permeabilized cells, isolated mitochondria)

• Technical validation strategy:

  • Verify protein conformation and modification status in both systems

  • Ensure all necessary cofactors and interaction partners are present

  • Test multiple independent methods to assess the same function

  • Validate antibodies and reagents for specificity across systems

• Temporal and spatial considerations:

  • Assess whether observed differences result from temporal dynamics not captured in static assays

  • Consider compartmentalization effects present in vivo but absent in vitro

  • Evaluate potential regulatory mechanisms active in cells but missing in purified systems

  • Develop time-resolved assays to capture dynamic behaviors

• Systematic troubleshooting framework: