Recombinant Uncinocarpus reesii Formation of crista junctions protein 1 (FCJ1)

What is Uncinocarpus reesii and why is it important in recombinant protein expression studies?

Uncinocarpus reesii is a nonpathogenic fungus phylogenetically related to Coccidioides species, with approximately 0.7% sequence divergence in the 18S ribosomal gene, representing 20-30 million years of evolutionary distance . Its importance in recombinant protein expression stems from:

• Its close evolutionary relationship to pathogenic Coccidioides species while remaining nonpathogenic

• The ability to express proteins with specific post-translational modifications similar to Coccidioides

• Significant biosafety advantages as it requires only BSL1 containment (compared to BSL3 for Coccidioides)

• Its capacity to produce glycosylated proteins with specific modifications like 3-O-methyl mannose moieties

What is Formation of crista junctions protein 1 (FCJ1) and what is its function?

FCJ1, also known as mitofilin in mammals, is a mitochondrial inner membrane protein that plays a crucial role in:

• Formation and maintenance of crista junctions (CJs), which are tubular invaginations connecting the inner boundary with the cristae membrane

• Determining mitochondrial cristae architecture and morphology

• Working antagonistically with subunits e and g of the F1FO-ATP synthase to modulate CJ formation

• Functioning as part of the MICOS complex (MItochondrial contact site and Cristae Organizing System), which is critical for establishing proper mitochondrial inner membrane topology

The protein contains several functional domains, with the C-terminal domain being essential for FCJ1 function, oligomer formation, and interactions with other proteins .

How does the structure of FCJ1 relate to its function in mitochondria?

FCJ1 structure-function relationship involves:

The C-terminal domain is particularly important as its absence leads to:

• Strong impairment of CJ formation

• Formation of irregular, stacked cristae

• Disruption of interactions with the TOB/SAM complex

When properly functioning, FCJ1 controls membrane curvature at specific sites, promoting the formation of tubular CJs instead of lamellar cristae formations .

What are the proven methods for transforming Uncinocarpus reesii for recombinant protein expression?

Effective transformation of U. reesii involves:

Transformation Protocol:

• Culture U. reesii on GYE agar (1% glucose, 0.5% yeast extract, 1.5% agar) at 30°C for 3 weeks to produce arthroconidia

• Generate protoplasts by digesting germ tubes with enzyme cocktail:

  • Lysing enzymes

  • Driselase

  • Recombinant coccidioidal chitinase 1

• Linearize the expression plasmid (e.g., pCE vector containing the gene of interest)

• Incubate U. reesii protoplasts with linearized plasmid in the presence of:

  • Polyethylene glycol (Mn 3350)

  • Calcium ions to facilitate DNA uptake

• Select transformants initially on GYE agar with 75 μg/ml hygromycin B

• Conduct subsequent passages (3×) on GYE agar with increased hygromycin B (100 μg/ml) to obtain stable transformed clones

• Confirm transformation by PCR screening using gene-specific primers

This methodology has been successfully employed for expressing various proteins, including coccidioidal antigens like BGL2 and CTS1 .

How can the heat shock protein (HSP60) promoter system be optimized for recombinant FCJ1 expression in U. reesii?

The HSP60 promoter system optimization involves:

Promoter System Optimization:

• Vector design: Use the pCE (coccidioidal protein expression) vector containing the heat shock protein (HSP60) promoter from Coccidioides posadasii

• Induction parameters:

  • Temperature shift from standard cultivation (30°C) to elevated temperature (typically 37-42°C)

  • Optimal induction time of 12-24 hours post-temperature shift

  • Fine-tuning of medium composition (nitrogen sources, carbon concentration)

• Expression enhancement strategies:

  • Codon optimization for U. reesii-preferred codons

  • Inclusion of endogenous signal sequences for proper protein targeting

  • Addition of purification tags (His6, FLAG) that don't interfere with protein function

The system has demonstrated successful expression of functional proteins with proper post-translational modifications, with reported expression levels sufficient for diagnostic applications (>75% sensitivity in serological tests) .

What purification strategies yield the highest recovery of functional recombinant FCJ1 from U. reesii cultures?

Effective purification strategies include:

Purification Workflow:

• Culture supernatant collection:

  • Harvest transformed U. reesii cultures after heat-shock induction

  • Remove mycelia by filtration

  • Clarify supernatant by centrifugation (10,000×g, 30 min)

• Initial concentration:

  • Ammonium sulfate precipitation (60-80% saturation)

  • Tangential flow filtration for larger volumes

• Chromatographic purification sequence:

  • Primary capture: Nickel affinity chromatography for His-tagged recombinant FCJ1

    • Binding buffer: 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole

    • Wash buffer: Same with 20-30 mM imidazole

    • Elution buffer: Same with 250 mM imidazole

  • Polishing step: Size exclusion chromatography

    • Buffer: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl

• Stabilization of purified protein:

  • Storage in Tris-based buffer with 50% glycerol

  • Aliquot and store at -20°C or -80°C for extended storage

  • Avoid repeated freeze-thaw cycles

This approach has yielded properly folded, glycosylated recombinant proteins from U. reesii with appropriate biological activity and antigenic properties .

How does FCJ1 interact with other components of the MICOS complex to establish crista junction architecture?

FCJ1 interactions within the MICOS complex involve:

MICOS Complex Interactions:

• FCJ1 forms part of the Mic60-subcomplex, which is sufficient for CJ formation

• The C-terminal domain of FCJ1 mediates interaction with:

  • Other FCJ1 molecules (homooligomerization)

  • The TOB/SAM complex of the outer membrane, linking CJs to the outer membrane

• FCJ1 antagonistically interacts with F1FO-ATP synthase subunits e and g to regulate:

  • CJ diameter (FCJ1 overexpression causes enlargement)

  • Cristae branching (increased with FCJ1 overexpression)

  • Membrane curvature at cristae regions

The functional significance of these interactions is demonstrated by phenotypic changes when FCJ1 is overexpressed:

• Increased CJ formation

• Branching of cristae

• Enlargement of CJ diameter

• Reduced levels of F1FO-ATP synthase supercomplexes

What methods are most effective for visualizing and quantifying structural changes in mitochondrial cristae following FCJ1 manipulation?

Advanced imaging approaches include:

Visualization and Quantification Techniques:

• Super-resolution light microscopy:

  • Stimulated emission depletion (STED) microscopy

  • Photoactivated localization microscopy (PALM)

  • Direct stochastic optical reconstruction microscopy (dSTORM)

  • Resolution: 20-50 nm for protein localization and distribution

• 3D electron microscopy approaches:

  • Transmission electron microscopy (TEM) with serial sectioning

  • Electron tomography for 3D reconstruction of mitochondrial ultrastructure

  • Focused ion beam-scanning electron microscopy (FIB-SEM)

  • Cryo-electron microscopy for near-native state visualization

• Quantitative analysis parameters:

  • CJ diameter measurements

  • Cristae density (number per mitochondrion)

  • Branching frequency

  • Cristae morphology classification (lamellar, tubular, or mixed)

  • Spatial distribution of CJs along the mitochondrial periphery

These methods allow researchers to detect subtle changes in mitochondrial ultrastructure following genetic manipulation of FCJ1, revealing how it controls cristae architecture .

How does the recombinant U. reesii FCJ1 protein compare functionally to its orthologs in other fungal species?

Comparative functional analysis reveals:

Functional complementation studies demonstrate that despite sequence divergence, the core function in cristae organization is preserved across species. The C-terminal domain shows the highest conservation, supporting its essential role in function .

What are the critical quality control parameters to assess the structural integrity of purified recombinant FCJ1?

Quality Control Parameters:

• Purity assessment:

  • SDS-PAGE: >95% purity with correct molecular weight (~70 kDa)

  • Western blot: Single band recognition with anti-FCJ1 antibodies

  • Mass spectrometry: Protein identification with >80% sequence coverage

• Structural integrity analysis:

  • Circular dichroism (CD) spectroscopy: Confirmation of secondary structure content

  • Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS): Assessment of oligomeric state and homogeneity

  • Thermal shift assay: Stability measurement (melting temperature)

• Functional validation:

  • Binding assays with known interaction partners (TOB complex components)

  • In vitro oligomerization assays

  • Membrane incorporation efficiency in liposome models

• Post-translational modification characterization:

  • Glycosylation analysis by mass spectrometry

  • Phosphorylation status assessment

  • Disulfide bond verification

Quality control standards should include threshold values for each parameter to ensure batch-to-batch consistency of the recombinant protein .

How can researchers design complementation experiments to validate the functional significance of specific FCJ1 domains?

Complementation Experimental Design:

• Generation of domain deletion/mutation constructs:

  • C-terminal domain deletion (essential for function)

  • Transmembrane domain mutations (affecting membrane anchoring)

  • Coiled-coil region modifications (influencing oligomerization)

  • Point mutations at conserved residues

• Expression system optimization:

  • Use of inducible promoters for tight expression control

  • Fluorescent protein tagging for localization studies

  • Quantitative expression level monitoring

• Functional readouts:

  • Mitochondrial morphology analysis by fluorescence microscopy

  • Cristae ultrastructure examination by electron microscopy

  • Biochemical assessment of MICOS complex assembly

  • Respiratory capacity and mitochondrial function tests

• Experimental controls:

  • Empty vector (negative control)

  • Wild-type FCJ1 (positive control)

  • Graduated expression levels to assess dose-dependency

This approach allows systematic assessment of structure-function relationships in FCJ1 and identification of critical residues for specific interactions or functions .

What strategies can address expression challenges when FCJ1 production levels are suboptimal in U. reesii?

Troubleshooting Strategies:

• Expression vector optimization:

  • Promoter strength adjustment (constitutive vs. inducible)

  • Codon optimization based on U. reesii codon usage bias

  • Inclusion of introns to enhance mRNA processing

  • Use of species-specific transcription terminators

• Host strain engineering:

  • Development of protease-deficient strains

  • Optimization of chaperone co-expression

  • Metabolic engineering to enhance protein production capacity

• Culture condition refinement:

  • Systematic temperature optimization during induction

  • Media composition adjustment (carbon source, nitrogen source)

  • pH optimization during growth and induction phases

  • Dissolved oxygen level monitoring and control

• Protein design modifications:

  • Addition of solubility-enhancing tags (MBP, SUMO)

  • Domain-based expression approach for challenging proteins

  • Signal peptide optimization for secretion efficiency

• Scalable cultivation strategies:

  • Fed-batch cultivation with controlled nutrient feeding

  • Perfusion culture systems for continuous harvesting

  • Solid-state fermentation optimization

Implementation of these strategies has resolved expression challenges for other complex fungal proteins, improving yields by 3-10 fold in challenging cases .

How has FCJ1 evolved among fungal species, and what does this reveal about mitochondrial structural adaptations?

Evolutionary Analysis of FCJ1:

FCJ1 evolutionary patterns reveal:

• The core C-terminal domain is highly conserved across fungi, reflecting its essential function in cristae formation

• The middle region shows greater sequence divergence while maintaining structural properties

• The N-terminal region contains species-specific adaptations related to membrane anchoring

Phylogenetic analysis suggests:

• FCJ1 likely evolved from a simpler ancestral protein involved in membrane organization

• Expansion of interaction capabilities occurred in parallel with increasing mitochondrial membrane complexity

• Gene duplication events in some lineages led to specialized FCJ1-like proteins with distinct functions

Comparing FCJ1 sequences from U. reesii, Coccidioides, and more distant fungi reveals selection pressures maintaining crucial functional domains while allowing adaptations in non-critical regions, possibly reflecting different metabolic requirements or environmental adaptations .

What genomic approaches can identify the regulatory elements controlling FCJ1 expression in U. reesii and related species?

Genomic and Regulatory Analysis Approaches:

• Comparative promoter analysis:

  • Identification of conserved transcription factor binding sites across related species

  • Motif discovery in intergenic regions upstream of FCJ1 orthologs

  • ChIP-seq analysis to identify transcription factors binding to FCJ1 promoter

• Epigenetic profiling:

  • ATAC-seq to map chromatin accessibility around the FCJ1 locus

  • Histone modification mapping (H3K4me3, H3K27ac) to identify active regulatory elements

  • DNA methylation analysis to detect possible epigenetic regulation

• Functional validation methods:

  • Reporter gene assays with promoter truncations and mutations

  • CRISPR-Cas9 genome editing to modify putative regulatory elements

  • RNA-seq under various conditions to determine expression patterns

• Regulatory network analysis:

  • Identification of co-regulated genes involved in mitochondrial function

  • Inference of transcription factor networks controlling mitochondrial biogenesis

  • Integration with metabolic modeling to link expression with function

These approaches can reveal how FCJ1 expression is coordinated with other mitochondrial components and adapted to different physiological conditions .

How does the nonpathogenic nature of U. reesii compare to pathogenic Coccidioides at the molecular level, and what implications does this have for FCJ1 function?

Comparative Pathogenicity Analysis:

The nonpathogenic nature of U. reesii compared to pathogenic Coccidioides involves:

This comparison reveals:

• Core mitochondrial functions (including FCJ1) are likely conserved between species

• Regulation of mitochondrial dynamics may differ in response to different environmental pressures

• U. reesii provides a safe model system to study fundamental aspects of mitochondrial biology relevant to both species

The understanding of these differences supports the use of U. reesii as both an expression system for pathogen proteins and as a model organism for studying conserved cellular processes without biosafety concerns .

How can recombinant U. reesii FCJ1 be utilized to study mitochondrial dysfunction in fungal and human disease models?

Research Applications in Disease Models:

• Fungal pathogenesis studies:

  • Expression of FCJ1 mutants to examine effects on mitochondrial function

  • Analysis of how mitochondrial morphology changes affect virulence in pathogenic fungi

  • Identification of potential antifungal targets in mitochondrial organization pathways

• Human disease modeling:

  • Study of FCJ1/mitofilin mutations associated with mitochondrial disorders

  • Examination of conserved mechanisms between fungal and human mitochondrial organization

  • Development of screening systems for compounds affecting cristae organization

• Experimental approaches:

  • Heterologous expression of human mitofilin variants in U. reesii

  • Mitochondrial isolation and functional assays (respiration, membrane potential)

  • High-content screening for compounds affecting cristae morphology

  • Proteomics analysis of altered protein-protein interactions in disease models

These applications leverage the biosafety advantages of U. reesii while providing insights into conserved mechanisms of mitochondrial organization relevant to disease .

What novel insights might be gained by combining cryo-electron tomography with recombinant FCJ1 in membrane reconstitution systems?

Advanced Structural Biology Approaches:

Combining cryo-electron tomography with in vitro reconstitution systems offers:

• Structural insights:

  • Near-atomic resolution of FCJ1 in membrane environments

  • Visualization of FCJ1 oligomerization states and conformational changes

  • Mapping of interaction interfaces with other MICOS components

  • Detection of membrane curvature effects induced by FCJ1

• Reconstitution system design:

  • Liposomes with defined lipid composition mimicking mitochondrial membranes

  • Incorporation of purified recombinant FCJ1 at controlled densities

  • Addition of other MICOS components to study complex assembly

  • Introduction of F1FO-ATP synthase to examine antagonistic interactions

• Dynamic analysis capabilities:

  • Time-resolved structural changes following protein addition

  • Effect of membrane potential on FCJ1 organization

  • Lipid-protein interactions at molecular resolution

  • Conformational changes in response to physiological triggers

This approach would bridge the gap between in vivo observations and molecular mechanisms, providing unprecedented insights into how FCJ1 physically shapes mitochondrial membranes .

What are the most promising directions for engineering U. reesii as an optimized expression system for complex mitochondrial membrane proteins?

Future Directions for Expression System Engineering:

• Genetic engineering advancements:

  • Development of CRISPR-Cas9 genome editing tools specifically for U. reesii

  • Creation of knockout strains for problematic proteases

  • Engineering of specialized glycosylation pathways for human-compatible modifications

  • Integration of inducible promoter systems with tighter regulation

• Cultivation technology improvements:

  • Design of specialized bioreactors for filamentous fungi

  • Development of continuous cultivation strategies with real-time monitoring

  • Optimization of downstream processing for membrane proteins

  • Scale-up protocols maintaining post-translational modifications

• Protein engineering approaches:

  • Design of fusion constructs facilitating membrane protein purification

  • Creation of reporter systems for rapid screening of expression conditions

  • Development of self-assembling membrane protein complexes

  • Engineering of pH-responsive solubility tags for membrane proteins

• Analytical method development:

  • Label-free quantification methods for membrane proteins

  • High-throughput functional assays for mitochondrial proteins

  • Automated image analysis for mitochondrial morphology assessment

  • Integration of systems biology approaches to optimize expression

These advancements would position U. reesii as a premiere expression system for mitochondrial membrane proteins, facilitating structural and functional studies of challenging targets like FCJ1 and other MICOS components .