Recombinant Lachancea thermotolerans Formation of crista junctions protein 1 (FCJ1)

What is FCJ1 protein and what is its fundamental role in mitochondrial structure?

FCJ1 (Formation of Crista Junctions protein 1) is a mitochondrial membrane protein specifically enriched in crista junctions (CJs). These junctions are critical architectural features of mitochondria that connect the inner boundary membrane to the cristae membrane. Research has demonstrated that FCJ1 plays an essential role in maintaining mitochondrial organization and function through the formation and maintenance of these crucial junction points .

The protein is also known by several synonyms including MIC60, KLTH0H09724g, MICOS complex subunit MIC60, and Mitofilin . In functional studies, cells lacking FCJ1 exhibit distinct morphological abnormalities including the absence of crista junctions and the formation of concentric stacks of inner membrane within the mitochondrial matrix .

How is recombinant Lachancea thermotolerans FCJ1 protein typically expressed and purified for research applications?

Recombinant full-length L. thermotolerans FCJ1 protein (C5E325) can be produced by expressing the mature protein sequence (amino acids 25-527) in E. coli expression systems. The protein is typically expressed with an N-terminal histidine tag to facilitate purification . The purification process generally employs affinity chromatography techniques that exploit the His-tag, followed by additional purification steps to achieve greater than 90% purity as determined by SDS-PAGE analysis .

The purified protein is commonly provided as a lyophilized powder in Tris/PBS-based buffer containing 6% Trehalose at pH 8.0. For experimental use, it is recommended to reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol (final concentration) for long-term storage at -20°C/-80°C .

What methodologies can researchers employ to study the functional impact of FCJ1 on mitochondrial morphology?

Researchers investigating the functional role of FCJ1 can employ several complementary approaches:

Genetic Manipulation Techniques:

• Gene deletion studies to create FCJ1-null mutants

• Controlled overexpression systems utilizing inducible promoters

• Site-directed mutagenesis to study specific functional domains

Microscopy Methods for Mitochondrial Ultrastructure Analysis:

• Transmission electron microscopy (TEM) to visualize crista junction architecture

• Electron tomography for 3D reconstruction of mitochondrial membranes

• Super-resolution fluorescence microscopy with FCJ1-fluorescent protein fusions

Biochemical Approaches:

• Co-immunoprecipitation to identify interaction partners

• Blue native gel electrophoresis to analyze membrane protein complexes

• Crosslinking mass spectrometry to map protein interaction interfaces

Studies have demonstrated that cells lacking FCJ1 exhibit dramatic ultrastructural changes, including complete absence of crista junctions and the formation of concentric stacks of inner membrane in the mitochondrial matrix. Conversely, overexpression of FCJ1 produces increased crista junction formation, branching of cristae, and enlargement of crista junction diameter .

How does FCJ1 functionally interact with F1FO-ATP synthase complexes and what experimental techniques can elucidate these interactions?

The relationship between FCJ1 and F1FO-ATP synthase appears to be antagonistic, with important implications for mitochondrial membrane architecture. Research has revealed that:

• Cells lacking FCJ1 show increased levels of F1FO-ATP synthase supercomplexes

• Overexpression of FCJ1 leads to reduced levels of F1FO supercomplexes

• Impairment of F1FO oligomer formation (through deletion of subunits e/g) causes crista junction diameter enlargement, reduction of cristae tip numbers, and promotes cristae branching

• FCJ1 and subunits e/g genetically interact

To investigate these interactions, researchers can employ:

Biochemical Approaches:

• Proximity labeling techniques (BioID, APEX) to identify proteins in close proximity to FCJ1

• Crosslinking coupled with mass spectrometry to map interaction interfaces

• Blue native PAGE to analyze effects on supercomplex formation

Biophysical Methods:

• Surface plasmon resonance to measure binding affinities

• Cryo-electron microscopy to visualize complex structures

• Fluorescence resonance energy transfer (FRET) to study protein-protein interactions in situ

Functional Assays:

• Mitochondrial respiration measurements in FCJ1 mutants

• ATP synthesis rate determination

• Membrane potential analyses using potentiometric dyes

The current model suggests that antagonism between FCJ1 and subunits e/g locally modulates the F1FO oligomeric state, thereby controlling membrane curvature of cristae to generate crista junctions and cristae tips .

What are the challenges in expressing and purifying functionally active Lachancea thermotolerans FCJ1 for structural studies?

Several challenges exist when producing recombinant L. thermotolerans FCJ1 for structural and functional studies:

Expression System Considerations:

• The optimal expression system must balance yield with proper folding

• E. coli is commonly used but may lack post-translational modifications

• Yeast expression systems may provide more native-like processing but lower yields

Protein Solubility and Stability Issues:

• As a membrane protein, FCJ1 presents solubility challenges

• Selection of appropriate detergents is critical for extraction and stabilization

• Maintaining protein stability during purification and concentration steps

Purification Complexity:

• Multi-step purification protocols are typically required to achieve >90% purity

• Removal of the His-tag may be necessary for certain structural studies

• Protein aggregation during concentration steps must be minimized

Storage and Handling:

• Lyophilization provides stability but may affect protein structure

• Preventing freeze-thaw damage requires addition of cryoprotectants (e.g., 6% trehalose)

• Reconstitution conditions must be optimized to maintain native conformation

For structural studies specifically, researchers must additionally consider:

• Protein homogeneity requirements for crystallization or cryo-EM

• The need for stable, monodisperse protein preparations

• Potential requirement for lipid addition to stabilize membrane domains

What approaches can be used to study the role of FCJ1 in the context of the MICOS complex and broader mitochondrial contact site organization?

FCJ1 (also known as MIC60) functions as part of the larger MICOS complex (Mitochondrial Contact Site and Cristae Organizing System). Understanding its role within this complex requires specialized approaches:

Protein Complex Analysis:

• Blue native PAGE to isolate intact MICOS complexes

• Complex immunoprecipitation followed by mass spectrometry

• Size exclusion chromatography to determine complex composition and stoichiometry

• Hydrogen-deuterium exchange mass spectrometry to map protein-protein interfaces

In vivo Interaction Studies:

• FRET/FLIM to study dynamic protein interactions

• Split-GFP complementation to visualize protein associations

• Proximity labeling techniques (BioID, APEX2) to map the FCJ1 interaction network

• Multi-color super-resolution microscopy to map spatial relationships

Functional Reconstitution:

• Reconstitution of minimal MICOS components in liposomes

• In vitro membrane tubulation/deformation assays

• Cryo-electron tomography of reconstituted systems

Comparative Genomic Approaches:

• Analysis of co-evolution patterns among MICOS components

• Identification of conserved interaction motifs across species

• Correlation of genomic features with mitochondrial ultrastructure differences

A proposed experimental workflow could include:

• Generation of L. thermotolerans strains with tagged MICOS components

• Purification of intact complexes under native conditions

• Compositional analysis by mass spectrometry

• Structural characterization by negative-stain EM or cryo-EM

• Functional reconstitution in artificial membrane systems

How might understanding FCJ1 function contribute to biotechnological applications involving Lachancea thermotolerans?

L. thermotolerans has gained attention as a promising organism for biotechnological applications, particularly in lactic acid production . Understanding FCJ1's role in mitochondrial function could benefit these applications in several ways:

Metabolic Engineering Opportunities:

• Modulation of FCJ1 expression could potentially alter respiration/fermentation balance

• Optimization of mitochondrial function may enhance stress tolerance

• Engineering mitochondrial architecture might improve carbon flux through desired pathways

Strain Improvement Strategies:

• FCJ1 modifications could be incorporated into strain development programs

• Combined with other genetic modifications to enhance lactic acid production

• Potential target for adaptive laboratory evolution approaches

Process Optimization Implications:

• Understanding FCJ1's role in stress response could inform fermentation parameters

• Mitochondrial function insights may guide media optimization

• Could contribute to development of high-cell density cultivation methods

The unique characteristics of L. thermotolerans, including its higher tolerance to acidic environments, broader substrate range, and potential for genetic engineering , could be further enhanced through targeted manipulation of mitochondrial function via FCJ1.

What emerging technologies could advance our understanding of FCJ1 structure and function?

Several cutting-edge technologies hold promise for deepening our understanding of FCJ1:

Advanced Structural Biology Approaches:

• Cryo-electron tomography of intact mitochondria to visualize FCJ1 in native context

• Integrative structural biology combining multiple data types (X-ray, NMR, EM, crosslinking)

• Single-particle cryo-EM of purified FCJ1-containing complexes

• Microcrystal electron diffraction for membrane protein structural determination

Next-Generation Functional Genomics:

• CRISPR-based screening to identify genetic interactions with FCJ1

• Base editing approaches for targeted mutagenesis of FCJ1

• Perturb-seq to link FCJ1 genetic variants to transcriptional responses

Advanced Imaging Techniques:

• Correlative light and electron microscopy (CLEM) to link protein dynamics to ultrastructure

• Super-resolution live-cell imaging of FCJ1 and associated proteins

• Expansion microscopy for enhanced visualization of mitochondrial substructures

• Label-free imaging methods to study native mitochondrial membrane dynamics

Systems Biology Integration:

• Multi-omic approaches correlating FCJ1 variants with metabolomic, proteomic, and phenotypic data

• Mathematical modeling of mitochondrial membrane dynamics

• Network analysis approaches to place FCJ1 in broader cellular context

These emerging technologies can help address current knowledge gaps and provide more comprehensive understanding of FCJ1's role in mitochondrial organization and function.

What controls and validation steps are essential when studying recombinant Lachancea thermotolerans FCJ1?

Rigorous experimental design for FCJ1 studies should include:

Expression and Purification Controls:

• Confirmation of protein identity by mass spectrometry

• Purity assessment by SDS-PAGE (target >90%)

• Western blot verification with anti-His antibodies

• Size exclusion chromatography to confirm monodispersity

Functional Validation:

• Circular dichroism to verify proper secondary structure

• Limited proteolysis to assess folding quality

• Thermal shift assays to determine protein stability

• Liposome binding assays to confirm membrane interaction capacity

Genetic Complementation Tests:

• Expression of recombinant FCJ1 in fcj1Δ yeast strains

• Rescue of mitochondrial morphology defects

• Restoration of respiratory growth phenotypes

• Verification of crista junction formation by electron microscopy

Interaction Validation:

• Confirmation of known protein interactions (e.g., with other MICOS components)

• Controls for non-specific binding in pull-down experiments

• Concentration-dependent binding assays

• Competition assays with known binding partners

How can researchers address potential inconsistencies between in vitro studies of purified FCJ1 and its in vivo function?

The translation between in vitro observations and in vivo function presents several challenges:

Reconstitution Approaches:

• Development of minimal membrane systems that recapitulate native environments

• Incorporation of FCJ1 into liposomes with defined lipid compositions

• Co-reconstitution with interaction partners to recreate functional complexes

• Use of giant unilamellar vesicles (GUVs) to study membrane deformation activities

Correlative Techniques:

• Parallel in vitro and in vivo mutagenesis studies

• Structure-guided functional assays in both systems

• Integration of data from multiple experimental approaches

• Development of assays that bridge the in vitro-in vivo divide

Validation Strategies:

• Engineered cell lines expressing FCJ1 variants identified in vitro

• Rescue experiments with structure-guided mutants

• In-cell crosslinking to verify interactions observed in vitro

• Microscopy validation of structural predictions

Computational Methods:

• Molecular dynamics simulations to predict protein-membrane interactions

• Integration of structural and functional data through modeling

• Systems biology approaches to predict network-level effects of FCJ1 perturbations

By employing these approaches, researchers can develop a more comprehensive understanding of FCJ1 function that reconciles in vitro observations with in vivo reality.

What is the recommended protocol for reconstituting lyophilized Lachancea thermotolerans FCJ1 protein for functional studies?

For optimal reconstitution of lyophilized L. thermotolerans FCJ1 protein:

• Initial Preparation:

  • Centrifuge the vial briefly to bring contents to the bottom

  • Allow the sealed vial to reach room temperature before opening

• Reconstitution Procedure:

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (50% recommended for long-term storage)

  • Mix gently until completely dissolved, avoiding vigorous vortexing

  • Allow protein to rehydrate fully for 30 minutes at room temperature

• Storage Considerations:

  • Aliquot into smaller volumes to avoid freeze-thaw cycles

  • Store working aliquots at 4°C for up to one week

  • Store long-term aliquots at -20°C/-80°C

  • Avoid repeated freeze-thaw cycles

• Quality Control:

  • Verify protein concentration by absorbance at 280 nm or Bradford assay

  • Confirm integrity by SDS-PAGE before experimental use

  • For sensitive applications, verify activity using appropriate functional assays