Recombinant Ajellomyces dermatitidis Formation of crista junctions protein 1 (FCJ1)

What is FCJ1 and what is its significance in mitochondrial structure?

FCJ1 (Formation of crista junctions protein 1) is a mitochondrial membrane protein specifically enriched at crista junctions (CJs), which are important sites connecting cristae to the inner boundary membrane. Studies have shown that cells lacking FCJ1 completely lack CJs and exhibit concentric stacks of inner membrane in the mitochondrial matrix . FCJ1 appears to function by antagonizing F1FO-ATP synthase supercomplex formation, thereby locally modulating membrane curvature to generate CJs . This protein is part of the larger MICOS (Mitochondrial Contact Site and Cristae Organizing System) complex, serving as a core component essential for maintaining proper mitochondrial inner membrane architecture.

What is the relationship between FCJ1 and mitochondrial F1FO-ATP synthase?

FCJ1 has an antagonistic relationship with the F1FO-ATP synthase complex, particularly with its subunits e and g (Su e/g). Research demonstrates that cells lacking FCJ1 show increased levels of F1FO-ATP synthase supercomplexes, while overexpression of FCJ1 leads to reduced levels of these supercomplexes . This relationship appears to be crucial for controlling membrane curvature within mitochondria. In FCJ1-deficient mitochondria, F1FO particles arrange in zipperlike structures with regular spacing (14-16 nm between particles), whereas this ordered arrangement is disrupted when both FCJ1 and Su e/g are deleted . The balance between FCJ1 and F1FO oligomerization appears to be a key determinant of cristae morphology.

What is the amino acid sequence and structural characteristics of Ajellomyces dermatitidis FCJ1?

The mature Ajellomyces dermatitidis FCJ1 protein (amino acids 32-665) has the following sequence:

SSTPNAAATPELSQKATNSTSTKPPGPNDPDVRSPASPSTGSTLHPETVSKPPQSPAVQGQTSPGSSVQPPEHEPSPPPPRPPPAPKTGLLRKLLYLFLTTGLAYAGGVWYSLRSDNFYDFFTEYIPYGEEAVLYLEERDFRSRFPSIARQINRRVSAPRDEGAQVMIPGRSGLSWKVAEEQQEASDVTKQGQHISATDANELTEETKVAEKAKEDVKSKPVAKKAEAAEPKSSPKVVEPHPAKAEENTSLEAPRQPVVPAAAAIEHLGLDNEDEPVVQDLVKVFNDIITVISADESASKFSVPIAKAKEELEKIGDRIVALKNDAQESAKEEIRNAQAALDKSAAELVRHINEVRAQDA AEFREEFESEREKISKSYQEKVTTELQRAHEVAEQRLRNELVEQAIELNRKFLADVKTLV ENEREGRLSKLAELTANVAELERLTAGWSDVIDINLRTQQLQVAVDSVRTTLENSEVPRP FIRELAAVKELASNDEVVAAAIASISPTAYQRGIPSPAQLVDRFRRVASEVRKASLLPEN AGITSHAASLVLSKVMLKKQGTPVGNDVESILTRTENLLEEGNFDEAAREMNSLQGWAKL LSKDWLADVRRVLEVKQALEVIETEARLRCLQVE

The protein contains several important structural features, including a transmembrane domain, coiled-coil regions that likely mediate protein-protein interactions, and domains responsible for membrane association. The protein's structure facilitates its localization to crista junctions, where it plays a crucial role in maintaining these structures.

How does deletion of FCJ1 affect mitochondrial ultrastructure?

Electron microscopy and tomography studies reveal that deletion of FCJ1 leads to dramatic changes in mitochondrial ultrastructure. FCJ1-deficient cells exhibit:

• Complete absence of crista junctions, which are normally abundant in wild-type mitochondria

• Formation of concentric, onion-like stacks of inner membrane within the mitochondrial matrix

• Arrangement of F1FO-ATP synthase particles in highly ordered zipperlike structures

• Loss of the normal branched architecture of cristae membranes

These observations demonstrate that FCJ1 is essential for the formation and maintenance of crista junctions. Without FCJ1, the normal connections between cristae and the inner boundary membrane are lost, resulting in altered mitochondrial membrane topology and organization. The ordered arrangement of F1FO particles in these abnormal structures further supports the antagonistic relationship between FCJ1 and F1FO oligomerization.

What experimental approaches can be used to study FCJ1 localization within mitochondria?

The localization of FCJ1 within mitochondrial subcompartments can be determined using quantitative immuno-electron microscopy. This involves:

• Chemical fixation of cells followed by preparation of cryosections

• Immunodecoration with antibodies against FCJ1 at carefully controlled concentrations

• Visualization using immunogold labeling, where gold particles mark the location of FCJ1

• Quantitative analysis of gold particle distribution across different mitochondrial regions

Using this approach, researchers have demonstrated that FCJ1 is specifically enriched at crista junctions, with lower amounts present in the planar parts of cristae . This distribution pattern differs from that of other mitochondrial proteins, including F1FO-ATP synthase subunits, which are predominantly found in cristae tips. The specific enrichment of FCJ1 at crista junctions correlates with its functional role in maintaining these structures.

How does overexpression of FCJ1 affect mitochondrial structure and function?

Controlled overexpression of FCJ1 produces several notable effects on mitochondrial structure and function:

• Increased number of crista junctions per mitochondrion (two- to threefold compared to control cells)

• Enhanced branching of cristae membranes

• Enlargement of crista junction diameter

• Reduced levels of F1FO-ATP synthase supercomplexes

These findings further support the role of FCJ1 in promoting crista junction formation and opposing F1FO oligomerization. The enlargement of crista junction diameter suggests that FCJ1 levels influence not only the number but also the architecture of these structures. The inverse relationship between FCJ1 overexpression and F1FO supercomplex levels reinforces the antagonistic relationship between these components in controlling mitochondrial membrane organization.

What is the functional interaction between FCJ1 and F1FO subunits e and g?

The genetic and functional interaction between FCJ1 and F1FO subunits e and g (Su e/g) is complex and critical for proper mitochondrial ultrastructure:

• Deletion of Su e/g impairs F1FO oligomer formation, causing enlargement of crista junction diameter, reduction of cristae tip numbers, and increased cristae branching

• Deletion of FCJ1 has opposite effects, leading to loss of crista junctions and increased F1FO oligomerization

• In double deletion mutants (∆fcj1/∆su g or ∆fcj1/∆su e), the ordered zipperlike arrangement of F1FO particles seen in ∆fcj1 mitochondria is disrupted

How is recombinant Ajellomyces dermatitidis FCJ1 protein produced and stored?

Recombinant Ajellomyces dermatitidis FCJ1 protein can be produced using the following approach:

• Expression system: The mature protein (amino acids 32-665) is expressed in E. coli with an N-terminal His tag

• Purification: The protein is purified using affinity chromatography and typically provided as a lyophilized powder

• Storage buffer: Commonly supplied in Tris/PBS-based buffer with 6% Trehalose at pH 8.0

For optimal storage and handling:

• Store the lyophilized powder at -20°C/-80°C upon receipt

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

• Add glycerol to a final concentration of 5-50% (typically 50% is recommended)

• Make aliquots to avoid repeated freeze-thaw cycles

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

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

Following these guidelines ensures maximum stability and activity of the recombinant protein for experimental applications.

What methods can be used to assess the purity and activity of recombinant FCJ1?

Ensuring the quality of recombinant FCJ1 requires multiple complementary approaches:

For purity assessment:

• SDS-PAGE analysis to confirm a single predominant band of the expected molecular weight with >90% purity

• Western blotting using anti-His tag or anti-FCJ1 specific antibodies

• Mass spectrometry for precise identification and detection of any modifications

For activity assessment:

• Binding assays to verify interaction with known partner proteins

• Structural integrity analysis using circular dichroism or other spectroscopic techniques

• Functional reconstitution into liposomes to test effects on membrane curvature

• Complementation assays in FCJ1-deficient cells or mitochondria

These methods collectively provide confidence in the structural integrity and functional capacity of the recombinant protein, ensuring its suitability for downstream experimental applications.

How can recombinant FCJ1 be used in membrane reconstitution studies?

Recombinant FCJ1 can be employed in membrane reconstitution studies to investigate its direct effects on membrane properties:

• Liposome preparation:

  • Generation of liposomes with mitochondrial-like lipid composition

  • Incorporation of purified FCJ1 at varying concentrations

  • Creation of labeled liposomes for visualization studies

• Membrane curvature analysis:

  • Electron microscopy to visualize FCJ1-induced membrane deformations

  • Dynamic light scattering to detect changes in liposome size distribution

  • Fluorescence microscopy of giant unilamellar vesicles containing FCJ1

• Interaction studies:

  • Co-reconstitution with other MICOS components

  • Analysis of how FCJ1 affects F1FO organization in membrane environments

  • Evaluation of lipid preferences and domain formation

These approaches can provide direct evidence for FCJ1's role in membrane shaping and organization, complementing in vivo studies and offering mechanistic insights into how FCJ1 contributes to crista junction formation.

What experimental systems are most suitable for studying FCJ1 function?

Several experimental systems can be employed to study different aspects of FCJ1 function:

• Yeast model systems:

  • Advantages: Genetic tractability, easy manipulation of FCJ1 and interacting partners

  • Applications: Effects of FCJ1 deletion/overexpression on mitochondrial morphology

  • Methods: Gene deletion, controlled expression, electron microscopy analysis

• Fungal pathogen models (for Ajellomyces-specific studies):

  • Advantages: Direct relevance to the organism of interest

  • Applications: Role of FCJ1 in pathogen biology and potential virulence

  • Methods: Gene manipulation, infection models, stress response studies

• In vitro reconstitution:

  • Advantages: Controlled environment, direct assessment of FCJ1 activity

  • Applications: Membrane-shaping properties, protein-protein interactions

  • Methods: Liposome reconstitution, binding assays, electron microscopy

• Biochemical approaches:

  • Advantages: Precise analysis of molecular interactions

  • Applications: Identifying binding partners, post-translational modifications

  • Methods: Co-immunoprecipitation, mass spectrometry, structural studies

The choice of system depends on the specific research question, with each approach offering complementary insights into FCJ1 function.

How does Ajellomyces dermatitidis FCJ1 compare to FCJ1/mitofilin homologs in other species?

FCJ1 belongs to a conserved family of proteins found across eukaryotes, with both similarities and differences between species:

Despite variations in size and some domain features, the core functions in maintaining cristae structure appear conserved across species. The Ajellomyces dermatitidis FCJ1 shares the fundamental property of localizing to and promoting the formation of crista junctions, though species-specific roles may exist, particularly in fungal pathogens like A. dermatitidis.

How does FCJ1 contribute to MICOS complex formation and function?

FCJ1 is a core component of the MICOS (Mitochondrial Contact Site and Cristae Organizing System) complex, which plays a critical role in cristae formation:

• As a central scaffolding protein, FCJ1 helps organize other MICOS components at crista junctions

• FCJ1 promotes the assembly of MICOS subcomplexes and their integration into a functional unit

• The interaction of FCJ1 with F1FO subunits represents a crucial regulatory mechanism controlling cristae architecture

The MICOS complex assembly process is tightly regulated, with FCJ1 playing a pivotal role in ensuring proper complex formation and localization. Disruption of FCJ1 affects the stability and distribution of other MICOS components, highlighting its importance as a core structural element of this complex.

What is the relationship between FCJ1 function and mating types in Ajellomyces dermatitidis?

While the primary function of FCJ1 relates to mitochondrial structure, Ajellomyces dermatitidis has interesting mating biology that may interact with mitochondrial functions:

• A. dermatitidis has "+" and "-" mating types that can produce fertile ascocarps when paired

• Clinical isolates can contain both mating types, suggesting mixed infections or dimorphic characteristics

• Mitochondrial function could potentially influence the morphological transitions between yeast and mold forms

How does the role of FCJ1 in mitochondrial structure compare to other cristae-shaping proteins?

FCJ1 is one of several proteins that shape mitochondrial cristae, each with distinct mechanisms and functions:

This comparison highlights the complementary roles of these proteins in shaping different regions of the mitochondrial inner membrane. While FCJ1 is specialized for crista junction formation and maintenance, other factors like F1FO subunits e and g shape the positive curvature of cristae tips. The coordinated action of these proteins ensures proper mitochondrial inner membrane architecture .

What emerging techniques could advance our understanding of FCJ1 function?

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

• Cryo-electron tomography:

  • High-resolution 3D visualization of FCJ1 in its native context

  • Mapping precise arrangement of FCJ1 molecules at crista junctions

  • Examining structural changes under different conditions

• Super-resolution microscopy:

  • Live-cell imaging of FCJ1 dynamics using techniques like PALM, STORM, or STED

  • Tracking FCJ1 movement and turnover in real-time

  • Correlating FCJ1 localization with mitochondrial function

• Proximity labeling proteomics:

  • Identifying proximal interactors in living cells

  • Temporal mapping of the FCJ1 interaction network during cellular stress

  • Discovery of new components of the MICOS complex

These advanced techniques can provide unprecedented insights into FCJ1's molecular mechanism, dynamic behavior, and integration into cellular physiology, potentially revealing new aspects of its function.

How might FCJ1 function be linked to fungal adaptation to environmental stresses?

The connection between FCJ1 and stress adaptation in fungi represents an intriguing area for future research:

• Hypoxic adaptation:

  • Investigation of FCJ1 expression and localization during oxygen limitation

  • Analysis of whether FCJ1-dependent cristae remodeling facilitates respiratory adaptation

• Thermal stress response:

  • Examination of FCJ1's role in mitochondrial stability during temperature shifts

  • Assessment of whether FCJ1 contributes to the thermal dimorphism of A. dermatitidis

• Nutrient limitation:

  • Study of FCJ1-dependent mitochondrial remodeling during carbon source shifts

  • Evaluation of FCJ1's role in mitochondrial autophagy during starvation

Understanding these connections could reveal how mitochondrial structural adaptations contribute to fungal survival in diverse environments, including during host infection, potentially offering insights into A. dermatitidis pathogenicity.

What is the potential for studying post-translational modifications of FCJ1?

Understanding FCJ1 post-translational modifications (PTMs) requires a multi-faceted approach:

• Mass spectrometry-based identification:

  • Enrichment of FCJ1 using immunoprecipitation or affinity purification

  • Analysis by LC-MS/MS to identify modification sites

  • Quantitative proteomics to compare modification levels under different conditions

• Site-directed mutagenesis:

  • Mutation of identified modification sites to assess functional consequences

  • Creation of phosphomimetic mutations to simulate constitutive modification

  • Expression of mutant proteins in FCJ1-deficient backgrounds

• Modification-specific antibodies:

  • Development of antibodies that specifically recognize modified forms of FCJ1

  • Immunoblotting to detect changes in modification status under different conditions

These approaches can reveal regulatory mechanisms that control FCJ1 function and mitochondrial membrane architecture in response to cellular signals, offering new insights into the dynamic regulation of mitochondrial structure.