Recombinant Ashbya gossypii Mitochondrial inner membrane organizing system protein AFR743W (AFR743W)

How does the MINOS complex contribute to mitochondrial membrane organization?

The MINOS complex (Mitochondrial Inner Membrane Organizing System) consists of six subunits that are all inner membrane proteins exposed to the intermembrane space . This complex is crucial for:

• Maintaining the characteristic architecture of the inner mitochondrial membrane

• Forming and stabilizing cristae junctions

• Facilitating connections between the inner and outer mitochondrial membranes

Research has demonstrated that MINOS independently interacts with both preprotein translocases of the outer mitochondrial membrane - the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM) . This interaction suggests that MINOS plays a role beyond membrane architecture maintenance, also contributing to protein biogenesis pathways, particularly for β-barrel proteins of the outer membrane .

How does Ashbya gossypii AFR743W compare to homologs in other species?

While exact comparative data for AFR743W across multiple species is limited in the provided search results, we can infer relationships based on functional conservation. The MINOS complex is highly conserved across eukaryotes, from fungi to mammals, indicating the fundamental importance of this system in mitochondrial biology.

MIC10 proteins share functional roles across species but may have evolved species-specific adaptations. In many organisms, MIC10 interacts with components like mitofilin/Fcj1 to maintain cristae architecture . Future comparative genomics and structural studies would be valuable to fully elucidate the evolutionary relationships between AFR743W and its homologs.

What experimental approaches can be used to study the interaction network of AFR743W?

Studying the interaction network of AFR743W requires a multi-faceted approach:

Biochemical Approaches:

• Co-immunoprecipitation (Co-IP): Using antibodies against tagged recombinant AFR743W to pull down interaction partners, followed by mass spectrometry.

• Crosslinking Mass Spectrometry: Chemical crosslinking followed by mass spectrometry to identify transient or weak interactions.

• Blue Native PAGE: To preserve native protein complexes and determine the size and composition of MINOS subcomplexes containing AFR743W.

Structural Biology Approaches:

• Cryo-electron microscopy: For visualization of the entire MINOS complex architecture.

• X-ray crystallography: Using purified recombinant AFR743W protein to determine its high-resolution structure .

Genetic Approaches:

• Yeast two-hybrid screening: To identify protein-protein interactions.

• CRISPR-Cas9 genome editing: For creating specific mutations in the AFR743W gene to study their effect on protein interactions.

The dissection of MINOS interactions with TOM and SAM complexes has revealed that MINOS binds to both translocases independently, and specifically binds to the SAM complex via the conserved polypeptide transport-associated domain of Sam50 .

How does AFR743W contribute to mitochondrial biogenesis and protein import?

AFR743W, as part of the MINOS complex, plays a significant role in mitochondrial protein biogenesis beyond membrane architecture:

• Interaction with Import Machinery: MINOS independently interacts with both the TOM and SAM complexes, which are essential for protein import into mitochondria .

• β-barrel Protein Biogenesis: Research indicates that MINOS plays a crucial role specifically in the biogenesis of β-barrel proteins destined for the outer mitochondrial membrane .

• Membrane Contact Sites: AFR743W/MIC10 likely contributes to the formation of contact sites between inner and outer membranes, which are important for coordinated protein import.

The exact molecular mechanism by which AFR743W facilitates these processes requires further investigation, but the current data suggests a model where MINOS serves as a scaffold that brings together protein import machineries and maintains the spatial organization necessary for efficient protein biogenesis.

What implications does AFR743W dysfunction have on cellular metabolism in Ashbya gossypii?

Dysfunction of AFR743W would be expected to have cascading effects on multiple cellular processes:

• Disrupted Mitochondrial Morphology: Loss of proper cristae junction formation, leading to abnormal mitochondrial ultrastructure.

• Impaired Respiratory Function: Altered cristae architecture could impact the organization of respiratory chain complexes.

• Metabolic Reprogramming: A. gossypii is used industrially for riboflavin production , and mitochondrial dysfunction could shift metabolic flux toward alternative pathways.

• Growth and Development Effects: MINOS dysfunction could impact the filamentous growth pattern characteristic of A. gossypii.

• Stress Response Activation: Mitochondrial dysfunction typically triggers cellular stress responses that may further alter metabolism.

Given that A. gossypii has been engineered for various biotechnological applications including utilizing different carbon sources , AFR743W dysfunction could significantly impact these bioengineering efforts by altering core mitochondrial functions.

What are the optimal conditions for expressing and purifying recombinant AFR743W?

Based on available information about commercially produced recombinant AFR743W:

Expression System:

• Escherichia coli is the preferred heterologous expression system for recombinant AFR743W .

• The full-length protein (amino acids 1-82) with an N-terminal His-tag has been successfully expressed .

Purification Protocol:

• Affinity Chromatography: Using Ni-NTA or similar matrices to capture the His-tagged protein.

• Buffer Conditions: Tris/PBS-based buffer, pH 8.0 with 6% Trehalose for stability .

• Storage: The purified protein is typically stored in the presence of 50% glycerol at -20°C/-80°C for long-term storage .

Critical Considerations:

• Avoid repeated freeze-thaw cycles as this may affect protein stability and function .

• For working aliquots, storage at 4°C for up to one week is recommended .

• Reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

What analytical methods are most suitable for characterizing recombinant AFR743W?

Purity Assessment:

• SDS-PAGE can confirm protein purity (>90% purity is typically achieved for commercial preparations) .

• Western blotting with anti-His antibodies can verify the identity of the recombinant protein.

Structural Characterization:

• Circular dichroism (CD) spectroscopy to assess secondary structure.

• Analytical ultracentrifugation to determine oligomerization state.

• Mass spectrometry for molecular weight verification and post-translational modification analysis.

Functional Characterization:

• Membrane binding assays to test interaction with lipids.

• In vitro reconstitution with other MINOS components to assess complex formation.

• Electron microscopy to visualize membrane remodeling activity.

What approaches can be used to study AFR743W interactions with other MINOS components?

In vitro Binding Assays:

• Surface Plasmon Resonance (SPR): To measure binding kinetics between purified AFR743W and other MINOS components.

• Microscale Thermophoresis (MST): For detecting interactions and determining binding affinities.

• Isothermal Titration Calorimetry (ITC): To measure thermodynamic parameters of binding interactions.

Reconstitution Systems:

• Liposome Reconstitution: Incorporating purified AFR743W and interaction partners into artificial membranes.

• Nanodiscs: For studying membrane protein interactions in a more native-like environment.

Visualization Techniques:

• Negative Stain EM: To visualize complex formation between AFR743W and other MINOS components.

• FRET Assays: Using fluorescently labeled components to detect proximity and interactions.

The independent binding of MINOS to both TOM and SAM complexes suggests that similar techniques could be employed to study how AFR743W specifically contributes to these interactions.

How can recombinant AFR743W be used in structural biology studies?

Recombinant AFR743W can be leveraged for various structural biology approaches:

X-ray Crystallography:

• The availability of highly purified recombinant protein (>90% purity) makes it suitable for crystallization trials.

• The relatively small size (82 amino acids) is advantageous for crystallization.

• Co-crystallization with interacting partners can provide insights into complex formation.

NMR Spectroscopy:

• The small size of AFR743W makes it amenable to solution NMR studies.

• Can provide information about dynamics and conformational changes upon interaction with binding partners.

Cryo-EM Studies:

• For visualization of AFR743W within the larger MINOS complex.

• Particularly useful for studying membrane-associated conformations.

Computational Structural Biology:

• Molecular dynamics simulations to study AFR743W interaction with lipid membranes.

• Homology modeling using structures of homologous proteins from other species.

How can A. gossypii serve as a model system for studying mitochondrial membrane organization?

A. gossypii offers several advantages as a model system:

Experimental Tractability:

• A. gossypii is a filamentous fungus with well-established genetic manipulation tools .

• Its genome has been fully sequenced and annotated, facilitating genomic studies.

Biotechnological Relevance:

• A. gossypii is used industrially for riboflavin production , making findings potentially relevant to biotechnology applications.

• Engineering efforts have already established protocols for manipulating metabolic pathways .

Evolutionary Insights:

• Comparative studies between A. gossypii and other fungi can provide evolutionary context.

• As a filamentous fungus, it offers a different cellular organization compared to unicellular yeast models.

Research Approaches:

• Genetic Manipulation: Using CRISPR-Cas9 or traditional homologous recombination.

• Live-Cell Imaging: For visualizing mitochondrial dynamics and structure.

• Metabolic Analysis: Connecting mitochondrial structure to metabolic output.

• Electron Microscopy: For ultrastructural analysis of mitochondrial morphology.

What are the emerging techniques for studying AFR743W function in vivo?

Several cutting-edge approaches could advance our understanding of AFR743W function:

CRISPR-Based Approaches:

• CRISPRi/CRISPRa: For tunable repression or activation of AFR743W expression.

• Base Editing: For introducing specific point mutations without double-strand breaks.

• Prime Editing: For precise genome editing to study specific domains or residues.

Advanced Imaging Techniques:

• Super-Resolution Microscopy: For visualizing AFR743W localization and dynamics at nanoscale resolution.

• Correlative Light and Electron Microscopy (CLEM): To connect protein localization with ultrastructural features.

• Live-Cell Single-Molecule Tracking: For studying dynamics of individual AFR743W molecules.

Systems Biology Approaches:

• Multi-omics Integration: Combining transcriptomics, proteomics, and metabolomics to understand the systems-level impact of AFR743W.

• Metabolic Flux Analysis: To determine how AFR743W impacts metabolic pathways.

Comparative Studies:

• Leveraging the growing body of research on MINOS complexes across species to identify conserved mechanisms and species-specific adaptations.

How might AFR743W research contribute to understanding human mitochondrial diseases?

Although AFR743W is from A. gossypii, research on this protein can provide valuable insights for human health:

Conserved Mitochondrial Mechanisms:

• The MINOS complex is highly conserved across eukaryotes.

• Fundamental principles of mitochondrial membrane organization discovered in A. gossypii may apply to human mitochondria.

Disease Models:

• Mutations in human MINOS components (including MICOS10/MIC10) have been associated with mitochondrial disorders.

• A. gossypii could serve as a model system for testing hypotheses about disease mechanisms.

Therapeutic Approaches:

• Understanding the basic biology of MINOS may suggest novel therapeutic targets.

• Recombinant proteins could be used for high-throughput screening of compounds that stabilize MINOS function.