Recombinant Xenopus tropicalis FUN14 domain-containing protein 1 (fundc1)

How is recombinant Xenopus tropicalis FUNDC1 typically produced and purified?

Recombinant Xenopus tropicalis FUNDC1 is typically expressed in E. coli expression systems, using a His-tag or GST-tag fusion for purification purposes. The protein is commonly supplied as a lyophilized powder for stability and can be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, it's recommended to add glycerol (final concentration of 50%) and store in aliquots at -20°C to -80°C. The purified protein typically achieves greater than 90% purity as determined by SDS-PAGE .

What is the phylogenetic relationship of Xenopus tropicalis FUNDC1 to other vertebrate FUNDC1 proteins?

FUNDC1 contains a conserved FUN14 domain which is present in nearly all three domains of living organisms (eukaryotes, archaea, and bacteria). Phylogenetic analysis reveals that the FUN14 domain-containing protein family is highly conserved across species, suggesting fundamental biological importance. In contrast to the BNIP3 domain-containing protein family (another mitophagy receptor family) which is present only in animals, the broader distribution of FUN14 domain-containing proteins indicates their more ancient evolutionary origin . Western clawed frog (Xenopus tropicalis) FUNDC1 serves as an important model in comparative studies due to its position in vertebrate evolution.

What are the primary functions of FUNDC1 in cellular physiology?

FUNDC1 is a mitochondrial receptor located in the outer mitochondrial membrane (OMM) that governs the mitophagy process. It plays essential roles in:

• Mitophagy regulation: FUNDC1 is a receptor that mediates selective mitochondrial autophagy, particularly under hypoxic conditions.

• Mitochondrial dynamics: FUNDC1 regulates mitochondrial fission/fusion by interacting with DRP1 (promoting fission) and OPA1, functioning as a critical link between mitochondrial dynamics and mitophagy .

• Calcium homeostasis: FUNDC1 binds to IP3R2 (inositol 1,4,5-trisphosphate type 2 receptor) to modulate ER Ca²⁺ release into mitochondria and cytosol. Disruption of this interaction lowers Ca²⁺ levels in mitochondria and cytosol, which can lead to mitochondrial dysfunction .

• MAMs formation: FUNDC1 is localized in mitochondria-associated ER membranes (MAMs) and plays a role in their formation and stability .

While most functional studies have been conducted in mammalian systems, the high conservation of FUNDC1 suggests similar roles in Xenopus tropicalis.

How does FUNDC1 interact with the autophagy machinery during mitophagy?

In mammalian systems, FUNDC1 interacts with LC3 (a key autophagy protein) directly through its typical LIR (LC3-interacting region) motif Y(18)-x-x-L(21). During hypoxia-induced mitophagy, FUNDC1 undergoes dephosphorylation at key residues, which enhances its interaction with LC3 and promotes mitophagy .

The detailed mechanism in Xenopus tropicalis would require specific investigation, but given the conservation of the protein, similar interactions are likely to occur. Methodologically, researchers can examine these interactions through co-immunoprecipitation assays, fluorescence microscopy with tagged proteins, and mutation studies of the predicted LIR motif in the Xenopus FUNDC1.

What is the relationship between FUNDC1 and mitochondrial fission/fusion machinery?

FUNDC1 serves as a critical regulator of mitochondrial dynamics by interacting with both fission and fusion machinery:

• Interaction with DRP1: Under hypoxic conditions, FUNDC1 recruits DRP1 (Dynamin-related protein 1) to drive mitochondrial fission. This interaction occurs at the hydrophilic region of FUNDC1 .

• Interaction with CANX: FUNDC1 initially aggregates onto the mitochondrial membrane to interact with endoplasmic reticulum (ER) calcium-binding protein (CANX) under hypoxia. As mitophagy proceeds, FUNDC1 dissociates from CANX and recruits DRP1 .

• Regulation of mitochondrial morphology: Deletion of FUNDC1 in cells can provoke mitochondrial elongation and increased numbers of elongated mitochondria, thereby preventing mitophagy .

These interactions position FUNDC1 as a "springboard" between mitochondrial dynamics and mitophagy, coordinating both processes to maintain mitochondrial quality control.

How is FUNDC1 expression regulated at the transcriptional level?

FUNDC1 expression is regulated by the PGC-1α/NRF1 transcriptional axis:

• PGC-1α regulation: The peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) positively regulates FUNDC1 expression. Knockdown of PGC-1α reduces both protein and mRNA levels of FUNDC1, and this effect can be rescued by reintroducing PGC-1α .

• NRF1-dependent transcription: Nuclear respiratory factor 1 (NRF1) binds to the FUNDC1 promoter and is responsible for FUNDC1 expression. The binding can be demonstrated through electrophoretic mobility shift assay (EMSA) and ChIP-PCR analysis .

• Specificity of regulation: This regulation is specific to PGC-1α, as knockdown of the homolog PGC-1β has no effect on FUNDC1 expression .

Methodologically, researchers can investigate transcriptional regulation through promoter reporter assays, ChIP-seq, and gene expression analysis following modulation of transcription factors.

What post-translational modifications regulate FUNDC1 activity in mitophagy?

FUNDC1 activity is tightly regulated through several post-translational modifications:

• Phosphorylation: FUNDC1 is regulated through reversible phosphorylation at several key sites. Dephosphorylation of these sites during hypoxia enhances FUNDC1's interaction with LC3 and promotes mitophagy .

• Ubiquitylation: FUNDC1 is regulated through ubiquitylation and degradation by MARCH5, an E3 ubiquitin ligase .

These modifications provide a dynamic regulatory mechanism that allows cells to fine-tune mitophagy in response to various stressors, particularly hypoxia. Experimental approaches to study these modifications include phospho-specific antibodies, ubiquitylation assays, and mutagenesis of key residues to prevent or mimic modifications.

How does FUNDC1 contribute to calcium homeostasis between ER and mitochondria?

FUNDC1 plays a crucial role in calcium homeostasis through its interactions with ER calcium channels:

• Binding to IP3R2: FUNDC1 localizes in MAMs by binding to ER-resided inositol 1,4,5-trisphosphate type 2 receptor (IP3R2) .

• Modulation of calcium release: FUNDC1 modulates ER Ca²⁺ release into mitochondria and cytosol. Ablation of FUNDC1 disrupts MAMs and reduces Ca²⁺ levels in both mitochondria and cytosol, while overexpression increases these Ca²⁺ levels .

• Regulation of ER calcium levels: FUNDC1 ablation increases Ca²⁺ levels in ER, whereas FUNDC1 overexpression lowers ER Ca²⁺ levels .

• Interaction with FBXL2: FUNDC1 interacts with FBXL2, which is involved in calcium homeostasis. FUNDC1 deficiency accelerates palmitic acid-induced degradation of FBXL2 and decelerates degradation of IP3R3 .

This regulation of calcium flux is critical for maintaining mitochondrial function, as proper calcium levels are essential for various mitochondrial processes including ATP production and apoptosis regulation.

What are the optimal storage and handling conditions for recombinant Xenopus tropicalis FUNDC1?

For optimal results when working with recombinant Xenopus tropicalis FUNDC1:

• Short-term storage: Store at 2-8°C for 1-2 weeks.

• Long-term storage: Store at -20°C to -80°C in aliquots to avoid repeated freeze-thaw cycles.

• Lyophilized form: The protein is often supplied as a lyophilized powder, which should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• Cryoprotectants: Addition of glycerol (final concentration 50%) is recommended for long-term storage. Some preparations include trehalose (6%) and mannitol (5%) as protectants before lyophilization .

• Reconstitution buffer: Typically uses Tris/PBS-based buffer, pH 8.0 .

Following these guidelines will help maintain protein stability and activity for experimental applications.

What experimental approaches can be used to study FUNDC1's role in mitophagy in Xenopus models?

Several experimental approaches can be employed to study FUNDC1's role in mitophagy in Xenopus models:

• Mitophagy assays: Using fluorescent reporters such as mt-Keima (a pH-sensitive fluorescent protein targeted to mitochondria) to monitor mitochondrial delivery to lysosomes.

• Electron microscopy: To visualize mitochondrial morphology and mitophagosomes at the ultrastructural level.

• Co-immunoprecipitation: To identify FUNDC1-interacting proteins in Xenopus cells or tissues.

• CRISPR/Cas9-mediated gene editing: To generate FUNDC1 knockout or mutant Xenopus models to study loss-of-function phenotypes.

• Hypoxia treatment: To induce mitophagy and study FUNDC1's response, as it's known to play a key role in hypoxia-induced mitophagy.

• Oxygen consumption rate (OCR) assays: To assess mitochondrial function in FUNDC1-deficient versus wild-type samples.

• Measurement of mitochondrial DNA copy number: To assess mitochondrial content and turnover.

These approaches can be complemented with biochemical assays such as Western blotting to monitor FUNDC1 levels and phosphorylation status under various conditions.

How can recombinant FUNDC1 be used in structural biology studies?

Recombinant FUNDC1 can be utilized in various structural biology approaches:

For these applications, high-purity recombinant protein is essential, and the His-tagged version of Xenopus tropicalis FUNDC1 with >90% purity would be suitable for initial studies .

What are the key differences between Xenopus tropicalis FUNDC1 and mammalian FUNDC1?

While the search results don't provide a direct comparison between Xenopus tropicalis and mammalian FUNDC1, we can infer some key points:

Methodologically, researchers can perform sequence alignments, functional complementation studies (expressing Xenopus FUNDC1 in mammalian cells lacking endogenous FUNDC1), and comparative binding studies to elucidate these differences.

How can comparative studies of FUNDC1 across species contribute to understanding its evolution?

Comparative studies of FUNDC1 across species can provide valuable insights into its evolution:

• Evolutionary origin: The FUN14 domain-containing protein family is present in nearly all three domains of life (eukaryotes, archaea, and bacteria), unlike other mitophagy receptor families like BNIP3 which are present only in animals . This suggests an ancient evolutionary origin for FUNDC1.

• Functional conservation and diversification: By comparing FUNDC1 functions across species, researchers can identify core conserved functions versus species-specific adaptations.

• Regulatory mechanisms: Comparing transcriptional and post-translational regulatory mechanisms across species can reveal how FUNDC1 regulation has evolved.

• Interaction networks: Identifying differences in FUNDC1-interacting proteins across species can help understand the evolution of mitophagy pathways.

These comparative approaches can utilize techniques such as phylogenetic analysis, cross-species functional complementation, and comparative genomics to trace the evolutionary history of FUNDC1 and its functional networks.

How can FUNDC1 be targeted in research on mitochondrial diseases and therapeutic approaches?

FUNDC1's role in mitochondrial quality control makes it a potential target for research on mitochondrial diseases:

• Modulation of mitophagy: Since FUNDC1 is a key regulator of mitophagy, enhancing or inhibiting its activity could potentially modulate the clearance of damaged mitochondria in disease states.

• Cardiovascular applications: FUNDC1 has been shown to play a role in cardiac function. Loss of FUNDC1 in mice accentuates high-fat diet-induced cardiac remodeling, functional and mitochondrial anomalies, cell death, and calcium overload . This suggests potential applications in cardiovascular disease research.

• Targeting protein-protein interactions: Small molecules or peptides that modulate FUNDC1's interactions with partners like IP3R2, FBXL2, or LC3 could provide therapeutic approaches for diseases involving mitochondrial dysfunction.

• Gene therapy approaches: In models with FUNDC1 deficiency, restoring FUNDC1 expression could potentially ameliorate mitochondrial dysfunction and associated pathologies.

Methodologically, research can employ in vitro screening of compounds that modulate FUNDC1 activity, followed by validation in cellular and animal models of mitochondrial diseases.

What methodological challenges exist in studying FUNDC1's role in the mitochondria-associated ER membrane (MAM)?

Studying FUNDC1's role in MAMs presents several methodological challenges:

• MAM isolation: MAMs represent contact sites between mitochondria and ER, making their isolation technically challenging. Subcellular fractionation protocols need to be optimized to obtain pure MAM fractions.

• Visualization: MAMs are dynamic structures at the nanoscale, requiring super-resolution microscopy or electron microscopy for proper visualization.

• Functional assays: Measuring calcium transfer at MAMs requires sophisticated calcium imaging techniques with high spatial and temporal resolution.

• Protein complex analysis: FUNDC1 interacts with multiple proteins at MAMs, such as IP3R2. Capturing these interactions in their native state requires techniques like proximity labeling or in situ crosslinking.

• Species differences: MAM composition and dynamics may differ between Xenopus and mammalian systems, requiring species-specific optimization of techniques.

To overcome these challenges, researchers can employ approaches such as proximity ligation assays, FRET-based sensors for protein-protein interactions, and newly developed MAM-specific reporters.

How does the interaction between FUNDC1 and FBXL2 contribute to mitochondrial calcium homeostasis and cellular function?

The interaction between FUNDC1 and FBXL2 plays a crucial role in maintaining mitochondrial calcium homeostasis:

• FBXL2-IP3R3 regulation: FBXL2, an F-box protein component of an SCF ubiquitin ligase complex, binds to IP3R3 and targets it for degradation. This helps regulate calcium release from the ER .

• FUNDC1-FBXL2 interaction: Mass spectrometry and co-immunoprecipitation analyses have revealed an interaction between FUNDC1 and FBXL2. Truncated mutants of F-box (Delta-F-box) disengage FBXL2 interaction with FUNDC1 .

• Effect on calcium homeostasis: FUNDC1 deficiency accelerates palmitic acid-induced degradation of FBXL2 and decelerates degradation of IP3R3, leading to calcium overload in mitochondria .

• Physiological impact: In mouse models, loss of FUNDC1 accentuates high-fat diet-induced cardiac remodeling, functional and mitochondrial anomalies, and cell death, which correlates with a rise in IP3R3 and calcium overload .

This interaction provides a molecular link between mitophagy receptor FUNDC1 and calcium homeostasis regulation, suggesting that FUNDC1's functions extend beyond its direct role in mitophagy to broader aspects of mitochondrial physiology.