Recombinant Xenopus laevis Mitochondrial fission factor homolog B (mff-b), partial

What is the role of Mitochondrial Fission Factor (MFF) in cellular biology?

MFF is a critical regulator of mitochondrial dynamics, specifically involved in mitochondrial fission processes. Studies reveal that MFF functions as a key factor controlling mitochondrial morphology, quality control, and apoptosis. In mammalian systems, MFF has been identified as an essential regulator of mitochondrial functions through its interactions with other proteins involved in mitochondrial homeostasis . The protein plays a fundamental role in maintaining mitochondrial health by facilitating the division of mitochondria, which is necessary for removing damaged portions of the organelle and ensuring proper mitochondrial distribution during cell division.

What are the known protein interaction partners of MFF in vertebrate systems?

In mammalian systems, MFF has been identified as an interaction partner with several proteins involved in mitochondrial dynamics. Most notably, research has demonstrated that MFF interacts with:

• TBC1D15 - In mammalian cells, Fis1 (another mitochondrial fission protein) acts as a mitochondrial receptor for recruiting TBC1D15, which is associated with regulation of mitochondrial morphology .

• TRAF3 - Studies have identified TRAF3 as a novel binding partner of MFF in B lymphocytes. This interaction was demonstrated through co-immunoprecipitation and GST pull-down assays .

The TRAF3-MFF interaction has functional significance, as "co-expression of TRAF3 and MFF resulted in decreased phosphorylation and ubiquitination of MFF as well as decreased ubiquitination of TRAF3" . This suggests that MFF may be involved in post-translational modification networks that regulate mitochondrial fission processes.

What expression systems are recommended for producing recombinant Xenopus laevis mff-b?

For expression of recombinant Xenopus proteins, the Xenopus oocyte system offers significant advantages. This homologous expression system provides the appropriate cellular machinery for proper folding and post-translational modifications of Xenopus proteins. Based on methodologies developed for other Xenopus proteins:

"We present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes" .

This approach can be adapted for mff-b expression by:

• Generating cDNA constructs with appropriate affinity tags

• Microinjecting cRNA into Xenopus oocytes

• Allowing expression for 2-4 days

• Performing affinity purification with appropriate detergents

Alternatively, bacterial or insect cell expression systems can be utilized for producing recombinant mff-b, though these may require optimization of expression conditions and refolding protocols to ensure proper protein structure.

What purification strategies yield the highest purity and activity of recombinant mff-b?

Based on successful approaches with other mitochondrial membrane proteins from Xenopus:

• Affinity purification using well-positioned tags is highly effective. For example:

  • "The method was validated for four human and one murine transporter... [revealing] the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly" .

• A recommended purification workflow includes:

  • Membrane fraction isolation from expressing cells

  • Solubilization using mild detergents (CHAPS has been effective for mitochondrial proteins: "Mitochondrial lysates were cleared by centrifugation at 10,000 g for 20 minutes at 4°C" )

  • Affinity chromatography using appropriate tags (SBP-6xHis tags have been successfully used: "Cleared mitochondrial lysates were subsequently incubated with the Streptavidin-Sepharose beads to immunoprecipitate TRAF3-SBP-6xHis" )

  • Size exclusion chromatography for removing aggregates and ensuring homogeneity

• Activity preservation requires careful buffer optimization:

  • Maintaining pH between 7.2-7.4

  • Including stabilizing agents such as glycerol (5-10%)

  • Adding reducing agents like DTT (1 mM) to prevent oxidation of cysteine residues

How can the mitochondrial fission activity of recombinant mff-b be measured in vitro?

Several complementary approaches can be employed to assess the mitochondrial fission activity of recombinant mff-b:

• Microscopy-based morphological analysis:

  • Expressing mff-b in cells and quantifying changes in mitochondrial network morphology

  • Parameters to measure include mitochondrial fragmentation index, network connectivity, and organelle size distribution

• Biochemical interaction assays:

  • Pull-down assays to measure binding with known interaction partners

  • For example, methods similar to those used for other mitochondrial proteins: "For ubiquitination analysis, 293T cells were co-transfected with LPC-HA-Ub and pUB-TRAF3-SBP-6xHis, pUB-Myc-MFF or an empty lentiviral vector pUB-Thy1.1"

• Mitochondrial functional assessments:

  • Measuring changes in mitochondrial membrane potential

  • Assessing mitochondrial ROS production: "For mitochondrial superoxide analysis, mouse splenic B cells were washed with PBS and stained with 1 μM of MitoSOX Red for 30 minutes at 37°C"

  • Quantifying changes in mitochondrial respiration

What techniques are most effective for studying mff-b interactions with other mitochondrial proteins?

Based on successful approaches documented in the literature:

• Co-immunoprecipitation assays:

  • Particularly effective for detecting native protein complexes

  • Example protocol: "Mitochondrial proteins were immunoprecipitated with Streptavidin (SA)-Sepharose beads. Immunoprecipitates of TRAF3-SBP-6xHis (SA IP) from the mitochondrial proteins were analyzed by immunoblotting and used to identify TRAF3-interacting proteins by LC-MS/MS-based sequencing"

• GST pull-down assays:

  • Useful for confirming direct protein-protein interactions

  • "TRAF3 specifically interacted with MFF as demonstrated by co-immunoprecipitation and GST pull-down assays"

• Proximity labeling methods:

  • BioID or APEX2-based approaches for identifying neighboring proteins in the mitochondrial outer membrane

• Fluorescence-based interaction assays:

  • FRET (Förster Resonance Energy Transfer) for measuring protein-protein interactions in live cells

  • BiFC (Bimolecular Fluorescence Complementation) for visualizing protein complex formation

How can mff-b be utilized to study mitochondrial dynamics in Xenopus development?

Xenopus embryos provide an excellent model system for studying developmental processes due to their experimental tractability:

• Microinjection of modified mff-b constructs:

  • "Xenopus embryos are ideal for studying gene function during embryogenesis by simple microinjection of mRNAs, antisense morpholinos, or genome editing constructs, because a well-defined cell fate map allows easy tissue-restricted manipulation"

  • Tissue-specific promoters can direct expression to specific cell lineages

• CRISPR/Cas9 genome editing:

  • "CRISPR/Cas9 gene editing is very effective in Xenopus, both for transient biallelic mutations in F0 embryos (X. laevis and X. tropicalis)"

  • This approach can be used to generate mff-b mutations or tagged versions of the endogenous protein

• Time-lapse imaging of mitochondrial networks:

  • Fluorescently tagged mff-b can be used to track mitochondrial dynamics during key developmental transitions

  • The large size of Xenopus cells facilitates high-resolution imaging

What are the challenges in distinguishing the function of mff-b from other mitochondrial fission factors in Xenopus?

Several experimental challenges must be addressed:

• Genetic redundancy:

  • Due to Xenopus laevis being allotetraploid, there may be multiple copies of mff genes

  • "The original Xenopus laevis is allotetraploid with larger embryos, whereas the more recently adopted Xenopus tropicalis is diploid"

  • Researchers must carefully design experiments to account for this genetic complexity

• Functional overlap:

  • Multiple proteins contribute to mitochondrial fission (e.g., Fis1, Drp1, MFF)

  • "Fis1 acts as a mitochondrial receptor in the recruitment of mitochondrial morphology protein in mammalian cells"

  • Careful experimental design is needed to isolate mff-b-specific effects

• Experimental approach recommendations:

  • Use combined knockdown/knockout strategies targeting multiple factors

  • Employ rescue experiments with specific mutations to map functional domains

  • Utilize both X. laevis and X. tropicalis for comparative studies: "X. laevis will continue to be the preferred model system for proteome analysis, as it has already been for the cell cycle or the analysis Wnt signaling dynamics"

What are common pitfalls in working with recombinant mitochondrial membrane proteins from Xenopus?

Researchers should be aware of several technical challenges:

• Protein solubility and stability issues:

  • Mitochondrial membrane proteins often have hydrophobic domains that can cause aggregation

  • Recommendation: "Total cellular proteins were lysed in 1% CHAPS Lysis Buffer containing 1x Phosphatase Inhibitors (Pierce) and 1 mM NEM"

  • Testing multiple detergents and buffer conditions is critical for maintaining native structure

• Expression level variability:

  • Expression in heterologous systems may yield variable results

  • The choice between X. laevis and X. tropicalis can impact results: "embryos remain experimentally very attractive because they are larger, easier to manipulate, and also because they yield about five-fold more material per embryo, an asset for biochemical work"

• Post-translational modifications:

  • Ensure the expression system can reproduce native modifications

  • Consider that "co-expression of TRAF3 and MFF resulted in decreased phosphorylation and ubiquitination of MFF"

  • Modifications may be critical for proper function and interactions

How can structural analysis of recombinant mff-b be optimized?

For structural characterization of recombinant mff-b:

• Electron microscopy approaches:

  • "Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters... revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly"

  • This approach can reveal oligomeric state and general structural features

• Crystallization strategies:

  • 2D crystallization has been successful for membrane proteins from Xenopus: "we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein"

  • Detergent screening is critical for finding conditions that maintain native structure

• Protein engineering considerations:

  • Strategic placement of affinity tags to minimize functional interference

  • Use of fusion partners to enhance solubility

  • Construct design to remove flexible regions that may impede crystallization