Recombinant Schizosaccharomyces pombe Formation of crista junctions protein C3E7.05c (SPBC3E7.05c)

What is SPBC3E7.05c protein and what is its role in Schizosaccharomyces pombe?

SPBC3E7.05c (also known as mic60 or Mitofilin) is a critical protein in Schizosaccharomyces pombe that functions as a subunit of the MICOS (mitochondrial contact site and cristae organizing system) complex. This protein plays an essential role in the formation and maintenance of crista junctions, which are the highly curved neck regions that connect the inner boundary membrane to the cristae membranes in mitochondria .

To study this protein:

• Begin with subcellular fractionation to isolate mitochondria from S. pombe cultures using differential centrifugation

• Confirm protein localization using fluorescent tagging (GFP fusion constructs) and confocal microscopy

• For ultrastructural analysis, employ transmission electron microscopy to visualize crista junction architecture in wild-type and mic60-depleted cells

Research has shown that Mic60 possesses membrane-shaping activity, deforming liposomes into thin membrane tubules. This activity is central to its function in creating the highly curved membrane structures at crista junctions .

How is recombinant SPBC3E7.05c protein expressed and purified for research purposes?

The recombinant full-length SPBC3E7.05c protein can be produced using the following methodological approach:

Expression System:

• Expression in E. coli using a His-tag fusion construct

• The typical expression region spans amino acids 39-550 of the mature protein

Purification Protocol:

• Transform E. coli with expression vector containing SPBC3E7.05c cDNA

• Induce protein expression with IPTG at optimal temperature (typically 18-25°C)

• Harvest cells and lyse using sonication or pressure-based methods

• Purify using affinity chromatography with Ni-NTA resin

• Perform size exclusion chromatography for further purification

• Concentrate and store in a Tris/PBS-based buffer with 6% Trehalose (pH 8.0)

Storage Considerations:

• Store at -20°C/-80°C

• For working aliquots, store at 4°C for up to one week

• Avoid repeated freeze-thaw cycles

• For long-term storage, add glycerol to a final concentration of 50%

What experimental approaches can be used to study the membrane-shaping activity of Mic60/SPBC3E7.05c?

Several sophisticated methodologies can be employed to investigate the membrane-shaping activity of Mic60:

In vitro membrane deformation assays:

• Liposome tubulation assays: Prepare synthetic liposomes with a composition mimicking the mitochondrial inner membrane. Incubate with purified Mic60 and observe membrane deformation using electron microscopy. This approach has demonstrated that Mic60 can deform liposomes into thin membrane tubules .

• Cryo-electron microscopy: Use cryo-EM to visualize the molecular details of Mic60-induced membrane curvature at nanometer resolution.

Structure-function analysis:

• Generate targeted mutations in specific domains of Mic60, particularly:

  • The membrane-binding site formed by the amphipathic helix

  • The mitofilin domain that regulates membrane-shaping activity

  • The coiled-coil domain involved in oligomerization

• Yeast mutation studies: Introduce mutations into the endogenous mic60 gene in S. pombe using CRISPR-Cas9 or traditional homologous recombination techniques. Analyze mitochondrial ultrastructure in these mutants by electron microscopy to correlate specific residues with membrane deformation activity .

Quantitative membrane interaction experiments:

• Surface plasmon resonance (SPR): Measure binding kinetics between purified Mic60 constructs and immobilized membrane mimetics.

• Fluorescence resonance energy transfer (FRET): Monitor protein-membrane interactions in real-time using fluorescently labeled Mic60 and lipid membranes.

Research has shown that the membrane-binding activity of Mic60 is modulated by its interaction with Mic19, which binds to the mitofilin domain and affects membrane shaping .

How does the Mic60-Mic19 complex form and function at the molecular level?

The Mic60-Mic19 complex forms through specific molecular interactions that are critical for proper crista junction formation:

Complex formation mechanism:

• The C-terminal mitofilin domain of Mic60 directly interacts with the C-terminal CHCH (coiled-coil-helix-coiled-coil-helix) domain of Mic19

• This interaction occurs via a conserved interface spanning approximately 660 Ų

• The mitofilin domain consists of α-helices that form a three-helical bundle with the CHCH domain of Mic19

Functional roles of the complex:

• Tetramerization: Mic19 promotes Mic60 tetramerization, which is critical for its function

• Regulation of membrane-shaping: The mitofilin domain negatively regulates the membrane-shaping activity of Mic60, while Mic19 binding to this domain modulates this activity

• Formation of molecular struts: The complex traverses crista junctions as molecular struts, controlling junction architecture and function

Experimental validation methods:

• Co-immunoprecipitation: To confirm protein-protein interactions in vivo

• X-ray crystallography: To resolve the atomic structure of the complex

• Mutational analysis: Introducing point mutations in the interaction interface disrupts complex formation and impairs crista junction formation, confirming the functional importance of the interaction

The Mic60-Mic19 complex represents a fundamental building block of the MICOS complex and is conserved from yeast to humans, highlighting its evolutionary importance in mitochondrial membrane architecture.

What are the structural insights into crista junction formation by the Mic60-Mic19 complex?

Recent structural studies have provided significant insights into how the Mic60-Mic19 complex shapes crista junctions:

Key structural features:

• Tetrameric assembly: The central coiled-coil domain of Mic60 forms an elongated, bow tie-shaped tetrameric assembly spanning approximately 17.5 nm

• Membrane interaction sites:

  • The mitofilin domain dimerizes to expose a crescent-shaped membrane-binding site

  • This site has convex curvature specifically tailored to interact with the highly curved crista junction neck

  • A second membrane-binding site is formed by the amphipathic helix located between the coiled-coil and mitofilin domains

• Molecular strut configuration:

  Feature	Dimensions/Properties
  Length of CC tetramer	~17.5 nm
  Orientation	Traverses the crista junction
  Membrane contacts	Simultaneous binding to inner boundary and cristae membranes
  Number of tetramers per junction	Estimated 2-3 tetramers
  Molecular architecture	Arch dome-like assembly vaulting the entry into cristae

Functional implications:

• The molecular dimensions of the complex directly influence the uniform diameters of circular or slit-like crista junctions

• The model explains how Mic60-Mic19 can simultaneously interact with both the inner boundary membrane and cristae membrane

• The structure suggests how the complex might form contact sites with the outer mitochondrial membrane via interactions with SAM and TOM complexes

These structural insights provide a mechanistic understanding of how the Mic60-Mic19 complex creates and maintains the highly curved membrane architecture at crista junctions, which is essential for proper mitochondrial function.

What experimental design considerations are important when studying the regulatory mechanisms of Mic60 activity?

When investigating the regulatory mechanisms controlling Mic60 activity, researchers should consider several key experimental design elements:

1. Controlling for post-translational modifications:

• Phosphorylation status may regulate Mic60 activity

• Design experiments to detect PTMs using:

  • Mass spectrometry-based phosphoproteomics

  • Phospho-specific antibodies

  • Phosphomimetic and phospho-null mutations

2. Membrane composition effects:

• Specific phospholipids have been implicated in cristae membrane organization and the generation of membrane curvature at crista junctions in cooperation with MICOS

• Experimental design should include:

  • Systematic variation of lipid composition in membrane mimetics

  • Analysis of lipid-binding properties of Mic60

  • Investigation of lipid-dependent conformational changes

3. Protein-protein interaction context:

• Mic60 function is modulated by interactions with other proteins, particularly Mic19

• Experimental considerations include:

  Approach	Application	Considerations
  In vitro reconstitution	Defined component analysis	May miss cellular factors
  In vivo studies	Physiological context	Higher complexity
  Mutational analysis	Structure-function relationships	Point mutations vs. domain deletions
  Crosslinking methods	Transient interactions	Chemical specificity

4. Temporal regulation:

• Mic60 activity may be regulated at different cell cycle stages or in response to metabolic changes

• Design elements should include:

  • Synchronized cell populations

  • Metabolic perturbation experiments

  • Live-cell imaging with temporal resolution

5. Validating findings across species:

• Evolutionary conservation suggests fundamental importance

• Cross-species experimental designs can confirm conserved regulatory mechanisms

• Consider testing findings in both S. pombe and higher eukaryotes

By carefully considering these experimental design elements, researchers can develop robust approaches to elucidate the complex regulatory mechanisms controlling Mic60 activity in crista junction formation and maintenance.

How can CRISPR-Cas9 genome editing be optimized for studying SPBC3E7.05c function in S. pombe?

CRISPR-Cas9 offers powerful approaches for studying SPBC3E7.05c function, but requires optimization for S. pombe's unique characteristics:

Optimization strategy for S. pombe:

• Guide RNA design:

  • Select target sites with minimal off-target effects

  • Optimize guide RNA sequence for S. pombe codon usage

  • Target conserved functional domains identified in structural studies (coiled-coil, mitofilin domains)

• Delivery method:

  • Plasmid-based expression of Cas9 and gRNA

  • Ribonucleoprotein (RNP) complex delivery via electroporation

  • Coordinate with the cell cycle by synchronizing cultures before transformation

• Repair template design:

  • For precise mutations: Include ~500-800 bp homology arms

  • For domain replacements: Consider codon optimization

  • For tagging: Ensure the tag doesn't interfere with critical domains

Validation approaches:

• Sequencing to confirm edits

• Western blotting to verify protein expression

• Electron microscopy to assess mitochondrial ultrastructure

• Growth assays to evaluate functional consequences

Key applications:

• Generate domain-specific mutants to dissect Mic60 function

• Create fluorescent protein fusions at endogenous loci for live imaging

• Introduce human disease-associated variants to study conservation of function

This approach allows for precise genetic manipulation to investigate the consequences of specific mutations in SPBC3E7.05c on mitochondrial structure and function.

What are the implications of SPBC3E7.05c research for understanding mitochondrial diseases in humans?

Research on SPBC3E7.05c in S. pombe provides valuable insights into human mitochondrial diseases through evolutionary conservation of function:

Translational relevance:

• The MICOS complex structure and function are highly conserved from yeast to humans

• Disruptions in cristae architecture are associated with numerous human diseases

• S. pombe provides a tractable model system to study fundamental processes

Mitochondrial diseases linked to MICOS dysfunction:

• Neurodegenerative disorders

• Mitochondrial encephalopathy

• Cardiomyopathies

• Age-related diseases

Mechanistic parallels:

• Human MIC60/Mitofilin shares structural features with S. pombe Mic60, including:

  • The membrane-shaping amphipathic helix

  • The coiled-coil domain for oligomerization

  • The regulatory mitofilin domain

• Mutations affecting similar domains in humans and yeast produce comparable phenotypes in mitochondrial ultrastructure

Therapeutic insights:

• Compounds that stabilize Mic60-mediated membrane curvature might have therapeutic potential

• Structure-based drug design targeting the Mic60-Mic19 interface could modulate crista junction formation

• Gene therapy approaches restoring MICOS function may be developed based on insights from yeast models

By leveraging the molecular understanding gained from S. pombe research, scientists can develop more targeted approaches to understanding and potentially treating human mitochondrial diseases resulting from MICOS dysfunction.

How can high-throughput approaches be applied to study the SPBC3E7.05c interactome?

Modern high-throughput techniques offer powerful ways to comprehensively map the SPBC3E7.05c interactome:

Mass spectrometry-based approaches:

• Proximity-dependent biotin identification (BioID):

  • Fuse a biotin ligase (BirA*) to SPBC3E7.05c

  • Proteins in close proximity become biotinylated

  • Isolate biotinylated proteins using streptavidin and identify by mass spectrometry

  • Particularly useful for identifying transient or weak interactions

• Quantitative affinity purification-mass spectrometry (AP-MS):

  • Use epitope-tagged SPBC3E7.05c as bait

  • Employ SILAC or TMT labeling for quantitative comparison

  • Compare interactomes under different conditions (e.g., nutrient starvation, cell cycle stages)

Genetic interaction screening:

• Synthetic genetic array (SGA) analysis:

  • Cross a query strain (SPBC3E7.05c mutant) with an ordered array of viable deletion mutants

  • Analyze growth phenotypes of double mutants to identify genetic interactions

  • This approach has been successfully applied in S. pombe for other genes

• CRISPR interference (CRISPRi) screens:

  • Generate a genome-wide gRNA library targeting S. pombe genes

  • Identify synthetic interactions with SPBC3E7.05c using depletion phenotypes

Data integration approach:

• Combine physical and genetic interaction data

• Incorporate structural information about interaction interfaces

• Use computational approaches to predict functional relationships

Such comprehensive interactome mapping would provide valuable insights into the functional context of SPBC3E7.05c and potentially identify novel players in crista junction formation and maintenance.