Recombinant Schizosaccharomyces pombe Succinate dehydrogenase [ubiquinone] cytochrome b small subunit, mitochondrial (sdh4)

What is Schizosaccharomyces pombe and why is it used as a model organism?

Schizosaccharomyces pombe, commonly known as fission yeast, is a unicellular eukaryotic organism that has emerged as a powerful model system for studying various cellular processes. S. pombe belongs to the division Ascomycota, the largest and most diverse group of fungi. It was first isolated from various fermented beverages and is naturally found in tree exudates, plant roots, soil, and fermented fruits .

S. pombe has become a preferred model organism for several reasons. First, its genome was fully sequenced in 2002, becoming the sixth eukaryotic organism to achieve this milestone. Approximately 70% of its genes have orthologs in humans, including many genes involved in human diseases, making it valuable for biomedical research . Additionally, S. pombe has contributed significantly to our understanding of DNA damage repair mechanisms, with numerous assays developed to study outcomes of mitotic recombination, a major repair mechanism for DNA double-strand breaks and stalled or collapsed replication forks . The subcellular localization of almost all proteins in S. pombe has been mapped using green fluorescent protein tagging, further enhancing its utility in research .

What is the role of succinate dehydrogenase in mitochondrial function?

Succinate dehydrogenase (SDH) is a critical enzyme that functions in both the tricarboxylic acid (TCA) cycle and the electron transport chain. In the TCA cycle, SDH catalyzes the oxidation of succinate to fumarate. Simultaneously, in the electron transport chain (complex II), it transfers electrons to ubiquinone (coenzyme Q).

The complete SDH complex typically consists of four subunits: SDHA (flavoprotein), SDHB (iron-sulfur protein), and SDHC and SDHD (membrane anchor proteins). The sdh4 gene specifically encodes the smallest subunit (equivalent to SDHD in mammals), which is a hydrophobic protein embedded in the inner mitochondrial membrane. This subunit is essential for anchoring the catalytic components to the membrane and for proper electron transfer to ubiquinone.

In S. pombe mitochondria, the respiratory chain components work together to generate ATP through oxidative phosphorylation. Studies have shown that S. pombe mitochondria exhibit antimycin A-sensitive oxygen uptake activity when utilizing various substrates including succinate, which involves the SDH complex . Understanding sdh4's role helps elucidate the mechanisms of cellular respiration and energy production in this model organism.

What techniques are available for genetic manipulation of S. pombe?

S. pombe offers several sophisticated techniques for genetic manipulation, making it an excellent model for studying genes like sdh4. A simple modification of the transformation procedure for directing integration of genomic sequences has been shown to increase transformation efficiency by 5-fold when using antibiotic-based dominant selection markers .

Additionally, the removal of the pku70+ and pku80+ genes, which encode DNA end-binding proteins required for the non-homologous end joining DNA repair pathway, can increase gene targeting efficiency by approximately 16-fold (to around 75-80%) . This is particularly useful for genes that are typically difficult to target.

Several vector systems have been developed to facilitate genetic studies in S. pombe:

• pINTL and pINTK vectors use ura4+ selection to direct disruptive integration of leu1+ and lys1+ respectively

• pINTH vectors exploit nourseothricin resistance to detect targeted disruption of a hygromycin B resistance-conferring hphMX6 cassette

• Multi-copy expression vectors that use resistance to nourseothricin or kanamycin/G418 for selection in prototrophic hosts

These tools collectively enhance the versatility of S. pombe as a model system for studying genes like sdh4.

How can recombinant sdh4 protein be expressed and purified?

Expression and purification of recombinant S. pombe sdh4 protein requires specialized techniques due to its hydrophobic nature and mitochondrial localization. The following methodology outlines a general approach:

• Vector Selection and Construction:

  • Choose an appropriate expression vector compatible with either E. coli or yeast expression systems

  • Include a purification tag (His, GST, or FLAG) to facilitate subsequent purification

  • For the S. pombe sdh4, consider using the full 85 amino acid sequence as mentioned in the search results

• Expression System Selection:

  • For membrane proteins like sdh4, yeast expression systems (particularly S. cerevisiae) often yield better results than bacterial systems

  • Alternative systems include insect cells or mammalian cells for proteins requiring eukaryotic post-translational modifications

• Optimization of Expression Conditions:

  • Test various induction conditions (temperature, inducer concentration, duration)

  • For S. pombe proteins, the urg1 promoter system allows rapid induction within 30 minutes, comparable to the S. cerevisiae GAL induction system

• Membrane Protein Purification:

  • Cell lysis using mechanical disruption methods

  • Membrane fraction isolation through differential centrifugation

  • Solubilization using appropriate detergents (typically DDM, LDAO, or Triton X-100)

  • Affinity chromatography based on the fusion tag

  • Size exclusion chromatography for final purification

• Quality Assessment:

  • SDS-PAGE and Western blotting to confirm identity

  • Mass spectrometry to verify protein integrity

  • Functional assays to confirm activity

When working specifically with S. pombe sdh4, researchers should note that the protein must maintain its native conformation to retain functionality, and detergent selection is critical for preserving membrane protein structure during purification.

What methods can be used to study sdh4 localization in S. pombe?

Several methods can be employed to study the subcellular localization of sdh4 in S. pombe:

• Fluorescent Protein Tagging:

  • Green Fluorescent Protein (GFP) tagging is particularly effective, as demonstrated by the comprehensive protein localization study in S. pombe

  • For mitochondrial proteins like sdh4, construct a C-terminal GFP fusion (N-terminal tags may interfere with mitochondrial targeting)

  • Express the fusion protein using either the native promoter (for physiological expression levels) or an inducible promoter system like urg1

• Immunofluorescence Microscopy:

  • Generate antibodies against the sdh4 protein or use antibodies against epitope tags

  • Fix cells with formaldehyde or methanol and perform immunostaining

  • Counterstain with DAPI to visualize nuclei and use mitochondrial markers (like MitoTracker) for colocalization studies

• Subcellular Fractionation and Western Blotting:

  • Isolate mitochondria from S. pombe cells using differential centrifugation

  • Perform protease protection assays to determine membrane topology

  • Conduct Western blotting with sdh4-specific antibodies

  • As shown in research on mitochondrial alcohol dehydrogenase in S. pombe, enzyme assays with Triton X-100 treatment can confirm matrix localization of proteins

• Electron Microscopy with Immunogold Labeling:

  • Prepare thin sections of S. pombe cells for transmission electron microscopy

  • Use antibodies conjugated to gold particles for precise localization

  • This technique provides high-resolution images of protein localization within mitochondrial subcompartments

• Proximity Labeling Techniques:

  • Fuse sdh4 to enzymes like BioID or APEX2 that biotinylate nearby proteins

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

  • This approach reveals the protein interaction network of sdh4 in its native context

By combining these techniques, researchers can comprehensively characterize the localization, topology, and interactions of the sdh4 protein in S. pombe mitochondria.

How does sdh4 contribute to respiratory function in S. pombe?

Sdh4 plays a crucial role in the respiratory function of S. pombe by serving as an essential component of Complex II (succinate dehydrogenase) in the mitochondrial electron transport chain. Its contributions can be understood through several key aspects:

Understanding sdh4's contribution to respiratory function provides valuable insights into mitochondrial energy metabolism and the adaptation of S. pombe to various growth conditions.

What are the key differences between sdh4 in S. pombe and other model organisms?

The sdh4 protein exhibits notable differences between S. pombe and other model organisms, reflecting evolutionary adaptation and specialized functions:

Key differences include:

These differences make comparative studies of sdh4 across species valuable for understanding mitochondrial evolution, gene transfer, and adaptation of respiratory functions.

How can we study the dual genomic presence of sdh4 and its expression patterns?

Some organisms demonstrate the phenomenon of having functional copies of sdh4 in both the mitochondrial and nuclear genomes. Studying this dual genomic presence and the resulting expression patterns requires sophisticated approaches:

• Genome Analysis and Comparison:

  • Conduct whole-genome sequencing to confirm the presence of sdh4 copies in both genomes

  • Perform sequence comparison to identify differences between nuclear and mitochondrial versions

  • Analyze surrounding genomic regions to understand the evolutionary context

  • Similar analyses in Populus revealed that nuclear copies of sdh4 had acquired N-terminal mitochondrial targeting presequences from pre-existing genes

• Transcriptional Analysis:

  • Employ RNA-Seq to quantify expression levels from both genomic copies

  • Use variant-specific primers in RT-PCR to distinguish transcripts from each copy

  • Apply nascent RNA capture techniques to measure transcription rates

  • Studies in Populus demonstrated that both nuclear and mitochondrial copies of sdh4 were expressed in multiple organs

• RNA Editing Analysis:

  • Investigate RNA editing events in mitochondrial transcripts

  • Compare edited mitochondrial transcripts with nuclear transcripts

  • Research has shown that RNA editing occurs in mitochondrial copies of sdh4 in some species

• Protein Isoform Characterization:

  • Use mass spectrometry to distinguish protein products from different genomic copies

  • Develop isoform-specific antibodies for Western blotting and immunoprecipitation

  • Apply ribosome profiling to measure translation rates of each transcript

• Regulatory Element Analysis:

  • Characterize promoters and regulatory elements controlling each copy

  • Perform ChIP-seq to identify transcription factors binding to nuclear copy promoters

  • Analyze mitochondrial transcription and processing mechanisms

• Tissue-Specific and Condition-Responsive Expression:

  • Measure expression of both copies across different tissues/cell types

  • Analyze expression changes under various stresses and metabolic conditions

  • Create reporter constructs with copy-specific promoters to visualize expression patterns

• Functional Complementation Experiments:

  • Selectively inactivate each genomic copy and assess phenotypic consequences

  • Perform cross-species complementation to test functional equivalence

  • Use the S. pombe genetic manipulation techniques described earlier to create specific genomic modifications

This multi-faceted approach provides comprehensive insights into the evolutionary significance, regulation, and functional implications of maintaining dual genomic copies of sdh4.

What are the molecular mechanisms of electron transfer involving sdh4 in S. pombe?

The molecular mechanisms of electron transfer involving sdh4 in S. pombe succinate dehydrogenase (Complex II) involve sophisticated protein-protein and protein-cofactor interactions:

Understanding these molecular mechanisms provides insights into mitochondrial energy conversion efficiency and may reveal targets for metabolic engineering or therapeutic interventions in related human diseases.

How can we design experiments to study the evolution of mitochondrial to nuclear gene transfer of sdh4?

Designing experiments to study the evolution of mitochondrial to nuclear gene transfer of sdh4 requires innovative approaches spanning comparative genomics, molecular biology, and evolutionary analysis:

• Comparative Genomic Analysis:

  • Sample diverse species across phylogenetic lineages to identify patterns of sdh4 gene location

  • Sequence both nuclear and mitochondrial genomes to detect potential duplicate copies

  • Construct phylogenetic trees to establish evolutionary relationships of sdh4 genes

  • Previous research in Populus identified both nuclear and mitochondrial copies of sdh4, with evidence of coexpression

• Experimental Evolution Approaches:

  • Design long-term evolution experiments with S. pombe under conditions that might favor gene transfer

  • Apply selective pressures that could benefit from nuclear encoding (e.g., mitochondrial stress)

  • Periodically sequence genomic DNA to detect spontaneous transfer events

  • Create reporter systems to detect functional nuclear integration of mitochondrial genes

• Functional Analysis of Transferred Genes:

  • Identify naturally transferred sdh4 genes in related species

  • Analyze acquisition of targeting sequences and regulatory elements

  • Studies have shown that transferred genes can gain N-terminal mitochondrial targeting presequences from pre-existing genes

  • Test functionality through complementation experiments in S. pombe sdh4 mutants

• Synthetic Biology Approaches:

  • Artificially transfer the mitochondrial sdh4 gene to the nucleus with various targeting sequences

  • Assess expression, protein import, and functional integration

  • Systematically modify the synthetic nuclear gene to determine minimum requirements for functional transfer

  • Apply the advanced S. pombe genetic manipulation techniques to integrate and express the transferred gene

• RNA Editing and Processing Analysis:

  • Compare RNA editing patterns between mitochondrial and nuclear sdh4 transcripts

  • Investigate changes in codon usage and adaptation following transfer

  • Research has documented RNA editing in mitochondrial copies of sdh4 in some species

• Computational Modeling:

  • Develop models of selection pressure for gene transfer

  • Simulate evolutionary trajectories under various conditions

  • Predict intermediate states in the transfer process

• Analysis of Transfer Mechanisms:

  • Test hypotheses about RNA-mediated vs. DNA-mediated transfer

  • Investigate the role of nuclear insertions of mitochondrial DNA (NUMTs)

  • Study the involvement of DNA repair machinery in integration events

These experimental approaches would provide comprehensive insights into the evolutionary process of mitochondrial gene transfer to the nucleus, a fundamental aspect of eukaryotic evolution with implications for understanding organellar genome reduction and nuclear-mitochondrial communication.

What methodologies can be used to investigate sdh4 interactions with other components of the respiratory chain?

Investigating the interactions between sdh4 and other components of the respiratory chain requires sophisticated biochemical, biophysical, and genetic approaches:

• Protein Crosslinking and Mass Spectrometry:

  • Apply chemical crosslinkers to stabilize transient protein-protein interactions

  • Perform in vivo crosslinking in intact S. pombe mitochondria

  • Digest crosslinked complexes and analyze by mass spectrometry

  • Map interaction sites at amino acid resolution

• Co-immunoprecipitation and Pull-down Assays:

  • Generate antibodies against sdh4 or use epitope-tagged versions

  • Perform co-immunoprecipitation from solubilized mitochondrial membranes

  • Identify interacting partners by Western blotting or mass spectrometry

  • Verify direct interactions using recombinant proteins in pull-down assays

• Blue Native PAGE Analysis:

  • Isolate intact respiratory complexes and supercomplexes using mild detergents

  • Separate complexes by Blue Native PAGE

  • Identify complex composition using 2D electrophoresis or mass spectrometry

  • Compare complex assembly in wild-type and sdh4 mutant strains

• Cryo-Electron Microscopy:

  • Purify intact Complex II or respiratory supercomplexes

  • Determine high-resolution structures by cryo-EM

  • Map the position of sdh4 and its interactions with neighboring subunits

  • Identify conformational changes associated with different functional states

• Genetic Interaction Mapping:

  • Create a library of S. pombe strains with mutations in respiratory chain components

  • Perform synthetic genetic array analysis with sdh4 mutations

  • Identify genetic interactions suggesting functional relationships

  • Use the advanced S. pombe genetic techniques to create precise mutations

• Proximity Labeling Techniques:

  • Fuse sdh4 to promiscuous biotin ligases (BioID) or peroxidases (APEX)

  • Allow in vivo labeling of proteins in close proximity to sdh4

  • Purify biotinylated proteins and identify by mass spectrometry

  • Map the spatial environment of sdh4 in the mitochondrial membrane

• Förster Resonance Energy Transfer (FRET):

  • Create fluorescent protein fusions with sdh4 and potential interaction partners

  • Measure FRET efficiency in live S. pombe cells

  • Quantify interaction strength and dynamics

  • Assess the effects of metabolic conditions on interaction patterns

• Hydrogen-Deuterium Exchange Mass Spectrometry:

  • Expose purified complexes to deuterated water

  • Monitor the rate of hydrogen-deuterium exchange by mass spectrometry

  • Identify protected regions indicating protein-protein interaction sites

  • Compare exchange patterns under different functional conditions

• Modular-Kinetic Analysis:

  • Perform respiratory measurements with different substrates

  • Apply specific inhibitors of respiratory complexes

  • Analyze kinetic parameters to deduce functional interactions

  • Similar approaches have revealed substrate-specific kinetic differences in S. pombe mitochondria

These methodologies would provide comprehensive insights into how sdh4 interacts with other respiratory chain components, advancing our understanding of mitochondrial energy metabolism and potentially revealing new therapeutic targets for mitochondrial disorders.