Recombinant Schizosaccharomyces pombe Uncharacterized protein C27E2.11c, mitochondrial (SPAC27E2.11c)

What is the current structural and functional annotation of SPAC27E2.11c?

SPAC27E2.11c is classified as an uncharacterized mitochondrial protein from Schizosaccharomyces pombe with UniProt accession number Q9UTA0. The protein has a defined amino acid sequence of ANSTAPYGNTTNSTGTTNGTNGTNTTTSSTATQSSAASITNFSSGAFVIAMIAVACSVMSL, with expression region spanning residues 21-81 . Based on sequence analysis, it contains features consistent with mitochondrial localization, including potential transmembrane domains suggesting membrane association within mitochondria.

For comprehensive structural characterization, researchers should implement a multi-method approach:

• Secondary structure prediction using bioinformatic tools like PSIPRED

• Transmembrane domain prediction with TMHMM or similar algorithms

• 3D structure modeling through homology approaches or AI-based prediction tools

• Experimental validation using circular dichroism spectroscopy or X-ray crystallography for definitive structural determination

What expression systems are optimal for producing recombinant SPAC27E2.11c?

Selection of expression systems should consider the unique challenges of mitochondrial membrane proteins:

For SPAC27E2.11c specifically, homologous expression in S. pombe often provides the most physiologically relevant form despite lower yields. Critical optimization parameters include codon usage, induction conditions, and appropriate solubilization methods for membrane proteins .

How can researchers design effective knockout experiments for SPAC27E2.11c in S. pombe?

A systematic approach to genetic manipulation includes:

Knockout strategies:

• Homologous recombination with selection markers like kanMX

• CRISPR-Cas9 gene editing with appropriate repair templates

• PCR-based gene replacement techniques

For validation, implement a multi-level confirmation approach:

• PCR verification of correct integration

• RT-qPCR confirmation of transcript elimination

• Western blot analysis to confirm protein absence

• Complementation assays to verify phenotype specificity

A critical consideration for mitochondrial proteins is essentiality assessment. If complete knockout produces lethal phenotypes, conditional systems may be necessary:

• Tetracycline-regulatable promoter replacement

• Auxin-inducible degron tagging for controlled depletion

• Temperature-sensitive allele generation

What are the most effective detection methods for studying SPAC27E2.11c?

For this uncharacterized protein, epitope tagging approaches are particularly valuable:

• C-terminal tagging with FLAG, HA, or Myc epitopes (though caution required if C-terminus is important for function)

• N-terminal tagging if C-terminal is critical (with consideration for mitochondrial targeting sequence)

• Fluorescent protein fusions for localization studies

• Split reporter systems for interaction studies

How might SPAC27E2.11c be involved in oxidative stress response mechanisms?

Based on studies of S. pombe stress signaling pathways, uncharacterized mitochondrial proteins may participate in redox homeostasis and stress response networks. Experimental evidence suggests potential connections to established stress response mechanisms:

• The Spc1/Sty1 MAPK cascade is activated in response to oxidative stress induced by H₂O₂, with mitochondrial proteins potentially serving as upstream sensors or downstream effectors .

• The Mpr1-Mcs4 phosphorelay system transmits oxidative stress signals, with Mpr1 serving as a histidine-containing phosphotransfer protein that interacts with the Mcs4 response regulator .

• In fission yeast, activation of Spc1 in response to oxidative stress leads to induction of various stress-response genes, such as gpd1⁺ and ctt1⁺, which encode enzymes involved in stress protection .

To test SPAC27E2.11c involvement in these pathways, researchers should:

• Measure sensitivity of deletion strains to H₂O₂ and other oxidative stressors

• Assess Spc1 MAPK phosphorylation in response to oxidative stress in wild-type versus mutant strains

• Examine potential physical interactions with known stress response components using co-immunoprecipitation

• Analyze transcriptional changes of stress-responsive genes in deletion strains

What is the relationship between SPAC27E2.11c and mitochondrial function?

Investigating the role of SPAC27E2.11c in mitochondrial function requires a multi-faceted approach:

Bioenergetic analysis:

• Oxygen consumption measurements using respirometry

• Membrane potential assessment using fluorescent probes (JC-1, TMRM)

• ATP production quantification

• Mitochondrial ROS measurements using mitochondria-targeted probes

Morphological assessment:

• Live-cell imaging of mitochondrial networks using fluorescent markers

• Electron microscopy for ultrastructural analysis

• Assessment of fusion/fission dynamics

• Quantification of mitochondrial mass and distribution

Expected experimental outcomes might include:

How can chemical genomics approaches reveal functions of SPAC27E2.11c?

Chemical genomics provides powerful tools for functional characterization of uncharacterized proteins through systematic analysis of chemical-genetic interactions. For SPAC27E2.11c, researchers could employ:

• Systematic screening of ΔSPAC27E2.11c strains against chemical libraries to identify compounds with enhanced toxicity or resistance compared to wild-type .

• Analysis of rapamycin sensitivity, as rapamycin targets the TOR pathway regulating cell growth and stress responses in S. pombe .

• Generation of chemical-genetic interaction profiles to place SPAC27E2.11c in functional networks by comparison with profiles of characterized genes.

Experimental design should include:

• Dose-response assays with various compounds

• Growth rate measurements under different stress conditions

• Transcriptional profiling after chemical treatment

• Epistasis analysis with known signaling pathway components

What approaches can resolve contradictory data about SPAC27E2.11c function?

When investigating uncharacterized proteins like SPAC27E2.11c, researchers often encounter seemingly contradictory results. Methodological approaches to reconcile such contradictions include:

• Context-dependent function analysis:

  • Testing under different growth conditions (minimal vs. rich media)

  • Cell cycle-specific analyses

  • Stress-specific phenotyping (oxidative, osmotic, temperature)

  • Genetic background dependence assessment

• Technical considerations:

  • Tag interference evaluation (comparing N- and C-terminal tags)

  • Expression level artifacts (comparing native vs. overexpression)

  • Acute vs. chronic depletion (knockdown vs. knockout)

  • Single-cell analysis to address population heterogeneity

• Resolution strategies:

  • Epistasis analysis to place the protein in pathways

  • Allele-specific effects through point mutations

  • Temporal control using degron systems

  • Complementation with orthologs from related species

How conserved is SPAC27E2.11c across species and what does this reveal about function?

Comparative genomics provides crucial evolutionary context for functional prediction:

• Conservation analysis:

  • High conservation in Schizosaccharomyces species suggests fundamental function

  • Presence/absence in other fungi indicates evolutionary importance

  • Identification of conserved domains may highlight functional motifs

• Functional transfer:

  • Study of characterized homologs in model organisms

  • Complementation studies to test functional conservation

  • Analysis of co-evolution with interacting partners

• Methodological approaches:

  • BLAST/PSI-BLAST for sequence-based homology detection

  • HMM-based searches for remote homologs

  • Structural homology detection when sequence similarity is low

  • Synteny analysis to identify positional homologs

If SPAC27E2.11c is involved in stress response pathways, comparative analysis may reveal evolutionary conservation of this function or potentially novel adaptations specific to fission yeast .

How does SPAC27E2.11c relate to the phosphorelay system in oxidative stress signaling?

The Mpr1-Mcs4 phosphorelay system in S. pombe represents a sophisticated mechanism for transmitting oxidative stress signals. Evidence suggests that this system operates as follows:

• The histidine-containing phosphotransfer protein Mpr1 transfers phosphoryl groups to the Mcs4 response regulator .

• Physical interaction between Mpr1 and Mcs4 increases under oxidative stress conditions, suggesting stress-induced association .

• The putative phosphorylation site, His-221, in Mpr1 is required for function in oxidative stress signaling to Spc1 .

• Deletion of mcs4 impairs Spc1 activation upon oxidative stress, whereas osmotic stress can still activate Spc1 in Δmcs4 cells .

To investigate potential relationships between SPAC27E2.11c and this phosphorelay system:

• Test physical interactions with Mpr1 or Mcs4 using co-immunoprecipitation

• Examine the phosphorylation status of pathway components in ΔSPAC27E2.11c strains

• Analyze epistatic relationships by creating double mutants with pathway components

• Compare transcriptional responses to oxidative stress between single and double mutants