Recombinant Zygosaccharomyces rouxii Genetic interactor of prohibitin 7, mitochondrial (GEP7)

What is the biological role of GEP7 in Zygosaccharomyces rouxii?

GEP7 (Genetic interactor of prohibitin 7, mitochondrial) is a mitochondrial protein in Z. rouxii that plays a role in cellular stress response pathways. Based on its interaction with prohibitins, GEP7 likely contributes to mitochondrial integrity maintenance and function under stress conditions . The protein's full length consists of 258 amino acids, with the expression region spanning positions 16-258 .

Z. rouxii is particularly notable for its extreme osmotolerance and halotolerance, allowing it to thrive in environments with high concentrations of salt and/or sugar that would be lethal to most other yeasts . GEP7 may contribute to this remarkable stress tolerance through mitochondrial homeostasis regulation, though direct experimental evidence connecting GEP7 to osmotolerance mechanisms requires further investigation.

How does Z. rouxii's genome compare to Saccharomyces cerevisiae, and what implications does this have for GEP7 function?

Z. rouxii shares a common evolutionary origin with Saccharomyces species but diverged before the whole-genome duplication event that shaped modern S. cerevisiae. As a member of the protoploid Saccharomycetaceae, Z. rouxii represents a lineage more closely related to the putative ancestral genome of S. cerevisiae . This evolutionary position provides valuable insights when studying protein function.

The genome of Z. rouxii displays several distinctive characteristics:

• Short centromeres

• Triplication of mating cassettes

• Limited number of spliceosomal introns

• Usage of the universal genetic code

• Single rDNA locus containing genes for 5S RNA molecules

These genomic features confirm Z. rouxii's position within the monophyletic origin of Saccharomycetaceae. For GEP7 research, this evolutionary context suggests that studying this protein may provide insights into ancestral mitochondrial functions that may have been modified or duplicated in post-genome duplication yeasts like S. cerevisiae.

What stress response pathways in Z. rouxii might involve GEP7?

GEP7 likely participates in multiple stress response pathways in Z. rouxii, particularly those involving mitochondrial function under challenging environmental conditions. Transcriptomic analysis of Z. rouxii under stress conditions reveals several potential pathways where GEP7 might function:

• High-temperature stress response: Z. rouxii demonstrates limited tolerance to elevated temperatures, with certain strains showing no growth at 40°C after 168 hours of cultivation . The response to high-temperature stress involves upregulation of heat shock factors like HSF1 (27.1-fold increase) and MSN4 (58.9-fold increase) . GEP7, as a mitochondrial protein, may interact with these pathways to maintain mitochondrial integrity during heat stress.

• Osmotic stress response: Z. rouxii's exceptional ability to survive in high sugar environments involves distinctive gene expression patterns that differ significantly from those observed in S. cerevisiae . GEP7 may participate in the mitochondrial aspects of this osmotic stress response.

• Glucose metabolism: Z. rouxii exhibits upregulation of high glucose receptor genes like GRT2 (12.0-fold) and hexokinase (HXK1, 8.3-fold increase) under certain stress conditions , suggesting interconnected pathways between glucose metabolism and stress responses where GEP7 might function.

What expression systems are available for producing recombinant Z. rouxii proteins?

While specific expression systems for Z. rouxii proteins like GEP7 are still being optimized, researchers can draw upon approaches developed for related yeasts:

• Heterologous expression in S. cerevisiae: Given the evolutionary relationship between Z. rouxii and S. cerevisiae, standard S. cerevisiae expression vectors can often be used, though optimization may be required for Z. rouxii proteins.

• Related Zygosaccharomyces expression systems: Systems developed for Z. bailii could be adapted for Z. rouxii protein expression. For Z. bailii, both chemical transformation (LiAc/PEG/ss-DNA protocol, yielding 3-5×10² clones/μg DNA) and electroporation methods (yielding 2-5×10³ clones/μg DNA) have been established .

• Native Z. rouxii expression: Evidence suggests that endogenous promoters from Z. rouxii, such as the TPI promoter, can yield significantly higher expression levels compared to S. cerevisiae promoters . The Z. rouxii TPI promoter has demonstrated 4-5 times higher activity than the S. cerevisiae equivalent .

• Plasmid-based systems: ARS1 chromosomal replication origin from S. cerevisiae has been shown to be recognized and maintained by Z. rouxii , offering a foundation for developing specialized expression vectors.

How can recombinant GEP7 protein be properly stored and handled for experimental use?

For optimal maintenance of recombinant Z. rouxii GEP7 protein activity and stability:

• Storage conditions: Store the purified protein at -20°C for short-term storage. For extended storage periods, maintain at -80°C .

• Buffer composition: The recommended storage buffer consists of a Tris-based buffer with 50% glycerol, specifically optimized for GEP7 protein stability .

• Handling precautions:

  • Avoid repeated freeze-thaw cycles as these significantly reduce protein activity

  • For routine work, maintain working aliquots at 4°C for up to one week

  • When thawing frozen aliquots, use rapid thawing techniques and maintain on ice during handling

• Quality verification: Before experimental use, verify protein integrity through SDS-PAGE and activity assays appropriate for mitochondrial proteins.

How can transcriptomic approaches be optimized to study GEP7 expression under different stress conditions?

Optimizing transcriptomic analyses for GEP7 expression in Z. rouxii requires:

• Experimental design considerations:

  • Include multiple stress conditions: temperature (30-40°C), osmotic pressure (varying sugar concentrations: 0-20% trehalose), pH variations, and combinations thereof

  • Implement time-course sampling to capture expression dynamics

  • Include appropriate Z. rouxii wild-type strains as controls

• RNA extraction optimization:

  • Standard yeast RNA extraction protocols require modification for Z. rouxii due to its robust cell wall, especially under stress conditions

  • Mechanical disruption (e.g., bead-beating) should be performed in the presence of RNase inhibitors

  • Validate RNA integrity using bioanalyzer before sequencing

• Sequencing approach:

  • Utilize strand-specific RNA-seq to differentiate sense and antisense transcription

  • Consider long-read sequencing for improved isoform detection

  • Implement spike-in controls for accurate quantification

• Bioinformatic analysis:

  • Employ tools specifically validated for non-conventional yeasts

  • Normalize data accounting for Z. rouxii's distinctive genomic features

  • Compare expression patterns with those of other genes known to respond to stress, such as HSF1 (heat shock factor) and MSN4 (stress-responsive transcriptional activator)

Based on existing research, GEP7 expression may show significant modulation under stress conditions, potentially correlating with the substantial up-regulation observed for stress-response genes (e.g., HSF1 showing 27.1-fold increase and MSN4 showing 58.9-fold increase under high temperature stress) .

What are the functional differences between GEP7 in Z. rouxii and homologous proteins in other yeast species?

Understanding the functional divergence between Z. rouxii GEP7 and its homologs requires:

• Comparative sequence analysis:

  • Multiple sequence alignment of GEP7 homologs from Z. rouxii, S. cerevisiae, and other yeasts

  • Identification of conserved domains and species-specific variations

  • Evolutionary rate analysis to detect sites under positive selection

• Complementation studies:

  • Express Z. rouxii GEP7 in homolog-knockout strains of S. cerevisiae

  • Assess restoration of mitochondrial function and stress tolerance

  • Compare with reciprocal experiments (S. cerevisiae homolog in Z. rouxii)

• Structural biology approaches:

  • Generate structural models of GEP7 from different species

  • Compare predicted binding surfaces and interaction domains

  • Validate through mutational analysis of key residues

• Physiological comparison:

  • Measure mitochondrial function parameters in different yeasts with and without functional GEP7

  • Examine growth and survival under various stress conditions

The differences likely reflect Z. rouxii's extreme stress tolerance capabilities, particularly its exceptional osmotolerance and halotolerance that exceed those of S. cerevisiae . These adaptations may be partially mediated through specialized functions of mitochondrial proteins like GEP7.

How can CRISPR-Cas9 gene editing be optimized for studying GEP7 function in Z. rouxii?

Developing an effective CRISPR-Cas9 system for Z. rouxii requires:

• Vector adaptation:

  • Modify existing yeast CRISPR vectors to incorporate Z. rouxii-optimized promoters

  • Consider using the Z. rouxii TPI promoter, which shows 4-5 times higher activity than S. cerevisiae equivalents

  • Adapt selection markers for Z. rouxii (e.g., antibiotic resistance genes for G418 or hygromycin at appropriate concentrations)

• Transformation optimization:

  • Electroporation methods yield higher efficiency (2-5×10³ clones/μg DNA) than chemical transformation (3-5×10² clones/μg DNA)

  • Use fructose rather than glucose in recovery media, as Z. rouxii is fructophilic

  • Allow 2-3 days at 30°C for transformant colonies to appear

• Guide RNA design:

  • Account for Z. rouxii's GC content and codon usage

  • Target conserved functional domains in GEP7

  • Design multiple gRNAs to increase editing efficiency

• Homology-directed repair templates:

  • Construct with longer homology arms (>500 bp) than typically used for S. cerevisiae

  • Include reporter genes or epitope tags for monitoring GEP7 expression and localization

• Validation strategies:

  • PCR-based genotyping

  • Western blotting for protein expression

  • Phenotypic assays focused on mitochondrial function and stress tolerance

What high-throughput screening methods are appropriate for identifying chemical modulators of GEP7 function?

Developing effective high-throughput screens for GEP7 modulators requires:

• Reporter system development:

  • Create fusion constructs linking GEP7 to fluorescent or luminescent reporters

  • Design stress-responsive promoter-reporter systems that reflect GEP7 activity

  • Develop Z. rouxii strains with varying GEP7 expression levels (knockout, wild-type, overexpression)

• Assay optimization:

  • Miniaturize growth and stress response assays to 384-well format

  • Establish temperature, osmotic, and oxidative stress conditions that produce robust, measurable phenotypes

  • Determine optimal timepoints for measurement based on Z. rouxii's growth characteristics

• Screening methodology:

  • Primary screen: Growth under multiple stress conditions (high temperature, high sugar)

  • Secondary screens: Mitochondrial function assays (membrane potential, respiration)

  • Counter-screens: General cytotoxicity assessment

• Data analysis approach:

  • Implement machine learning algorithms to identify patterns in multiparametric screens

  • Develop customized Z-score calculations accounting for Z. rouxii growth characteristics

  • Cluster compounds by mechanism based on phenotypic signatures

• Validation studies:

  • Dose-response curves for hit compounds

  • Direct binding assays using purified recombinant GEP7

  • Transcriptomic analysis of treated cells to determine pathway effects

This approach would allow for identification of both inhibitors and activators of GEP7 function, potentially leading to chemical tools for studying mitochondrial stress responses in Z. rouxii.

What are the optimal conditions for expressing recombinant GEP7 in heterologous systems?

Based on comparative analyses of expression systems for Zygosaccharomyces proteins, the following parameters are recommended:

For optimal expression of functional GEP7, inclusion of the complete expression region (amino acids 16-258) is essential . Expression systems should incorporate appropriate secretion signals if extracellular production is desired, though GEP7 as a mitochondrial protein may require specialized approaches for proper folding and activity.

How can researchers effectively purify GEP7 while maintaining its native conformation and activity?

A multi-step purification strategy is recommended to obtain functionally active GEP7:

• Cell disruption and initial extraction:

  • For Z. rouxii, use mechanical disruption methods (e.g., glass beads) in buffer containing protease inhibitors

  • Include mitochondrial isolation steps if extracting native GEP7

  • For recombinant His-tagged GEP7, perform lysis under native conditions (avoid harsh denaturants)

• Purification workflow:

  • Initial capture: Immobilized metal affinity chromatography (IMAC) for His-tagged protein

  • Intermediate purification: Ion exchange chromatography (suggested based on GEP7's theoretical pI)

  • Polishing: Size exclusion chromatography to remove aggregates and ensure monodispersity

• Buffer optimization:

  • Maintain Tris-based buffers similar to storage buffer (pH 7.5-8.0)

  • Include stabilizing agents (glycerol 10-50%)

  • Consider adding reducing agents (DTT or β-mercaptoethanol) to prevent oxidation

• Activity verification:

  • Develop an in vitro activity assay based on GEP7's interaction with prohibitins

  • Monitor mitochondrial function parameters in reconstitution experiments

  • Perform thermal shift assays to verify proper folding

• Storage conditions:

  • Store at -20°C for short-term or -80°C for long-term stability

  • Avoid repeated freeze-thaw cycles

  • Consider flash-freezing aliquots in liquid nitrogen

What controls are essential when studying GEP7's role in stress response pathways?

When investigating GEP7's function in stress response mechanisms, the following controls are critical:

• Genetic controls:

  • Wild-type Z. rouxii strains

  • GEP7 knockout mutants

  • GEP7 overexpression strains

  • Strains with mutations in known stress response genes (HSF1, MSN4, etc.)

  • S. cerevisiae with Z. rouxii GEP7 (for complementation studies)

• Environmental controls:

  • Temperature series (30°C, 35°C, 38°C, 40°C) to assess heat stress response

  • Sugar concentration gradients (0%, 5%, 10%, 20% trehalose) to evaluate osmotic stress response

  • Combined stressors versus single stressors

  • Time-course sampling to capture dynamic responses

• Molecular controls:

  • qPCR housekeeping genes validated specifically for Z. rouxii under stress conditions

  • Spike-in controls for RNA-seq experiments

  • Non-target proteins for interaction studies

  • Inactive GEP7 mutants (site-directed mutagenesis of key residues)

• Technical controls:

  • Biological replicates (minimum n=3) for all experiments

  • Technical replicates for methods with higher variability

  • Inter-strain comparison to account for strain-specific responses

  • Cross-validation using orthogonal methods for key findings

These controls are particularly important given Z. rouxii's distinctive stress response patterns, which differ significantly from the better-characterized S. cerevisiae responses .

How should researchers interpret conflicting data on GEP7 function across different experimental conditions?

When confronted with conflicting results regarding GEP7 function:

• Strain-specific variation assessment:

  • Z. rouxii displays significant strain-dependent variation in stress tolerance

  • Compare strain backgrounds and genetic markers

  • Consider genome sequencing to identify strain-specific polymorphisms in GEP7 or interacting genes

• Methodological reconciliation:

  • Evaluate differences in experimental conditions (temperature, media composition, stress duration)

  • Assess sensitivity and specificity of different assay methods

  • Consider whether conflicts reflect different aspects of GEP7's multifunctional nature

• Context-dependent function analysis:

  • GEP7 may have different roles depending on stress type and severity

  • Examine whether conflicting results occur under different stress conditions

  • Consider combinatorial stress effects versus single stressors

• Integration with systems biology:

  • Place conflicting results in pathway context

  • Use network analysis to identify condition-specific interaction partners

  • Develop predictive models that incorporate conditional dependencies

• Statistical approaches:

  • Perform meta-analysis across multiple studies

  • Implement Bayesian analysis to incorporate prior knowledge

  • Use principal component analysis to identify major sources of variation

What are the most promising future research directions for understanding GEP7's role in Z. rouxii biology?

Based on current knowledge gaps and technological developments, several high-priority research directions emerge:

• Structural biology approaches:

  • Determine GEP7's three-dimensional structure through X-ray crystallography or cryo-EM

  • Map interaction surfaces with prohibitins and other partners

  • Identify functional domains for targeted mutagenesis

• Systems-level analysis:

  • Perform comprehensive interactome mapping under different stress conditions

  • Integrate transcriptomic, proteomic, and metabolomic data

  • Develop computational models of GEP7's role in stress response networks

• Comparative evolutionary studies:

  • Analyze GEP7 sequence evolution across Zygosaccharomyces and related genera

  • Identify signatures of positive selection related to stress adaptation

  • Perform ancestral sequence reconstruction and functional testing

• Applied biotechnology:

  • Engineer GEP7 variants with enhanced stress protection capabilities

  • Develop Z. rouxii strains with modified GEP7 expression for industrial applications

  • Explore potential for transferring Z. rouxii stress tolerance mechanisms to other yeasts

• Stress biology integration:

  • Investigate GEP7's potential role in cross-protection between different stressors

  • Examine how GEP7 function integrates with known trehalose-mediated protection mechanisms

  • Study potential roles in chronological and replicative aging under stress