Recombinant Saccharomyces cerevisiae Vacuolar membrane protein FOSTERSB_4073 (FOSTERSB_4073)

How is recombinant FOSTERSB_4073 protein typically stored and what are the optimal storage conditions?

Recombinant FOSTERSB_4073 is optimally stored in a Tris-based buffer containing 50% glycerol at -20°C for regular storage or -80°C for extended preservation. For working with the protein, it's recommended to create aliquots to avoid repeated freeze-thaw cycles, which can compromise protein integrity. Working aliquots can be stored at 4°C for up to one week to maintain protein stability and activity .

What is known about the cellular localization and membrane topology of FOSTERSB_4073?

FOSTERSB_4073 is a vacuolar membrane protein in Saccharomyces cerevisiae, localizing specifically to the membrane of the yeast vacuole, which functions similarly to lysosomes in higher eukaryotes. Based on its amino acid sequence analysis, the protein contains regions with high hydrophobicity that form transmembrane domains, anchoring it within the vacuolar membrane. The topology appears to include both cytoplasmic and intra-vacuolar facing domains, with the cytoplasmic regions potentially involved in protein-protein interactions or signaling processes .

What are the recommended expression systems for producing recombinant FOSTERSB_4073?

The recommended expression system for FOSTERSB_4073 is Escherichia coli, which has been successfully used to produce the full-length protein (amino acids 1-314). E. coli provides several advantages for expression, including rapid growth, high protein yields, and well-established protocols for induction and harvesting. The protein is typically expressed with a histidine tag to facilitate purification using affinity chromatography. Alternative expression systems such as yeast or insect cells might be considered for projects requiring post-translational modifications, though these systems would necessitate optimization of expression conditions and codon usage .

What purification strategies are most effective for isolating FOSTERSB_4073 with high purity and yield?

For purifying FOSTERSB_4073, a multi-step approach is most effective:

• Affinity Chromatography: Utilizing the His-tag, nickel or cobalt affinity columns provide the initial purification step, capturing the recombinant protein from the crude lysate.

• Size Exclusion Chromatography: Following affinity purification, size exclusion chromatography helps remove aggregates and further purify the protein.

• Ion Exchange Chromatography: A final polishing step using ion exchange can be employed to achieve higher purity.

For membrane proteins like FOSTERSB_4073, the addition of appropriate detergents during cell lysis and throughout the purification process is crucial for maintaining protein solubility and preventing aggregation. Typical yields range from 2-5 mg per liter of bacterial culture when optimized conditions are employed .

How can researchers assess the quality and functionality of purified FOSTERSB_4073?

Multiple analytical methods should be employed to assess protein quality:

• SDS-PAGE: To evaluate purity and molecular weight

• Western Blotting: For specific detection using anti-His antibodies

• Circular Dichroism: To analyze secondary structure integrity

• Dynamic Light Scattering: To check for aggregation and homogeneity

• Functional Assays: Depending on the known biological activities of the protein

These quality control steps are essential before proceeding to experimental applications, as membrane protein functionality is particularly sensitive to purification conditions .

How can Trypan Blue staining be used to assess the impact of FOSTERSB_4073 on yeast cell viability?

Trypan Blue staining provides a direct method to assess cell viability in yeast strains expressing or lacking FOSTERSB_4073:

• Culture Preparation: Grow wild-type and FOSTERSB_4073-deficient yeast strains in appropriate medium (such as YEPD) to mid-log phase.

• Staining Procedure: Mix equal volumes of yeast cell suspension and 0.4% Trypan Blue dye. Living cells will exclude the dye, while dead cells will appear blue due to dye uptake.

• Quantification: Using disposable hemocytometers such as KOVA Glasstic slides, count both viable (unstained) and non-viable (blue) cells in defined grid areas.

• Analysis: Calculate cell concentration (cells/mL) and viability percentage for each strain under various experimental conditions.

This technique allows researchers to determine if FOSTERSB_4073 affects cellular resistance to stressors such as UV radiation, oxidative stress, or osmotic shock. The methodology can be adapted from protocols for yeast viability assessment, where strains with different genotypes are compared under controlled conditions .

What methods are recommended for studying the membrane integration and topology of FOSTERSB_4073?

To investigate membrane integration and topology of FOSTERSB_4073, researchers should consider these complementary approaches:

• Protease Protection Assays: Treating isolated vacuoles with proteases in the presence or absence of membrane-permeabilizing agents to determine which protein domains are accessible.

• Site-Directed Mutagenesis: Creating cysteine substitutions at various positions followed by accessibility labeling with membrane-permeable and impermeable sulfhydryl reagents.

• Fluorescence Microscopy: Using GFP fusion constructs at different positions to visualize localization and orientation.

• Computational Prediction: Employing algorithms that predict transmembrane domains based on hydrophobicity plots and amino acid composition.

For experimental verification, researchers often combine these approaches to generate a comprehensive model of membrane protein topology .

How can researchers design experiments to investigate potential protein-protein interactions involving FOSTERSB_4073?

To identify and characterize protein-protein interactions involving FOSTERSB_4073, researchers should implement a multi-faceted approach:

• Yeast Two-Hybrid Screening: Modified for membrane proteins using split-ubiquitin systems to detect interactions at the vacuolar membrane.

• Co-Immunoprecipitation: Using anti-His antibodies to pull down FOSTERSB_4073 complexes from solubilized yeast membranes, followed by mass spectrometry to identify binding partners.

• FRET Analysis: Employing fluorescently tagged proteins to detect proximity-based interactions in vivo.

• Crosslinking Studies: Using chemical crosslinkers followed by mass spectrometry to capture transient or weak interactions.

• Bimolecular Fluorescence Complementation (BiFC): To visualize interactions in living cells by reconstituting fluorescent protein fragments.

These methodologies should be applied under various physiological conditions to capture condition-specific interactions that may occur only under certain stresses or developmental stages .

What approaches can be used to investigate the functional role of FOSTERSB_4073 in vacuolar membrane dynamics?

Investigating FOSTERSB_4073's role in vacuolar membrane dynamics requires sophisticated experimental designs:

• Live-Cell Imaging: Using fluorescently tagged FOSTERSB_4073 combined with markers for vacuolar subdomains to track protein movement during processes like vacuole fusion or fission.

• CRISPR-Cas9 Gene Editing: Creating precise deletions or mutations in the FOSTERSB_4073 gene to assess phenotypic effects on vacuolar morphology and function.

• Lipidomic Analysis: Comparing membrane lipid composition between wild-type and FOSTERSB_4073-deficient strains to identify potential roles in lipid organization.

• Electron Microscopy: Employing immunogold labeling to precisely localize FOSTERSB_4073 within vacuolar membrane microdomains and examine ultrastructural changes in mutant strains.

• Functional Reconstitution: Incorporating purified FOSTERSB_4073 into artificial liposomes to study its intrinsic effects on membrane properties and dynamics.

These approaches collectively provide insights into how FOSTERSB_4073 contributes to vacuolar membrane structure, integrity, and function under various physiological conditions .

How should researchers design experiments to study the effects of stress conditions on FOSTERSB_4073 expression and function?

To investigate FOSTERSB_4073's response to stress conditions, researchers should implement the following experimental design:

• Stress Exposure Protocol:

  Stress Type	Conditions	Duration	Control
  Oxidative	1-5 mM H₂O₂	15-120 min	Untreated cells
  UV Radiation	5-20 J/m²	Immediate, 1h, 3h recovery	Foil-covered cells
  Osmotic	0.4-1.0 M NaCl	30-180 min	Standard medium
  pH Stress	pH 3.0, 5.0, 7.0, 9.0	1-4 hours	pH 5.5 (optimal)
  Nutrient Limitation	Nitrogen or carbon starvation	1-24 hours	Complete medium

• Analysis Methods:

  • qRT-PCR for transcript level changes

  • Western blotting for protein expression levels

  • Fluorescence microscopy for localization changes

  • Viability assays using Trypan Blue staining

  • Vacuolar pH and morphology assessment

• Strain Comparison: Compare wild-type, FOSTERSB_4073 deletion mutants, and complemented strains to distinguish direct from indirect effects.

This comprehensive approach allows researchers to determine if FOSTERSB_4073 plays a stress-responsive role in yeast cells, potentially participating in adaptive responses to environmental challenges .

What are the challenges in studying protein-lipid interactions involving FOSTERSB_4073 and how can they be addressed?

Studying protein-lipid interactions involving FOSTERSB_4073 presents several technical challenges that require specialized approaches:

• Challenges:

  • Maintaining native membrane environment during extraction

  • Distinguishing specific from non-specific lipid interactions

  • Capturing transient interactions

  • Determining functional significance of observed interactions

• Methodological Solutions:

  a) Lipidomic Analysis with Protein Crosslinking:

  • UV-activated lipid probes to capture direct interactions

  • Mass spectrometry to identify crosslinked lipid species

  b) Microscale Thermophoresis:

  • Label fluorescent lipids and measure binding affinities

  • Determine specificity through competition assays

  c) Native Nanodiscs:

  • Reconstitute FOSTERSB_4073 in nanodiscs with defined lipid composition

  • Assess protein function in controlled lipid environments

  d) Molecular Dynamics Simulations:

  • Model protein-lipid interactions based on structural data

  • Generate testable hypotheses about specific binding sites

• Mutational Analysis:

  • Create point mutations in predicted lipid-binding regions

  • Assess changes in lipid binding and protein function

This integrated approach helps overcome the inherent difficulties in studying membrane protein-lipid interactions while providing meaningful insights into how FOSTERSB_4073's function may be modulated by the lipid environment .

How can researchers quantitatively assess the impact of FOSTERSB_4073 mutations on cellular functions?

Quantitative assessment of FOSTERSB_4073 mutations requires systematic phenotypic analysis:

• Growth Rate Analysis:

  • Measure growth curves in liquid culture under various conditions

  • Calculate doubling times and lag phases

  • Compare growth on different carbon sources

• Viability Assays:

  • Trypan Blue exclusion to determine percentage of viable cells

  • Colony forming unit (CFU) counts after stress exposure

  • Flow cytometry with viability dyes for high-throughput analysis

• Vacuolar Function Tests:

  • Measure vacuolar pH using pH-sensitive fluorescent probes

  • Assess vacuolar fragmentation/fusion defects via microscopy

  • Quantify protein sorting efficiency to the vacuole

• Stress Response Metrics:

  • Survival rates following heat shock, oxidative stress, or osmotic stress

  • Gene expression changes of stress response pathways

  • Protein aggregation assessment using aggregation-prone reporters

For proper experimental design, researchers should include wild-type controls, deletion mutants, and point mutants affecting specific domains to comprehensively understand structure-function relationships .

What experimental design would best determine if FOSTERSB_4073 is involved in stress response pathways in yeast?

A comprehensive experimental design to investigate FOSTERSB_4073's role in stress response would include:

• Strain Preparation:

  • Wild-type S. cerevisiae (FostersB strain)

  • FOSTERSB_4073 deletion mutant (Δfostersb_4073)

  • Complemented strain (Δfostersb_4073 + plasmid-expressed FOSTERSB_4073)

  • Site-directed mutants affecting key domains

• Stress Exposure Protocol:

  • Apply multiple stress conditions (UV, oxidative, osmotic, thermal)

  • Use increasing stress intensities to establish dose-response relationships

  • Include both acute and chronic stress exposures

• Multidimensional Analysis:

  Analysis Type	Measurement	Timepoints	Expected Outcome if Involved
  Transcriptional	qRT-PCR of FOSTERSB_4073	0, 15, 30, 60, 120 min post-stress	Upregulation after stress
  Protein Level	Western blot	0, 30, 60, 120, 240 min	Changed protein abundance
  Localization	Fluorescence microscopy	Before, during, after stress	Redistribution within cell
  Viability	Trypan blue assay	3, 6, 24 hours post-stress	Decreased viability in mutant
  Interactome	Co-IP followed by MS	Before and after stress	Stress-specific interactions

• Pathway Analysis:

  • Transcriptomic analysis comparing wild-type and mutant strains

  • Epistasis analysis with known stress response genes

  • Chemical genetics using inhibitors of specific pathways

This experimental design provides multiple lines of evidence to determine FOSTERSB_4073's potential role in stress response pathways, allowing researchers to distinguish between direct involvement and secondary effects .

How does UV sensitivity testing with FOSTERSB_4073 compare methodologically to other stress response assays?

UV sensitivity testing of yeast strains expressing or lacking FOSTERSB_4073 involves specific methodological considerations compared to other stress assays:

• Methodological Differences:

  Aspect	UV Sensitivity Testing	Other Stress Assays (e.g., Oxidative, Osmotic)
  Exposure Method	Physical exposure via UV lamp with precise dosage (J/m²)	Chemical addition to media at specific concentrations
  Spatial Control	Requires physical shielding (foil) for protected controls	Typically uses separate culture vessels for treatment/control
  Exposure Duration	Usually brief (seconds to minutes) with long recovery	Often involves continuous exposure during growth
  Damage Mechanism	Primarily DNA damage (thymine dimers)	Varies by stressor (protein oxidation, membrane disruption)
  Recovery Analysis	Focus on DNA repair capacity and cell cycle checkpoints	Focus on adaptive responses and stress response pathways

• Specific Protocol for UV Testing:

  • Prepare lawn cultures of wild-type and FOSTERSB_4073 mutant yeast

  • Cover half of each plate with aluminum foil as internal control

  • Expose to UV radiation for precise duration (e.g., 5 minutes)

  • Remove foil and incubate under normal conditions

  • Collect samples from exposed and protected regions

  • Stain with Trypan Blue to assess cell viability

  • Count living (unstained) and dead (blue) cells using hemocytometers

• Data Analysis Considerations:

  • Calculate cell concentration (cells/mL) for each condition

  • Determine percentage viability (living cells/total cells × 100%)

  • Compare viability between strains and treatments

  • Analyze recovery kinetics over multiple timepoints

This methodology is particularly valuable for studying FOSTERSB_4073's potential role in UV stress response, DNA damage repair pathways, or cellular protection mechanisms .

What are the most promising research directions for understanding FOSTERSB_4073's biological significance?

Based on current knowledge, several high-priority research directions emerge for FOSTERSB_4073:

• Comprehensive Functional Characterization:

  • Systematic phenotypic analysis of deletion and point mutants

  • Investigation of condition-specific functions (stress, starvation, growth phase)

  • Determination of evolutionary conservation and divergence across yeast species

• Integration with Vacuolar Biology:

  • Defining FOSTERSB_4073's role in vacuolar pH maintenance

  • Investigating potential functions in vacuole fusion/fission dynamics

  • Examining interactions with other vacuolar membrane proteins

• Structural Biology Approaches:

  • Cryogenic electron microscopy to determine three-dimensional structure

  • Structure-function studies correlating domains with specific activities

  • Computational modeling of protein dynamics within the membrane

• Systems Biology Integration:

  • Transcriptomic and proteomic analyses under various conditions

  • Network analysis to position FOSTERSB_4073 in cellular pathways

  • Multi-omics approaches to understand regulatory mechanisms

These directions leverage complementary approaches to build a comprehensive understanding of FOSTERSB_4073's biological significance, potentially revealing new insights into vacuolar membrane biology and yeast cell physiology .

What contradictions or unexplained observations exist in the current literature regarding FOSTERSB_4073?

The current literature on FOSTERSB_4073 presents several unresolved questions and potential contradictions:

• Functional Annotation Gaps:

  • Despite its identification as a vacuolar membrane protein, specific molecular functions remain undefined

  • Tables for pathway involvement and protein function in database entries remain empty, suggesting limited functional characterization

• Sequence Anomalies:

  • The presence of an unidentified amino acid (marked as 'X' in the sequence) at position 72 requires clarification regarding its identity and significance

  • The unusual clustering of glutamic acid residues (EEEEEEE) suggests a potential functional domain that remains uninvestigated

• Evolutionary Context:

  • Relationship to other vacuolar membrane proteins across fungal species remains unexplored

  • Whether FOSTERSB_4073 represents a conserved or species-specific adaptation is unknown

• Methodological Limitations:

  • Current approaches for studying membrane proteins may not fully capture the native environment and interactions of FOSTERSB_4073

  • The reliance on recombinant expression in E. coli may obscure post-translational modifications important for function

Resolving these contradictions requires interdisciplinary approaches combining biochemistry, genetics, proteomics, and evolutionary biology to build a coherent understanding of FOSTERSB_4073's biological role .

How might investigating FOSTERSB_4073 contribute to broader understanding of membrane protein biology?

FOSTERSB_4073 represents an excellent model system for advancing membrane protein biology in several ways:

• Methodological Advancements:

  • Optimizing expression and purification protocols for challenging membrane proteins

  • Developing improved approaches for functional reconstitution in artificial membranes

  • Refining techniques for structural analysis of membrane proteins

• Fundamental Principles:

  • Understanding how sequence motifs determine membrane topology and protein-lipid interactions

  • Elucidating mechanisms of membrane protein quality control and trafficking

  • Investigating how membrane proteins respond to and mediate cellular stress responses

• Evolutionary Insights:

  • Examining how membrane proteins evolve while maintaining functional interactions with lipid bilayers

  • Understanding species-specific adaptations in vacuolar biology

  • Identifying conserved principles in membrane protein structure-function relationships

• Translational Applications:

  • Developing improved heterologous expression systems for membrane proteins

  • Informing the design of membrane-interacting therapeutic agents

  • Contributing to synthetic biology approaches for engineering cellular compartments