Recombinant Saccharomyces cerevisiae Vacuolar membrane protein VL3_4134 (VL3_4134)

What are the recommended storage conditions for recombinant VL3_4134?

Recombinant VL3_4134 should be stored at -20°C/-80°C, with an expected shelf life of 6 months for liquid formulations and 12 months for lyophilized forms. For working aliquots, storage at 4°C for up to one week is recommended. Repeated freeze-thaw cycles should be avoided to maintain protein integrity. For lyophilized powder, reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL is recommended, with the addition of 5-50% glycerol (final concentration) before aliquoting for long-term storage .

How should researchers reconstitute lyophilized VL3_4134 protein?

For optimal reconstitution:

• Briefly centrifuge the vial to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (the default recommendation is 50%)

• Create aliquots for long-term storage at -20°C/-80°C

What expression systems are available for producing recombinant VL3_4134?

Recombinant VL3_4134 can be produced using multiple expression systems:

The choice depends on the research requirements regarding protein folding, post-translational modifications, and experimental applications .

What methodological approaches can be used to study VL3_4134 localization in the vacuolar membrane?

To study VL3_4134 localization:

• Fluorescence microscopy techniques: Using GFP-fusion constructs similar to those used for studying Vph1-GFP distribution. This allows visualization of protein distribution across different domains of the vacuolar membrane under various conditions .

• Freeze-fracture electron microscopy (EM): This technique has been used to observe vacuolar membrane domain segregation, showing intramembrane particle (IMP)-rich and IMP-deficient domains. This approach would be valuable for determining if VL3_4134 localizes to specific membrane domains .

• Co-localization studies: Using fluorescent markers for different membrane domains (e.g., Fast DiI which has higher affinity for liquid-disordered (Ld) phases) to determine the phase preference of VL3_4134 .

• Immunogold labeling with electron microscopy: For high-resolution localization studies, particularly to determine if VL3_4134 localizes to membrane invaginations .

How might VL3_4134 be involved in vacuolar membrane domains and phase separation?

Based on studies of other vacuolar membrane proteins, VL3_4134 may participate in the formation of microdomains on the vacuolar membrane through phase separation processes. Research has shown that vacuolar membranes can segregate into different domains, particularly under stress conditions like heat shock or during stationary phase.

The methodological approach to investigate this would include:

• Observe VL3_4134-GFP distribution patterns under normal conditions versus stress conditions

• Use lipid dyes like Fast DiI to determine if VL3_4134 preferentially localizes to specific lipid phases

• Compare distribution patterns with known domain-forming proteins like Vph1, Zrc1, or Ybt1

• Analyze the amino acid sequence for motifs that might target it to specific membrane domains

• Create deletion mutants to identify regions responsible for domain localization

What is the potential relationship between VL3_4134 and vacuolar invagination formation?

Vacuolar membrane invaginations are critical structures that form under stress conditions. To investigate VL3_4134's role:

• Generate VL3_4134 deletion mutants and observe vacuolar morphology changes under stress conditions

• Compare the phenotype with other mutants known to affect invagination (e.g., hfl1Δ, atg8Δ, ivy1Δ)

• Test genetic interactions by creating double mutants (e.g., VL3_4134Δ/hfl1Δ or VL3_4134Δ/ivy1Δ)

• Determine if VL3_4134 localizes to the neck of invaginations using high-resolution microscopy

• Examine the function of ESCRT machinery in VL3_4134 mutants since ESCRT is necessary for invagination formation

These approaches would help determine if VL3_4134 functions similarly to Hfl1, which localizes to invagination necks and suppresses excessive invaginations, or if it has a distinct role in vacuolar dynamics .

How does the ESCRT machinery potentially interact with VL3_4134 in vacuolar membrane dynamics?

The ESCRT (Endosomal Sorting Complex Required for Transport) machinery is necessary for forming vacuolar invaginations. To study potential interactions with VL3_4134:

• Create double mutants between VL3_4134 and ESCRT components (e.g., VL3_4134Δ/vps23Δ, VL3_4134Δ/vps24Δ, VL3_4134Δ/vps4Δ)

• Perform epistasis analysis to determine the genetic relationship

• Conduct co-immunoprecipitation experiments to test for physical interactions

• Use fluorescence microscopy to determine if VL3_4134 co-localizes with ESCRT components during invagination formation

Based on studies with other vacuolar proteins, if ESCRT mutations are epistatic to VL3_4134 mutations (as they are to hfl1Δ and atg8Δ), this would suggest that VL3_4134 functions upstream of the ESCRT machinery in the regulation of vacuolar dynamics .

How should researchers design experiments to investigate VL3_4134's role in heat stress response?

When investigating VL3_4134's role in heat stress response, consider this experimental design approach:

• Define variables:

  • Independent variable: Heat stress conditions (e.g., 40.5°C for 2.5 hours)

  • Dependent variable: VL3_4134 localization, vacuolar morphology, or cell survival metrics

  • Controls: Wild-type cells, known vacuolar protein mutants (hfl1Δ, atg8Δ)

• Formulate testable hypotheses:

  • H0: VL3_4134 distribution does not change under heat stress

  • H1: VL3_4134 redistributes to specific membrane domains under heat stress

• Design treatments:

  • Vary heat stress intensity (37°C, 40.5°C, 42°C)

  • Vary duration (30min, 1h, 2.5h, 5h)

  • Include recovery periods at normal temperature

• Group assignment:

  • Between-subjects design comparing different yeast strains

  • Within-subjects design comparing the same strain under different conditions

• Measurement methods:

  • Fluorescence microscopy for protein localization

  • Electron microscopy for membrane structure analysis

  • Cell viability assays for functional consequences

This systematic approach follows established experimental design principles and will provide robust data on VL3_4134's stress response functions.

What statistical approaches are most appropriate for analyzing VL3_4134 localization data?

When analyzing VL3_4134 localization data, consider these statistical approaches:

• For microscopy quantification:

  • Use intensity profile analysis across membrane sections

  • Calculate coefficient of variation for intensity distribution

  • Determine percentage of protein in different membrane domains

• For comparing multiple experimental conditions:

  • ANOVA for comparing multiple groups, followed by appropriate post-hoc tests

  • Use non-parametric alternatives (Kruskal-Wallis) if normality assumptions are violated

• For correlation analyses:

  • Pearson correlation for relationships between VL3_4134 distribution and other quantitative variables

  • Multiple regression for identifying key predictors of localization patterns

• For time-course experiments:

  • Repeated measures ANOVA

  • Mixed effects models to account for random variation between samples

• Sample size considerations:

  • Power analysis based on effect sizes from pilot experiments

  • Typically analyze >100 cells per condition to account for cell-to-cell variability

These approaches ensure robust statistical inference while controlling for Type I and Type II errors .

How might VL3_4134 function in relation to cell growth and vacuolar expansion?

Vacuole size correlates with cell size, and vacuolar expansion is linked to cell growth. To investigate VL3_4134's potential role:

• Create VL3_4134 deletion or overexpression strains and measure:

  • Cell size distribution

  • Vacuolar volume (using 3D modeling and quantitative analysis)

  • Growth rates under different conditions

• Determine if VL3_4134 affects vacuolar occupancy (the proportion of cellular space occupied by the vacuole)

• Examine potential interactions with the actin cytoskeleton, as some vacuolar membrane proteins connect to actin filaments through NET family proteins

• Test if VL3_4134 expression correlates with expression of aquaporins (e.g., γ-TIP) that enable water uptake by the vacuole

If VL3_4134 regulates vacuolar expansion, genetic manipulation would be expected to alter both vacuolar and cellular size parameters, potentially affecting growth rates, particularly under stress conditions .

How can researchers investigate potential interactions between VL3_4134 and the Vps1 protein trafficking pathway?

Vps1 protein plays a crucial role in the retention of Golgi membrane proteins and the formation of vesicles that transport proteins to the prevacuolar compartment. To investigate potential interactions with VL3_4134:

• Create VL3_4134Δ/vps1Δ double mutants and observe vacuolar morphology and protein trafficking

• Track VL3_4134 localization in vps1Δ cells to determine if its trafficking to the vacuole is affected

• Design pulse-chase experiments to follow newly synthesized VL3_4134 in wild-type versus vps1Δ cells

• Use temperature-sensitive vps1 mutants combined with VL3_4134 fluorescent tagging to observe trafficking routes

• Perform protease digestion assays to determine if VL3_4134 appears on the plasma membrane in vps1Δ cells

These experiments would reveal whether VL3_4134 trafficking depends on the Vps1-mediated Golgi-to-vacuole pathway or if it follows an alternative route to the vacuolar membrane .

What approaches should be used to resolve contradictory data on VL3_4134 function in different experimental systems?

When facing contradictory data on VL3_4134 function:

• Standardize experimental conditions:

  • Control for yeast strain backgrounds (laboratory vs. industrial strains)

  • Standardize growth media and conditions

  • Use identical stress parameters and duration

• Validate with multiple methodologies:

  • Combine genetic, biochemical, and imaging approaches

  • Use both tagged and untagged versions of the protein

  • Verify key findings with different expression systems

• Perform epistasis analysis:

  • Create double mutants with genes in potentially related pathways

  • Determine genetic hierarchies to place VL3_4134 in functional networks

• Consider context-dependent functions:

  • Test function under multiple stress conditions

  • Examine cell-cycle dependent effects

  • Investigate nutrient-dependent regulation

• Analyze protein isoforms and modifications:

  • Identify potential post-translational modifications

  • Examine if different experimental systems produce variants

This systematic approach helps resolve apparent contradictions by identifying condition-specific functions or technical variables affecting experimental outcomes .

How does research on VL3_4134 contribute to understanding fundamental mechanisms of membrane organization?

Research on VL3_4134 may provide insights into several fundamental aspects of membrane biology:

• Membrane domain formation: Studying how VL3_4134 distributes between different membrane domains can elucidate mechanisms of phase separation in biological membranes and how proteins sort to these domains.

• Membrane curvature regulation: If VL3_4134 is involved in vacuolar invaginations, it may reveal mechanisms of membrane deformation and stabilization of highly curved membranes.

• Organelle size control: VL3_4134's potential role in vacuolar size regulation would contribute to understanding how cells control organelle dimensions to match cellular needs.

• Stress-induced membrane reorganization: By examining how heat and other stresses affect VL3_4134 distribution, researchers can understand general principles of how membranes adapt to environmental challenges .

What experimental approaches can determine if VL3_4134 has diagnostic or biotechnological applications?

To assess VL3_4134's potential applications:

• For diagnostic applications:

  • Develop antibodies against VL3_4134 with high specificity

  • Create quantitative assays (ELISA, RT-PCR) to detect expression levels

  • Test if expression correlates with specific yeast strains or conditions

  • Perform comparative analysis with human proteins (if homologs exist)

• For biotechnological applications:

  • Test if VL3_4134 manipulation affects stress tolerance in industrial yeast strains

  • Determine if VL3_4134 alterations improve protein production in yeast expression systems

  • Explore if VL3_4134's membrane-organizing properties could be utilized in synthetic biology applications

  • Investigate if VL3_4134 homologs in pathogenic fungi could be drug targets

These approaches would require rigorous validation across multiple experimental systems and extensive comparative analysis .

What are the current methodological limitations in studying VL3_4134 function?

Current limitations include:

• Membrane protein purification challenges:

  • Maintaining native structure during extraction from lipid environment

  • Achieving sufficient purity without aggregation

  • Preserving functional activity outside cellular context

• Visualization constraints:

  • Resolution limits for studying dynamic membrane processes

  • Potential artifacts from fluorescent protein tags

  • Challenges in correlating fluorescence with electron microscopy data

• Functional redundancy:

  • Potential compensatory mechanisms in knockout studies

  • Multiple proteins may have overlapping functions

  • Subtle phenotypes may be difficult to detect

• Time resolution:

  • Rapid membrane reorganization events may be missed

  • Challenges in synchronizing cells for temporal studies

Future methodological developments in cryo-electron tomography, super-resolution microscopy, and membrane protein handling techniques may address these limitations .

What novel experimental approaches might advance understanding of VL3_4134 function?

Emerging approaches that could advance VL3_4134 research include:

• CRISPR-based approaches:

  • Precise genome editing for endogenous tagging

  • CRISPRi for temporal control of expression

  • CRISPR screens to identify genetic interactions

• Advanced imaging techniques:

  • Light-sheet microscopy for long-term live imaging

  • Super-resolution microscopy (PALM/STORM) for nanoscale localization

  • Correlative light and electron microscopy (CLEM) for structure-function relationships

• Proteomics and interactomics:

  • BioID or APEX proximity labeling to identify interaction partners

  • Cross-linking mass spectrometry for structural analysis

  • Quantitative proteomics to identify regulatory modifications

• Membrane biophysics:

  • Reconstitution in giant unilamellar vesicles (GUVs)

  • Atomic force microscopy of membrane topography

  • Lipid mass spectrometry to characterize associated lipids

• Computational modeling:

  • Molecular dynamics simulations of membrane interactions

  • Systems biology approaches to place in regulatory networks

  • Structural prediction using AlphaFold or similar tools