Recombinant Saccharomyces cerevisiae Vacuolar membrane protein EC1118_1N9_3125g (EC1118_1N9_3125g)

What is the primary function of EC1118_1N9_3125g in Saccharomyces cerevisiae?

EC1118_1N9_3125g is a vacuolar membrane protein that likely plays a role in intravacuolar membrane dynamics. Based on homology with other vacuolar membrane proteins like Cvt17/Aut5p, it may be involved in intravacuolar membrane breakdown and autophagic processes . To determine its specific function, researchers should consider:

• Gene knockout studies using CRISPR-Cas9 or traditional homologous recombination

• Phenotypic characterization of knockout strains, particularly examining vacuolar morphology

• Complementation assays to confirm function

• Fluorescence microscopy using GFP-tagged versions to track localization during cellular processes

Current research suggests it may function similarly to other vacuolar membrane proteins that facilitate intravacuolar vesicle breakdown, though specific function verification requires experimental validation.

What expression systems are most effective for producing recombinant EC1118_1N9_3125g?

The most commonly used and effective expression system for EC1118_1N9_3125g is E. coli . The methodological approach includes:

• Cloning the full-length coding sequence (1-314 amino acids) into an appropriate expression vector

• Adding an N-terminal His-tag for purification purposes

• Transforming the construct into an E. coli expression strain

• Inducing protein expression under optimized conditions

• Purifying using immobilized metal affinity chromatography

The expressed protein typically achieves >90% purity as determined by SDS-PAGE . When working with this expression system, researchers should monitor protein folding, as transmembrane proteins can sometimes misfold in bacterial systems. Alternative expression systems to consider include:

What is the proper storage protocol for recombinant EC1118_1N9_3125g to maintain activity?

For optimal stability and activity maintenance of recombinant EC1118_1N9_3125g, follow these methodological guidelines:

• Store the lyophilized protein at -20°C/-80°C upon receipt

• For the reconstituted protein:

  • Use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 50% for long-term storage

  • Aliquot to avoid repeated freeze-thaw cycles

  • Store working aliquots at 4°C for up to one week

Storage buffer composition significantly affects stability:

• Optimal buffer: Tris/PBS-based buffer, pH 8.0, with 6% trehalose

• For long-term storage (-80°C): Add 50% glycerol as a cryoprotectant

Repeated freeze-thaw cycles dramatically reduce protein activity. Experimental data shows activity loss of approximately 15-20% per freeze-thaw cycle for similar membrane proteins.

What experimental design is most effective for studying EC1118_1N9_3125g protein-protein interactions?

To effectively study EC1118_1N9_3125g protein-protein interactions, researchers should implement a multi-technique experimental design:

• Yeast Two-Hybrid Screening:

  • Use the cytosolic domains of EC1118_1N9_3125g as bait

  • Screen against a yeast genomic library

  • Validate positive interactions with co-immunoprecipitation

• Proximity-Dependent Biotin Identification (BioID):

  • Generate a fusion of EC1118_1N9_3125g with BirA* biotin ligase

  • Express in yeast cells and induce biotinylation

  • Purify biotinylated proteins and identify by mass spectrometry

• Co-Immunoprecipitation with Quantitative Mass Spectrometry:

  • Generate strains expressing epitope-tagged EC1118_1N9_3125g

  • Use SILAC labeling to distinguish specific from non-specific interactions

  • Analyze data using specialized software like MaxQuant

Cross-validation of results using multiple methods is critical for membrane proteins due to their hydrophobic nature. Based on studies of similar proteins, potential interaction partners may include components of the vacuolar fusion machinery and cytoskeletal elements .

How can researchers design experiments to investigate EC1118_1N9_3125g's role in vacuolar membrane dynamics?

To investigate EC1118_1N9_3125g's role in vacuolar membrane dynamics, implement the following experimental design approach:

• Generate fluorescently-tagged versions of EC1118_1N9_3125g:

  • Create C-terminal GFP fusion constructs under native promoter

  • Verify proper localization and function

• Live-cell imaging experiments:

  • Use confocal microscopy to track protein movement during:

    • Normal growth conditions

    • Nutrient starvation (to induce autophagy)

    • Osmotic stress (to induce vacuolar fragmentation/fusion)

  • Measure vacuolar dynamics parameters (fusion rate, fission events)

• Genetic interaction studies:

  • Create double mutants with known vacuolar dynamics regulators

  • Assess synthetic genetic interactions

  • Quantify phenotypic effects on vacuolar morphology

• In vitro reconstitution assays:

  • Purify recombinant EC1118_1N9_3125g

  • Test effect on liposome fusion/fission in the presence of vacuolar SNAREs

  • Measure membrane curvature induction

By analogy with Yeb3p/Vac8p, EC1118_1N9_3125g may be involved in vacuolar inheritance and fusion processes, potentially serving as a link between vacuoles and the actin cytoskeleton . Researchers should pay particular attention to its distribution during cell division and its potential concentration in bands between clustered vacuoles, similar to patterns observed with Yeb3p-GFP .

What techniques are recommended for analyzing EC1118_1N9_3125g post-translational modifications?

For comprehensive analysis of EC1118_1N9_3125g post-translational modifications (PTMs), researchers should implement a multi-technique approach:

• Mass Spectrometry-Based PTM Mapping:

  • Purify native EC1118_1N9_3125g from yeast cells

  • Digest with multiple proteases to ensure complete sequence coverage

  • Analyze using high-resolution LC-MS/MS with HCD and ETD fragmentation

  • Apply specific enrichment strategies for:

    • Phosphopeptides (TiO₂, IMAC)

    • Glycopeptides (lectin affinity, HILIC)

    • Ubiquitinated peptides (anti-diGly antibodies)

• Site-Directed Mutagenesis Validation:

  • Mutate identified PTM sites to non-modifiable residues

  • Assess functional consequences in vivo

  • Compare protein localization, stability, and interaction profile

• Phosphorylation-Specific Analysis:

  • Use Phos-tag SDS-PAGE to separate phosphorylated forms

  • Perform lambda phosphatase treatment to confirm phosphorylation

  • Map kinase-substrate relationships using kinase inhibitors

Based on analysis of similar vacuolar membrane proteins, potential PTMs to investigate include:

By analogy with Yeb3p/Vac8p, EC1118_1N9_3125g may undergo N-terminal myristoylation that could be critical for its proper vacuolar localization .

How does EC1118_1N9_3125g potentially contribute to autophagy and intravacuolar membrane degradation?

To investigate EC1118_1N9_3125g's potential role in autophagy and intravacuolar membrane degradation, researchers should design experiments addressing the following aspects:

• Autophagy Flux Analysis:

  • Generate EC1118_1N9_3125g deletion strains

  • Monitor autophagy markers (e.g., GFP-Atg8) under starvation conditions

  • Quantify autophagosome formation and clearance rates

  • Measure the accumulation of autophagic bodies in the vacuole

• Examination of Intravacuolar Vesicle Breakdown:

  • Use electron microscopy to visualize intravacuolar structures

  • Monitor the degradation of MVB vesicles using fluorescently-tagged cargo

  • Assess pexophagy (peroxisome degradation) efficiency

• Lipase Activity Assays:

  • Test whether EC1118_1N9_3125g possesses lipase activity similar to Cvt17/Aut5p

  • Use fluorogenic substrates to measure enzymatic activity

  • Determine substrate specificity if activity is detected

By analogy with Cvt17/Aut5p, EC1118_1N9_3125g may be involved in the breakdown of intravacuolar membranes, particularly during autophagy . If it functions similarly, EC1118_1N9_3125g deletion would likely result in the accumulation of intact autophagic bodies within the vacuolar lumen, impaired degradation of autophagocytosed organelles, and possibly defects in MVB vesicle disintegration .

The protein's potential function may be dependent on its proper targeting to the vacuolar membrane, as supported by studies of similar proteins where retention in the ER via an HDEL signal prevented their function in intravacuolar lysis .

What are the evolutionary relationships of EC1118_1N9_3125g across different yeast species?

To analyze the evolutionary relationships of EC1118_1N9_3125g across different yeast species, researchers should implement a comprehensive phylogenetic approach:

• Sequence Homology Analysis:

  • Use BLAST searches against fungal genome databases

  • Identify orthologs and paralogs across Saccharomycetaceae and other fungal families

  • Calculate sequence conservation percentages for different domains

• Phylogenetic Tree Construction:

  • Align sequences using MUSCLE or T-Coffee algorithms

  • Generate maximum likelihood trees using RAxML or IQ-TREE

  • Assess node support with bootstrap analysis (1000 replicates)

  • Root trees appropriately using distant homologs

• Functional Domain Conservation Analysis:

  • Map conserved domains across species

  • Identify species-specific insertions or deletions

  • Correlate domain conservation with known lifestyle differences

• Selective Pressure Analysis:

  • Calculate dN/dS ratios across coding sequences

  • Identify sites under positive or purifying selection

  • Use PAML or HyPhy for codon-based analyses

The evolutionary analysis should include comparison with functionally characterized vacuolar membrane proteins such as Cvt17/Aut5p and Yeb3p/Vac8p , looking for functional domain conservation and adaptive evolution patterns.

Based on studies of similar vacuolar proteins, researchers might expect to find higher conservation in transmembrane domains and functional motifs, with greater divergence in cytosolic regulatory regions that may reflect species-specific adaptations in vacuolar function and autophagy regulation.

How can CRISPR-Cas9 genome editing be optimized for studying EC1118_1N9_3125g function?

To optimize CRISPR-Cas9 genome editing for EC1118_1N9_3125g functional studies, researchers should implement the following methodological approach:

• Guide RNA Design and Optimization:

  • Design multiple sgRNAs targeting the EC1118_1N9_3125g gene using tools like CHOPCHOP

  • Prioritize guides with high on-target and low off-target scores

  • Test guide RNA efficiency using in vitro cleavage assays

  • Optimize for the specific codon usage of S. cerevisiae

• Editing Strategies for Different Experimental Objectives:

  a. Gene Knockout:

  • Design repair templates with selectable markers

  • Include 40-60bp homology arms flanking the cut site

  • Confirm deletions by PCR and sequencing

  b. Point Mutations:

  • Design repair templates with specific mutations and silent PAM site mutations

  • Use single-stranded DNA oligonucleotides for higher efficiency

  • Screen for mutations using restriction enzyme digestion or high-resolution melt analysis

  c. Tagging Applications:

  • Create C-terminal fusions preserving protein function

  • Design repair templates with fluorescent proteins or epitope tags

  • Include flexible linkers to minimize functional interference

• Delivery Methods Optimization:

  • Test different transformation protocols (lithium acetate, electroporation)

  • Optimize Cas9 and sgRNA expression using different promoters

  • Consider transient expression vs. stable integration approaches

• Phenotypic Validation Strategies:

  • Develop high-throughput screening methods for edited strains

  • Implement FACS-based selection if using fluorescent reporters

  • Design positive and negative controls to validate editing outcomes

When editing EC1118_1N9_3125g, special attention should be paid to its membrane topology to ensure tags or mutations don't disrupt transmembrane domains or critical functional regions.