Recombinant Saccharomyces cerevisiae Vacuolar membrane protein C1Q_01198 (C1Q_01198)

Basic Research Questions

• What is the Vacuolar membrane protein C1Q_01198 and what are its fundamental properties?

  Vacuolar membrane protein C1Q_01198 is a protein identified in Saccharomyces cerevisiae (strain JAY291), commonly known as baker's yeast. According to UniProt database entry C7GLU4, it is classified as a transmembrane protein localized to the vacuolar membrane . The protein is part of the complex network of proteins involved in vacuolar function, which is critical for various cellular processes including protein degradation, ion homeostasis, and metabolite storage.

  Key Properties:

  • Source organism: Saccharomyces cerevisiae (strain JAY291)

  • UniProt accession number: C7GLU4

  • Subcellular localization: Vacuolar membrane

  • Purity when recombinantly produced: >85% (SDS-PAGE)

• How should researchers store Recombinant Saccharomyces cerevisiae Vacuolar membrane protein C1Q_01198?

  The stability and shelf life of this protein depend on several factors including storage state, buffer composition, and temperature. Based on manufacturer recommendations:

  Formulation	Storage Temperature	Shelf Life
  Liquid form	-20°C to -80°C	6 months
  Lyophilized	-20°C to -80°C	12 months
  Working aliquots	4°C	Up to 1 week

  Repeated freezing and thawing should be avoided as it significantly reduces protein stability and activity . For optimal preservation, aliquoting the protein solution before freezing is strongly recommended.

• What is the recommended reconstitution protocol for C1Q_01198?

  For optimal reconstitution of lyophilized protein:

  1. Briefly centrifuge the vial prior to opening to bring contents to the bottom

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

  3. For long-term storage, add glycerol to a final concentration of 5-50% (with 50% being the default recommendation)

  4. Create aliquots and store at -20°C to -80°C

  This protocol helps maintain protein integrity while minimizing degradation during freeze-thaw cycles.

Advanced Research Applications

• How can Vacuolar membrane protein C1Q_01198 be used as a model for studying human neurodegenerative diseases?

  Saccharomyces cerevisiae serves as an important model organism for studying neurodegenerative diseases due to conservation of fundamental cellular processes . Vacuolar membrane proteins like C1Q_01198 are particularly relevant because:

  1. The yeast vacuole is functionally equivalent to the mammalian lysosome, which is implicated in many neurodegenerative disorders

  2. Protein aggregation and mislocalization to the vacuole/lysosome are hallmarks of diseases like Alzheimer's and Parkinson's

  3. Vacuolar proteins participate in protein quality control mechanisms similar to those disrupted in neurodegenerative conditions

  Research approaches include:

  • Creating disease-relevant mutations in C1Q_01198 and studying effects on vacuolar function

  • Co-expressing C1Q_01198 with human disease proteins to identify functional interactions

  • Using C1Q_01198 as a reporter for vacuolar/lysosomal stress responses

  • Screening for compounds that restore normal vacuolar function in disease models

• What role might C1Q_01198 play in protein trafficking and cellular homeostasis?

  Based on research on similar vacuolar membrane proteins in S. cerevisiae, C1Q_01198 likely participates in protein trafficking pathways between the Golgi, endosomes, and vacuole. The VPS5/GRD2 gene, which encodes another vacuolar sorting protein, provides a potential model:

  1. Possible involvement in retrieval of membrane proteins from prevacuolar/late endosomal compartments to the Golgi apparatus

  2. May contribute to maintenance of vacuolar morphology

  3. Could participate in sorting of vacuolar proteins like carboxypeptidase Y

  4. Might function in complexes with other trafficking proteins

  Understanding C1Q_01198's role in these processes could provide insights into fundamental cellular homeostasis mechanisms and their disruption in disease states.

• How do recombination events affect the genetic diversity of yeast vacuolar membrane proteins?

  Genetic recombination contributes significantly to diversity in yeast proteins through several mechanisms:

  1. Meiotic recombination: During sexual reproduction, crossing over events can generate novel alleles

  2. Homologous recombination: Similar sequences can exchange genetic material during DNA repair

  3. Horizontal gene transfer: Though rare in yeast, can introduce novel genetic elements

  4. Internal recombination events: Can modify protein domain organization

  These mechanisms might specifically affect C1Q_01198 by:

  • Creating novel functional domains through recombination with other vacuolar proteins

  • Generating adaptive variations in response to environmental pressures

  • Introducing strain-specific variations that affect protein function or regulation

  Research in HIV-1 has shown how recombination can restrict evolutionary bottlenecks to minimal genome segments required for selective advantage, potentially preserving diversity in adjacent regions . Similar mechanisms may operate in yeast genes encoding vacuolar membrane proteins.

Data Analysis and Interpretation

• How can researchers address contradictory results in studies of Vacuolar membrane protein C1Q_01198?

  Contradictions in research data can arise from various sources. A structured approach to addressing such contradictions includes:

  1. Parameter-based contradiction analysis: Using the (α, β, θ) notation system where:

     • α represents the number of interdependent items

     • β represents the number of contradictory dependencies defined by domain experts

     • θ represents the minimal number of required Boolean rules to assess contradictions

  2. Controlled comparative experiments: Systematically varying experimental conditions while maintaining all other variables constant

  3. Multiple methodological approaches: Applying different techniques to verify the same hypothesis

  4. Metadata analysis: Examining experimental conditions that might explain divergent results

  When specifically analyzing contradictions in C1Q_01198 studies, researchers should document all experimental variables including:

  • Strain background

  • Growth conditions

  • Protein preparation methods

  • Analytical techniques

  • Environmental factors (pH, temperature, media composition)

• What bioinformatics approaches are most effective for predicting C1Q_01198 function?

  A comprehensive bioinformatics analysis of vacuolar membrane proteins should include:

  1. Sequence homology analysis:

     • BLAST searches against multiple databases

     • Multiple sequence alignments with known vacuolar proteins

     • Identification of conserved domains and motifs

  2. Structural prediction:

     • Transmembrane domain identification

     • Secondary and tertiary structure modeling

     • Prediction of post-translational modification sites

  3. Functional inference:

     • Gene Ontology term assignment

     • Protein-protein interaction network analysis

     • Metabolic pathway mapping

     • Co-expression data analysis

  4. Comparative genomics:

     • Ortholog identification across species

     • Evolutionary rate analysis

     • Synteny mapping

  These approaches can be combined to generate testable hypotheses about C1Q_01198 function based on computational predictions.

Experimental Troubleshooting

• What are common purification challenges for C1Q_01198 and how can they be addressed?

  Membrane proteins like C1Q_01198 present specific purification challenges:

  Challenge	Solution Strategy
  Poor solubility	Use appropriate detergents (DDM, LDAO, or MNG) for extraction
  Low expression	Optimize codon usage, culture conditions, and induction parameters
  Protein instability	Add stabilizing agents (glycerol 5-50%, specific lipids)
  Aggregation	Perform purification at lower temperatures (4°C)
  Contaminants	Implement multi-step purification combining affinity, ion exchange, and size exclusion
  Proteolytic degradation	Add protease inhibitor cocktails during extraction and purification

  Specific recommendations for C1Q_01198 include centrifuging the vial before opening, reconstituting to 0.1-1.0 mg/mL in deionized water, and adding glycerol for long-term storage .

• How can researchers validate the specificity of antibodies against C1Q_01198?

  Rigorous antibody validation is essential for reliable research outcomes. Key validation steps include:

  1. Western blot analysis:

     • Using wild-type yeast expressing C1Q_01198 (positive control)

     • Testing C1Q_01198 knockout strains (negative control)

     • Comparing signal with recombinant protein of known concentration

  2. Immunoprecipitation validation:

     • Mass spectrometry verification of pulled-down proteins

     • Testing for co-immunoprecipitation of known interacting partners

  3. Immunofluorescence controls:

     • Co-localization with known vacuolar membrane markers

     • Absence of signal in knockout strains

     • Peptide competition assays to confirm specificity

  4. Cross-reactivity assessment:

     • Testing against closely related proteins

     • Evaluation in different yeast strains

  Documentation of validation results should accompany all published research using antibodies against C1Q_01198 to ensure reproducibility.

• What reference genes should be used when studying C1Q_01198 expression under varying conditions?

  Selection of appropriate reference genes is crucial for accurate RT-qPCR analysis. For studies involving vacuolar membrane proteins in Saccharomyces cerevisiae, recommended reference genes include:

  Reference Gene	Stability Characteristics	Optimal Conditions
  TPI1	High stability during glucose perturbation	Glucose limitation studies
  FBA1	Stable under both glucose and ammonium perturbations	Multiple nutrient studies
  CDC19	Stable during glucose perturbation	Metabolic studies
  ACT1	Commonly used, stable under various conditions	General studies
  TDH3	Stable during ammonium perturbation	Nitrogen metabolism studies
  CCW12	Stable during ammonium perturbation	Cell wall/membrane studies

  These genes have been validated in dynamic transcriptional studies and outperform commonly used reference genes in determining expression profiles under specific experimental conditions .

Methodology Optimization

• What are the most effective genetic manipulation strategies for studying C1Q_01198 function?

  Saccharomyces cerevisiae offers powerful genetic tools for functional studies:

  1. CRISPR-Cas9 gene editing:

     • Enables precise mutations at the endogenous locus

     • Can introduce specific amino acid substitutions to study structure-function relationships

     • Allows creation of conditional alleles

  2. Fluorescent protein tagging:

     • C-terminal or N-terminal tagging for localization studies

     • Split fluorescent protein complementation for interaction studies

     • Photoactivatable tags for dynamic trafficking analysis

  3. Regulatable expression systems:

     • GAL1/10 promoter for glucose-regulated expression

     • TET-off/TET-on systems for doxycycline-dependent control

     • Degron-based systems for rapid protein depletion

  4. Synthetic genetic array (SGA) analysis:

     • Systematic creation of double mutants to identify genetic interactions

     • Screens for suppressors or enhancers of C1Q_01198 mutant phenotypes

  These approaches can be combined to comprehensively characterize C1Q_01198 function in various cellular contexts.

• How can researchers optimize experimental conditions for studying protein splicing in C1Q_01198?

  Based on studies of protein splicing in S. cerevisiae vacuolar membrane ATPase intein:

  1. Modulation strategies:

     • Amino acid substitutions at the -1 position can attenuate splicing initiation

     • Modifications of the intein penultimate residue affect branch resolution and C-terminal cleavage

  2. Environmental manipulation:

     • pH adjustments affect splicing efficiency

     • Temperature changes can modulate reaction rates

     • Addition of thiol reagents influences specific splicing steps

  3. Structural considerations:

     • Both insertion and deletion can be tolerated in the central region of inteins

     • These modifications may affect splicing efficiency or intein structure

  Researchers should systematically vary these parameters to identify optimal conditions for studying specific aspects of protein splicing in C1Q_01198.

• What are the best approaches for studying C1Q_01198 in the context of vacuolar membrane dynamics?

  Advanced imaging and biochemical approaches provide insights into membrane protein dynamics:

  1. Live-cell imaging techniques:

     • Fluorescence recovery after photobleaching (FRAP) to measure protein mobility

     • Single-particle tracking to follow individual protein molecules

     • Super-resolution microscopy for detailed localization studies

  2. Membrane fractionation approaches:

     • Density gradient centrifugation to separate membrane compartments

     • Immunoisolation of specific membrane subdomains

     • Protease protection assays to determine topology

  3. Protein dynamics studies:

     • Pulse-chase analysis to track protein trafficking pathways

     • Cycloheximide chase to measure protein turnover rates

     • Conditional mutants to study acute effects of protein depletion

  4. Reconstitution systems:

     • Liposome reconstitution to study function in defined lipid environments

     • Giant unilamellar vesicles (GUVs) for membrane curvature studies

     • Supported lipid bilayers for controlled interaction studies

  These approaches, combined with genetic manipulation strategies, provide a comprehensive toolkit for investigating C1Q_01198 function in vacuolar membrane dynamics.