Recombinant Saccharomyces cerevisiae UPF0479 membrane protein YLL067W-A (YLL067W-A)

What is UPF0479 membrane protein YLL067W-A?

UPF0479 membrane protein YLL067W-A is a protein encoded by the YLL067W-A gene in Saccharomyces cerevisiae (baker's yeast). It belongs to the "Uncharacterized Protein Family" (UPF), indicating that its specific function has not been fully determined. The protein consists of 160 amino acids and is integrated into cellular membranes, suggesting potential roles in membrane structure, transport, or signaling pathways .

What is the amino acid sequence of YLL067W-A?

The full amino acid sequence of YLL067W-A (1-160) is:
MMPAKLQLDVLRTLQSSARHGTQTLKNSNFLERFHKDRIVFCLPFFPALFLVPVQKVLQHLCLRFTQVAPYFIIQLFDLPSRHAENLAPLLASCRIQYTNCFSSSSNGQVPSIISLYLRVDLSPFYAKKFQIPYRVPMIWLDVFQVFFVFLVISQHSLHS

How is YLL067W-A classified within the S. cerevisiae proteome?

YLL067W-A is classified as a membrane protein within the UPF0479 family. In the context of yeast biology, it is part of the extensive network of proteins found in Saccharomyces cerevisiae, which is one of the most thoroughly studied eukaryotic model organisms . Its UniProt ID is P0CY01 , which places it within the international protein database system for reference and comparative analysis.

What are the optimal conditions for storing and reconstituting recombinant YLL067W-A protein?

For optimal storage and reconstitution of recombinant YLL067W-A protein, researchers should follow these methodological steps:

Storage:

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

• Aliquot reconstituted protein to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

Reconstitution Protocol:

• Centrifuge the vial briefly before opening 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% (recommended default: 50%)

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

What expression systems are most effective for producing recombinant YLL067W-A?

For structural and functional studies, researchers should evaluate the impact of expression system on protein folding and activity through quality control experiments.

How can researchers design experiments to study YLL067W-A function in S. cerevisiae?

When designing experiments to investigate YLL067W-A function in S. cerevisiae, researchers should employ a multi-faceted approach:

• Gene Disruption Studies:

  • Create YLL067W-A deletion strains using CRISPR-Cas9 or homologous recombination

  • Analyze phenotypic changes in growth, stress responses, and membrane integrity

  • Perform complementation studies with wild-type gene to confirm phenotype specificity

• Protein Localization Studies:

  • Generate fluorescent protein fusions (GFP, mCherry) to visualize subcellular localization

  • Perform co-localization with known organelle markers

  • Examine localization changes during different cell cycle stages, particularly during cytokinesis

• Interaction Network Analysis:

  • Utilize the His-tagged protein for affinity purification followed by mass spectrometry

  • Perform yeast two-hybrid screening or proximity labeling techniques

  • Validate interactions with co-immunoprecipitation studies

• Functional Assays:

  • Measure membrane integrity parameters in wild-type vs. knockout strains

  • Assess response to various stressors (osmotic, oxidative, temperature)

  • Monitor impact on cell cycle progression and cytokinesis using time-lapse microscopy

Proper experimental design should include appropriate controls and statistical analysis as described in standard experimental design principles .

What structural characteristics define YLL067W-A as a membrane protein?

YLL067W-A is classified as a membrane protein based on its hydrophobic domains that likely form transmembrane helices. As a multipass membrane protein, it contains multiple α-helical transmembrane domains (TMDs) that span the lipid bilayer . The protein's insertion into membranes follows the general biogenesis pathway:

• Cotranslational insertion via the Sec61 translocon complex in the endoplasmic reticulum

• Recognition of hydrophobic signal sequences by the signal recognition particle (SRP)

• Lateral movement of transmembrane segments through the Sec61 lateral gate into the lipid bilayer

• Assembly and folding within the membrane environment

The specific topology (orientation of N-terminus and C-terminus relative to the membrane) would require experimental verification through techniques such as protease protection assays or reporter fusion constructs.

What methodological approaches can determine the membrane topology of YLL067W-A?

Determining the membrane topology of YLL067W-A requires a combination of computational prediction and experimental verification:

Integration of results from multiple approaches provides the most reliable topological model for further functional studies.

What role might YLL067W-A play in yeast membrane organization or cytokinesis?

Given that S. cerevisiae utilizes asymmetric division through budding and has specific membrane reorganization during cell division , YLL067W-A might be involved in membrane-related processes during the cell cycle:

• Membrane Remodeling During Budding:

  • Potential role in membrane curvature at the bud site

  • Involvement in lipid distribution between mother and daughter cells

  • Possible function in establishing membrane asymmetry

• Cytokinesis Processes:

  • Potential association with the actomyosin ring at the bud neck

  • Possible involvement in septum formation during cytokinesis

  • Membrane fusion/fission events during cell separation

• Membrane Trafficking:

  • Facilitation of vesicle transport to growing buds

  • Maintenance of organelle inheritance during cell division

  • Coordination of membrane expansion during growth

Research approaches to explore these roles should include localization studies during different cell cycle phases, interaction analysis with known cytokinesis proteins, and phenotypic characterization of deletion mutants during budding and cytokinesis.

How can YLL067W-A be used as a model for studying membrane protein biogenesis?

YLL067W-A represents an excellent model for investigating fundamental aspects of membrane protein biogenesis for several reasons:

• Expression in Model Organism:

  • S. cerevisiae is a well-established eukaryotic model with extensive genetic tools

  • Allows for both in vivo and in vitro studies of membrane insertion

• Multipass Membrane Protein Characteristics:

  • Undergoes typical insertion via the Sec61 translocon complex

  • Contains multiple transmembrane domains requiring coordinated insertion

  • Represents challenges common to many membrane proteins

• Manageable Size and Complexity:

  • At 160 amino acids , it's amenable to various structural and biochemical analyses

  • Simpler than many mammalian membrane proteins while maintaining eukaryotic features

Experimental approaches utilizing YLL067W-A as a model could include:

These approaches would contribute valuable insights to the broader understanding of membrane protein biology.

What techniques are most effective for studying YLL067W-A interactions with the lipid bilayer?

Understanding YLL067W-A interactions with the lipid bilayer requires specialized techniques that maintain native-like membrane environments:

• Fluorescence-Based Approaches:

  • Fluorescence resonance energy transfer (FRET) between protein and labeled lipids

  • Environment-sensitive fluorescent probes at protein-lipid interfaces

  • Fluorescence recovery after photobleaching (FRAP) for lateral mobility assessment

• Biophysical Characterization:

  • Differential scanning calorimetry to measure thermodynamic parameters

  • Surface plasmon resonance with immobilized protein or model membranes

  • Electron paramagnetic resonance (EPR) spectroscopy with spin-labeled residues

• Lipid-Specific Interaction Analysis:

  • Lipidomics analysis of co-purifying lipids

  • Reconstitution in defined lipid compositions

  • Chemical crosslinking to specific lipid species

• Computational Approaches:

  • Molecular dynamics simulations of protein-lipid interactions

  • Coarse-grained modeling of membrane embedding

  • Identification of potential lipid binding sites

These methodologies provide complementary information about how YLL067W-A interacts with its membrane environment, which may be crucial for understanding its function.

How does the membrane environment influence YLL067W-A structure and function?

The membrane environment can significantly impact YLL067W-A structure and function through multiple mechanisms:

Research into these environmental effects would help determine whether YLL067W-A has a primarily structural role or serves more dynamic functions within cellular membranes.

What are the challenges in determining the high-resolution structure of YLL067W-A?

Obtaining high-resolution structural information for membrane proteins like YLL067W-A presents several significant challenges:

• Expression and Purification Barriers:

  • Achieving sufficient expression levels for structural studies

  • Maintaining protein stability during detergent-based purification

  • Preventing aggregation and maintaining native conformation

• Structural Determination Challenges:

These technical challenges explain why many membrane proteins, including those in the UPF category, remain structurally uncharacterized despite their biological importance.

What methodological approaches can resolve contradictory data about YLL067W-A function?

When confronted with contradictory data regarding YLL067W-A function, researchers should implement a systematic resolution approach:

• Critical Evaluation of Experimental Conditions:

  • Compare expression systems and tags used in different studies

  • Analyze differences in purification methods and detergents

  • Consider variations in buffer conditions and temperature

  • Evaluate cellular contexts of functional assays

• Independent Validation with Multiple Techniques:

• Structured Scientific Collaboration:

  • Establish collaborations between laboratories with different expertise

  • Implement standardized protocols and reagents

  • Conduct blind testing procedures when appropriate

  • Share raw data and analytical methods

This systematic approach helps identify sources of discrepancies and builds a more robust understanding of protein function consistent with the principles of rigorous experimental design .

How might post-translational modifications affect YLL067W-A function and interactions?

Post-translational modifications (PTMs) could significantly impact YLL067W-A function through several mechanisms:

Comprehensive characterization of PTMs would provide valuable insights into regulatory mechanisms controlling YLL067W-A in different cellular contexts or stress conditions.