Recombinant Saccharomyces cerevisiae Putative uncharacterized membrane protein YLR311C (YLR311C)
Description
Introduction to YLR311C
Saccharomyces cerevisiae, commonly known as baker's yeast, has served as a crucial eukaryotic model organism in molecular biology and genomics research for decades. Its genome was the first eukaryotic genome to be completely sequenced, providing an essential foundation for functional genomic studies across eukaryotes. Despite this extensive characterization, approximately 20% of its genes remain functionally uncharacterized, including the YLR311C gene.
YLR311C represents an open reading frame (ORF) that encodes a putative uncharacterized membrane protein in S. cerevisiae. The "YLR" designation in its systematic name indicates its location on chromosome XII of the yeast genome, while the numeric part specifies its relative position within that chromosome . The "C" suffix indicates that the gene is transcribed from the complementary strand. The protein has been annotated in several genomic databases including the Saccharomyces Genome Database (SGD), where it is categorized among proteins with undetermined function .
As part of ongoing efforts to characterize the complete yeast proteome, recombinant forms of the YLR311C protein have been developed to facilitate biochemical and functional studies. These recombinant versions provide researchers with purified protein material for experimental investigations aimed at elucidating its biological role and structural characteristics.
Current Classification Status
YLR311C is classified as a "putative uncharacterized membrane protein," reflecting both its predicted cellular localization and the current limited understanding of its function. This classification is derived from bioinformatic analyses of its amino acid sequence, which suggests membrane-spanning domains characteristic of integral membrane proteins . The term "putative" acknowledges that this functional assignment is based on computational predictions rather than direct experimental evidence.
Protein Identifiers and Database Entries
The YLR311C protein is cataloged in multiple protein databases with the following identifiers:
| Database | Identifier | Description |
|---|---|---|
| UniProt | Q06158 | Putative uncharacterized membrane protein YLR311C |
| SGD | YLR311C | Putative protein of unknown function |
| Aliases | L8543.10 | Alternative gene identifier |
These database entries primarily note the uncharacterized nature of the protein, reflecting the current state of knowledge regarding its function and cellular role .
Expression and Purification Systems
The commercially available recombinant YLR311C protein is produced using bacterial expression systems, specifically Escherichia coli . This approach allows for the large-scale production of the protein for research purposes. The recombinant form encompasses the full-length protein (amino acids 1-115) fused to an N-terminal histidine (His) tag to facilitate purification .
The His-tag fusion enables efficient purification through affinity chromatography, allowing the protein to be isolated from bacterial cell lysates with high purity. According to product specifications, the purified recombinant protein typically exhibits purity greater than 90% as determined by SDS-PAGE analysis .
Applications of Recombinant YLR311C
The recombinant YLR311C protein is primarily produced for research applications, with SDS-PAGE analysis being the main documented use . Other potential applications may include:
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Generation of antibodies for immunological detection
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Structural studies to determine three-dimensional conformation
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Interaction studies to identify binding partners
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Functional assays to elucidate biochemical activities
Position in the S. cerevisiae Genome
YLR311C is located within the central region of chromosome XII in S. cerevisiae. This positioning is significant in the context of genome architecture, as research indicates that essential genes and more conserved elements tend to be concentrated in the central regions of yeast chromosomes, as opposed to the more variable subtelomeric regions .
Genomic analyses of S. cerevisiae have revealed distinct patterns of conservation and variability across chromosomal regions. A comprehensive study of 94 yeast strains found that the central regions of chromosomes typically contain more conserved genes, including essential genes and those encoding tRNAs and other RNA species . The positioning of YLR311C in a central chromosomal region rather than a subtelomeric region may suggest evolutionary conservation, though its essentiality has not been explicitly documented.
Conservation and Evolutionary Significance
While detailed information about the evolutionary conservation of YLR311C across fungal species is limited in the available search results, its genomic location provides some context. The central regions of S. cerevisiae chromosomes typically harbor genes that are more conserved across yeast strains and potentially across related species .
Unlike essential genes that are required for cell viability, YLR311C does not appear to be classified as essential based on available information . This suggests that while it may have a functional role in cellular processes, its absence is not lethal under standard laboratory conditions. This non-essential nature is common among many membrane proteins that may serve specialized or condition-specific functions.
Membrane Localization and Possible Functions
Based on sequence analysis and classification, YLR311C is predicted to be a membrane protein, suggesting potential involvement in membrane-associated processes . These might include:
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Transport of molecules across membranes
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Signal transduction
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Maintenance of membrane structure or integrity
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Response to environmental stresses affecting membranes
Relationship to Other Yeast Membrane Proteins
S. cerevisiae contains numerous membrane proteins with characterized functions, including transporters, channels, receptors, and structural elements. In contrast to well-characterized membrane proteins like the hexose transporters or ATP/ADP translocators mentioned in the research literature, YLR311C remains functionally uncharacterized .
In yeast metabolism, membrane proteins play crucial roles in determining the balance between fermentative and respiratory glucose metabolism. While YLR311C is briefly mentioned in literature discussing these metabolic pathways, its specific role, if any, in these processes is not elucidated .
Research Context in Yeast Functional Genomics
The study of proteins like YLR311C is part of broader efforts to complete the functional annotation of the S. cerevisiae genome. While approximately 80% of yeast genes have assigned functions, the remainder, including YLR311C, represent knowledge gaps in our understanding of eukaryotic cell biology .
The development of functional genomic technologies has accelerated the characterization of previously uncharacterized genes. These approaches include:
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Transcriptomic analyses to identify expression patterns
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Proteomic studies to determine interaction partners
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Metabolomic assessments to identify affected metabolic pathways
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Systematic gene deletion studies to observe phenotypic effects
While such comprehensive analyses would be valuable for understanding YLR311C function, the available search results do not provide specific information from such studies for this particular protein.
Techniques for Characterizing Uncharacterized Proteins
Several experimental approaches can be employed to characterize proteins of unknown function like YLR311C:
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Gene deletion or disruption: Creating knockout strains to observe resulting phenotypes under various conditions.
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Localization studies: Using fluorescent tags to determine subcellular localization.
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Protein-protein interaction assays: Identifying binding partners through techniques such as yeast two-hybrid screens or co-immunoprecipitation.
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Expression profiling: Monitoring gene expression under different conditions to identify regulation patterns.
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Heterologous expression: Expressing the protein in different host systems to observe functional effects.
The commercially available recombinant YLR311C protein provides a resource for biochemical and structural studies that might complement these approaches .
Methodological Considerations for Membrane Proteins
Membrane proteins present distinct challenges for characterization compared to soluble proteins. Their hydrophobic nature can complicate expression, purification, and structural analyses. The recombinant production of YLR311C with a His-tag represents an approach to overcome some of these challenges, providing purified protein for experimental investigation .
For functional studies, techniques such as reconstitution into artificial membrane systems or expression in membrane-protein-deficient yeast strains might provide insights into the protein's activity and role in cellular processes.
Product Specs
Please note: We will prioritize shipping the format we currently have in stock. However, if you have a specific format preference, please indicate it in your order notes. We will accommodate your request to the best of our ability.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance as additional fees may apply.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.
Target Background
STRING: 4932.YLR311C
Q&A
What methods are most effective for confirming the subcellular localization of YLR311C in S. cerevisiae?
The subcellular localization of YLR311C can be determined using several complementary approaches:
Fluorescent protein tagging: A GFPdeg fusion protein approach has proven effective for identifying novel mitochondrially localized proteins. This technique uses a fluorescent protein that is rapidly degraded in the cytoplasm but protected when localized to organelles. Expression of YLR311C-GFPdeg fusion proteins followed by fluorescence microscopy observation can confirm mitochondrial or other organelle localization .
Prediction tools validation: While computational tools like DeepLoc-1.0 can predict protein localization, experimental validation is essential. For YLR311C, predictions should be verified through empirical methods, particularly as many uncharacterized proteins in yeast lack classical N-terminal localization signals .
Subcellular fractionation: Isolation of different cellular compartments (mitochondria, endoplasmic reticulum, etc.) followed by Western blot detection can provide biochemical evidence of YLR311C localization.
Co-localization with known markers: Fluorescence microscopy using organelle-specific dyes or markers in conjunction with tagged YLR311C provides additional confirmation of subcellular localization.
How can researchers determine if YLR311C contains transmembrane domains consistent with its putative membrane protein classification?
Determining the membrane topology of YLR311C involves:
Computational prediction: Tools like TMHMM, Phobius, and TOPCONS can predict transmembrane domains based on hydrophobicity profiles and amino acid composition.
Protease protection assays: By exposing isolated membrane fractions to proteases, researchers can identify which portions of YLR311C are protected (embedded in the membrane) versus exposed (accessible to proteolytic digestion).
Glycosylation site mapping: Introduction of artificial glycosylation sites followed by detection of glycosylation patterns can reveal which portions of the protein face the lumen/cytoplasm.
Cysteine accessibility methods: Substituting amino acids with cysteines followed by labeling with membrane-permeable or impermeable reagents can map protein topology relative to the membrane.
What genetic manipulation strategies are most appropriate for studying the function of YLR311C in S. cerevisiae?
Several genetic approaches can be employed to study YLR311C function:
Gene deletion/knockout: CRISPR-Cas9 or homologous recombination-based deletion of YLR311C followed by phenotypic analysis under various conditions.
Controlled expression systems: Using inducible promoters (e.g., GAL1, TET) to modulate YLR311C expression levels and study dosage effects.
Site-directed mutagenesis: Introduction of specific mutations to identify crucial residues for protein function.
Protein tagging strategies: Addition of epitope tags or fluorescent proteins to track expression, localization, and interactions without disrupting function.
When designing these experiments, it's essential to include appropriate controls and ensure the experimental design follows systematic procedures similar to those described for other yeast studies :
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Define independent variables (e.g., YLR311C expression levels) and dependent variables (e.g., growth rate, stress resistance)
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Formulate specific, testable hypotheses
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Design treatments that manipulate the independent variable
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Assign experimental groups properly (between-subjects or within-subjects designs)
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Measure dependent variables with appropriate controls
What growth conditions and phenotypic assays would be most informative for revealing YLR311C function?
To characterize YLR311C function, researchers should consider:
Carbon source utilization: Testing growth on different carbon sources (glucose, galactose, xylose, etc.) may reveal metabolic roles, especially if YLR311C is involved in alternative carbon source utilization pathways similar to those described for xylose metabolism .
Growth phase-specific expression: Analyzing expression during different growth phases (exponential, diauxic shift, stationary) can provide functional insights. Many uncharacterized mitochondrial proteins are upregulated during the postdiauxic shift when mitochondrial development occurs .
Stress response assays: Examining growth under various stressors (oxidative, osmotic, temperature) can reveal roles in stress tolerance.
Mitochondrial function assays: If localized to mitochondria, measuring oxygen consumption, membrane potential, and ROS production in wild-type versus YLR311C mutant strains.
Metabolomic profiling: Comparing metabolite levels between wild-type and YLR311C mutant strains can identify affected metabolic pathways.
What approaches can determine if YLR311C interacts with other proteins to form functional complexes?
Several complementary methods can identify protein-protein interactions involving YLR311C:
Affinity purification-mass spectrometry (AP-MS): Using tagged YLR311C to pull down interaction partners followed by mass spectrometry identification.
Yeast two-hybrid screening: Systematic screening for binary interactions between YLR311C and other yeast proteins.
Proximity labeling: BioID or APEX2 fusion proteins can identify proteins in the vicinity of YLR311C in living cells.
Co-immunoprecipitation: Using antibodies against YLR311C or its tagged version to precipitate the protein and its interacting partners.
Genetic interaction screening: Synthetic genetic array (SGA) analysis to identify genes that genetically interact with YLR311C, suggesting functional relationships.
How can researchers distinguish between direct and indirect protein interactions with YLR311C?
Distinguishing direct from indirect interactions requires:
In vitro binding assays: Using purified recombinant proteins to test direct binding without cellular intermediaries.
Crosslinking coupled with mass spectrometry: Chemical crosslinking of closely associated proteins followed by mass spectrometry can identify direct interaction interfaces.
Förster Resonance Energy Transfer (FRET): Measuring energy transfer between fluorescently labeled proteins can confirm close proximity in living cells.
Surface Plasmon Resonance (SPR): Quantitative measurement of binding affinities between purified proteins.
Nuclear Magnetic Resonance (NMR): Structural characterization of protein-protein interfaces at atomic resolution.
What can evolutionary analysis reveal about YLR311C function and conservation across fungal species?
Evolutionary analysis provides important functional insights through:
Phylogenetic profiling: Determining the presence/absence of YLR311C homologs across fungal species can indicate functional importance and evolutionary origin.
Sequence conservation analysis: Identifying conserved domains or motifs that suggest functional regions.
Evolutionary rate analysis: Measuring selective pressures (dN/dS ratios) across the protein sequence to identify functionally constrained regions.
Synteny analysis: Examining conservation of gene order around YLR311C to identify functionally related gene clusters.
Many uncharacterized proteins in S. cerevisiae are "emerging genes" that exist only in this species, suggesting recent evolutionary origins . If YLR311C falls into this category, it may represent a species-specific adaptation worth investigating from both functional and evolutionary perspectives.
How does the expression pattern of YLR311C correlate with other genes during different growth phases and conditions?
Expression pattern analysis includes:
Transcriptomic profiling: RNA-seq or microarray analysis under different conditions can reveal co-regulated gene networks.
Growth phase-specific expression: Many mitochondrial proteins show upregulation during the postdiauxic shift when cells transition from fermentation to respiration .
Stress response expression: Examining YLR311C expression under various stressors can indicate functional roles.
Promoter analysis: Identifying transcription factor binding sites in the YLR311C promoter can reveal regulatory mechanisms.
The table below shows a hypothetical expression pattern comparison of YLR311C with known genes during different growth phases:
| Growth Phase | YLR311C Expression | Glycolytic Genes | Mitochondrial Genes | Stress Response Genes |
|---|---|---|---|---|
| Log Phase | Low | High | Low | Low |
| Diauxic Shift | Increasing | Decreasing | Increasing | Moderate |
| Post-diauxic | High | Low | High | Moderate |
| Stationary | Moderate | Low | Moderate | High |
How can systems biology approaches integrate multiple data types to elucidate YLR311C function?
Systems biology integrates:
Multi-omics data integration: Combining transcriptomics, proteomics, metabolomics, and interactomics data to place YLR311C in cellular networks.
Flux balance analysis: Computational modeling of metabolic networks to predict the impact of YLR311C perturbation on cellular metabolism.
Bayesian network analysis: Inferring causal relationships between YLR311C and other cellular components.
Genome-scale models: Incorporating YLR311C into existing genome-scale metabolic models of S. cerevisiae to predict functional roles.
Network analysis: Identifying functional modules and pathways where YLR311C plays a role based on various interaction data.
What structural biology approaches can reveal the molecular function of YLR311C?
Structural characterization involves:
X-ray crystallography or cryo-EM: Determining the three-dimensional structure of purified YLR311C to infer function from structural features.
Homology modeling: Using related proteins with known structures to model YLR311C structure if experimental determination is challenging.
Molecular dynamics simulations: Predicting protein dynamics and potential ligand binding sites.
Structural proteomics: Limited proteolysis coupled with mass spectrometry to map structural domains.
Structure-guided mutagenesis: Using structural insights to design mutations that test functional hypotheses.
How can researchers overcome difficulties in expressing and purifying membrane proteins like YLR311C?
Membrane protein expression and purification challenges can be addressed through:
Expression systems optimization: Testing different promoters, host strains, and growth conditions to maximize expression.
Fusion partners: Using solubility-enhancing tags (MBP, SUMO) to improve expression and folding.
Detergent screening: Systematic testing of detergents for optimal solubilization while maintaining native structure.
Nanodiscs or liposomes: Reconstituting purified YLR311C into membrane mimetics for functional studies.
Cell-free expression systems: Using cell-free systems specifically designed for membrane protein expression.
What approaches can resolve contradictions between computational predictions and experimental observations of YLR311C?
Resolving discrepancies requires:
Improved computational methods: Using ensemble approaches that combine multiple prediction algorithms.
Experimental validation: Designing experiments specifically to test conflicting predictions.
Domain-specific analysis: Analyzing protein domains separately when whole-protein analysis yields contradictory results.
Integration of multiple data types: Using orthogonal experimental approaches to build consensus.
Iterative refinement: Using experimental results to improve computational models in a feedback loop.
How can CRISPR-based technologies enhance our understanding of YLR311C function beyond traditional knockout approaches?
Advanced CRISPR applications include:
CRISPRi/CRISPRa: CRISPR interference or activation to modulate YLR311C expression without complete deletion.
Base editing: Introducing specific nucleotide changes to study the effect of single amino acid substitutions.
Prime editing: Precise genome editing to introduce specific mutations or tags at the endogenous locus.
CRISPR screening: Genome-wide screens to identify genetic interactions with YLR311C.
Perturb-seq: Combining CRISPR perturbations with single-cell RNA-seq to profile transcriptional responses to YLR311C modulation.
What high-throughput experimental approaches can accelerate the functional characterization of YLR311C and similar uncharacterized proteins?
High-throughput methods include:
Pooled fitness assays: Competitive growth of mutant collections under hundreds of conditions to identify phenotypes.
Synthetic genetic interaction mapping: Systematic creation of double mutants to identify genetic relationships.
Proteome-wide interaction mapping: High-throughput methods to map all protein-protein interactions involving YLR311C.
Metabolic profiling: Untargeted metabolomics to identify metabolites affected by YLR311C perturbation.
Automated microscopy: High-content screening of cellular phenotypes in response to YLR311C manipulation.
