Recombinant Saccharomyces cerevisiae Uncharacterized membrane protein YLR050C (YLR050C)

What is YLR050C and what are its structural properties?

YLR050C is an uncharacterized membrane protein from Saccharomyces cerevisiae (strain ATCC 204508 / S288c) with 161 amino acids and a molecular mass of approximately 19.1 kDa . The complete amino acid sequence is: MKLGHREQQFYLWYFIVHIPITIFIDSSVVIPAKWQLGIAQKVVSDHIAKQHDFLLSEKPEWLYWFVVLELVLQLPLFVYFVNKFWNSSELQVNTNSRLKKWLRIYGWNASLTTLICIVVIFKRGYIPYDVLKTSLSMTQKCQLASVYLPTFLIPLRLCFV . The protein is classified as a membrane protein, suggesting it contains hydrophobic domains that span or associate with cellular membranes. Structural analysis would likely reveal multiple transmembrane domains characteristic of integral membrane proteins, though detailed structural studies remain to be conducted.

As with many membrane proteins, structural characterization presents significant challenges due to difficulties in crystallization and protein stability outside of the membrane environment. Researchers typically employ computational prediction tools to analyze potential structural features before pursuing experimental characterization.

How is YLR050C typically expressed and purified for research?

Recombinant YLR050C protein is commonly expressed in E. coli expression systems, with His-tag modifications to facilitate purification . Commercial sources provide recombinant full-length YLR050C protein with His-tags, suggesting this is a viable approach for obtaining research quantities . When expressing membrane proteins like YLR050C, researchers must address challenges related to protein folding, toxicity to host cells, and maintaining native conformation during extraction from membranes.

The purification process typically involves cell lysis, membrane fraction isolation, detergent solubilization, and affinity chromatography using the attached His-tag. Optimization of detergent type and concentration is crucial for maintaining protein stability and function during purification. For functional studies, researchers may need to reconstitute the purified protein into artificial membrane systems such as liposomes or nanodiscs.

What genetic context information is important when studying YLR050C?

YLR050C has been mentioned in genomic studies, including one investigating cis,cis-muconic acid production in Saccharomyces cerevisiae, where a silent mutation (Leu112Leu) was identified . This suggests that YLR050C may be subject to genetic variation across different yeast strains or under different selective pressures. When designing experiments, researchers should consider the specific strain background and potential genetic interactions that might influence YLR050C function or expression.

Silent mutations, though not changing the amino acid sequence, can affect mRNA stability, translation efficiency, or co-translational folding. The Leu112Leu mutation in YLR050C might influence these processes despite not altering the protein sequence itself. Additionally, consideration of nearby genomic features and potential regulatory elements is essential for comprehensive functional studies.

What experimental approaches are most effective for characterizing uncharacterized membrane proteins like YLR050C?

Characterizing uncharacterized membrane proteins requires multifaceted approaches. For YLR050C, researchers should consider gene deletion or knockdown studies to observe resulting phenotypes under various growth conditions. High-throughput screening methods using the yeast deletion collection can identify conditions where YLR050C becomes essential or detrimental.

Protein-protein interaction studies using techniques such as yeast two-hybrid, co-immunoprecipitation, or proximity labeling (BioID) can identify potential binding partners, providing functional context. The BenM biosensor approach, similar to what was used in studies of other yeast proteins, could be adapted to detect interactions specific to YLR050C . Additionally, subcellular localization studies using fluorescent protein fusions or immunofluorescence microscopy can determine where YLR050C functions within the cell.

For direct functional characterization, biochemical assays testing various potential substrates should be designed based on bioinformatic predictions or proteomic data. Structural studies using techniques optimized for membrane proteins, such as cryo-electron microscopy or X-ray crystallography with lipidic cubic phase crystallization, can provide insight into molecular function.

How can researchers investigate potential functional pathways involving YLR050C?

To investigate potential pathways involving YLR050C, researchers should employ systematic functional genomics approaches. Synthetic genetic array (SGA) analysis can identify genes that exhibit synthetic lethality or suppression when combined with YLR050C deletion, revealing functional relationships and pathway connections. Transcriptomic profiling using RNA-seq comparing wild-type and YLR050C deletion strains under various conditions can identify differentially expressed genes, suggesting regulatory relationships.

Metabolomic analyses comparing wild-type and YLR050C mutant strains can detect changes in metabolite levels, particularly focusing on membrane-related lipids or potential transported substrates. Stable isotope labeling experiments can track metabolic flux changes associated with YLR050C function. Interestingly, YLR050C was mentioned in a study related to cis,cis-muconic acid production, suggesting potential involvement in aromatic compound metabolism .

Researchers should design experiments to test YLR050C's response to environmental stressors (temperature, pH, osmotic stress) and chemical perturbations to identify conditions where its function becomes critical. Comprehensive phenotypic profiling across hundreds of conditions can provide insight into functional contexts where YLR050C plays important roles.

What methodologies are appropriate for studying membrane topology and integration of YLR050C?

Determining membrane topology of YLR050C requires specialized techniques for membrane proteins. Protease protection assays can differentiate between cytoplasmic and extracytoplasmic domains by selective degradation of accessible regions. This approach involves isolating membrane fractions containing YLR050C and treating with proteases in the presence or absence of membrane-disrupting detergents.

Site-directed mutagenesis of predicted transmembrane regions coupled with functional assays can validate the importance of specific domains. Creating a library of YLR050C variants with single cysteine residues at different positions allows for accessibility studies using membrane-impermeable sulfhydryl reagents, providing detailed topological information.

Fusion reporter systems using GFP or enzymatic reporters (PhoA, LacZ) at various positions can determine which regions face different cellular compartments. Glycosylation mapping, where potential N-glycosylation sites are introduced throughout the protein, can identify luminal domains, as glycosylation occurs only on the luminal side of the endoplasmic reticulum.

Computational prediction tools should complement experimental approaches, with hydropathy analysis identifying potential transmembrane segments and evolutionary conservation analysis highlighting functionally important regions.

How can researchers interpret silent mutations in YLR050C, such as the Leu112Leu observed in previous studies?

The Leu112Leu silent mutation in YLR050C identified in a study on cis,cis-muconic acid production requires careful interpretation . Silent mutations may affect gene expression through several mechanisms despite not changing the amino acid sequence. Researchers should analyze codon usage bias, as rare codons can slow translation and affect co-translational protein folding, which is particularly important for membrane proteins like YLR050C.

Investigators should examine potential effects on mRNA secondary structure and stability, as nucleotide changes can alter RNA folding energies and affect translational efficiency or mRNA degradation rates. Computational tools can predict how the mutation changes mRNA structure. Additionally, researchers should investigate whether the mutation lies within or affects regulatory elements such as splicing enhancers or miRNA binding sites within the coding sequence.

Comparative genomics approaches examining the conservation of synonymous sites across related yeast species can reveal whether specific codons are under selection pressure despite encoding the same amino acid. Experimental validation through targeted mutagenesis and gene expression analysis would be necessary to determine the functional significance of this silent mutation.

What potential role might YLR050C play in metabolic pathways based on current evidence?

While YLR050C remains largely uncharacterized, its mention in a study focused on cis,cis-muconic acid production in Saccharomyces cerevisiae suggests a potential connection to aromatic compound metabolism . The silent Leu112Leu mutation was identified alongside mutations in genes involved in shikimate and aromatic amino acid biosynthesis pathways. This correlation warrants investigation into possible roles in these metabolic networks.

Researchers should conduct targeted metabolomic analysis comparing wild-type and YLR050C deletion strains, focusing on aromatic compounds, their precursors, and derivatives. Stable isotope labeling experiments using 13C-labeled glucose can trace carbon flux through aromatic amino acid pathways in the presence and absence of functional YLR050C. Additionally, expression analysis of YLR050C under conditions that induce or repress aromatic amino acid biosynthesis could reveal regulatory relationships.

Since YLR050C is a membrane protein, it might function as a transporter for metabolic intermediates or endproducts of aromatic amino acid pathways. Transport assays using radioactively labeled or fluorescently tagged substrates would be appropriate for testing this hypothesis. Genetic interaction mapping with known components of aromatic amino acid metabolism could further establish functional connections.

YLR050C Protein Properties

The protein properties table summarizes the basic characteristics of YLR050C as documented in the literature and protein databases . This information provides the foundation for experimental design and bioinformatic analysis. The relatively small size of 161 amino acids suggests a compact structure, potentially with multiple transmembrane helices typical of small membrane transporters or channels.

Reported Mutations in YLR050C

This table highlights the reported Leu112Leu silent mutation in YLR050C identified during a study on improving cis,cis-muconic acid production in Saccharomyces cerevisiae . While the mutation doesn't change the amino acid sequence, it may affect gene expression through various mechanisms, making it worthy of investigation in studies focused on YLR050C function and regulation.

Recommended Experimental Approaches for YLR050C Characterization

This methodological table provides researchers with a roadmap for systematic characterization of YLR050C . The complementary approaches address different aspects of protein function, from cellular context to molecular mechanism, enabling comprehensive functional elucidation of this uncharacterized membrane protein.