Recombinant Saccharomyces cerevisiae Uncharacterized protein YIL029C (YIL029C) is a genetically engineered version of a conserved but functionally uncharacterized protein encoded by the YIL029C gene in S. cerevisiae. Despite its unknown molecular role, YIL029C has been implicated in cellular stress responses, vacuolar function, and cell wall integrity. This protein is part of the yeast core genome, present in all 94 examined S. cerevisiae strains, and has a paralog, YPR071W, arising from a single-locus duplication . Recombinant YIL029C is produced for functional studies, leveraging heterologous expression systems to explore its biochemical properties and interactions .
Gene locus: YIL029C (Chromosome IX, S. cerevisiae S288c).
Protein length: 142 amino acids (UniProt ID: P40538).
Conservation: Present in all S. cerevisiae strains analyzed, classified as a core gene .
Cellular role: Linked to propionic acid resistance, vacuolar acidification, and cell wall β-glucan synthesis .
Phenotypic traits: Deletion confers sensitivity to 4-(N-(S-glutathionylacetyl)amino) phenylarsenoxide (GSAO) .
YIL029C is required for maximal tolerance to weak acid stress (e.g., propionic acid) via the Rim101 pathway. Key findings:
Rim101 dependency: YIL029C deletion strains exhibit cytosolic acidification and impaired vacuolar function under propionic acid stress .
Transcriptional regulation: Co-expressed with KNH1 (cell wall β-1,6-glucan synthesis gene) under Rim101p control .
Genetic interaction with KNH1 suggests a role in cell wall remodeling during stress .
Co-regulated with BAG7 (Rho GTPase-activating protein) and CWP1 (mannoprotein) .
Recombinant YIL029C is produced in E. coli with an N-terminal His tag for purification .
YIL029C participates in a network enriched for vacuolar and ubiquitin-related processes. Key interactors identified via STRING-DB :
| Interactor | Function | Interaction Score |
|---|---|---|
| SSM4 | ERAD-associated E3 ubiquitin ligase; targets misfolded proteins | 0.994 |
| STE24 | CAAX prenyl protease; processes a-factor and regulates membrane proteins | 0.994 |
| KNH1 | Cell wall β-1,6-glucan synthesis; co-regulated under propionic acid | 0.833 |
| RIM101 | pH-responsive transcription factor; activates stress-response genes | 0.443 |
| VNX1 | Vacuolar cation/H⁺ antiporter; regulates ion homeostasis | 0.539 |
Rim101 Pathway Integration
Genetic Interaction with EPL1
Industrial Relevance
KEGG: sce:YIL029C
STRING: 4932.YIL029C
YIL029C is an uncharacterized protein in Saccharomyces cerevisiae (baker's yeast) with a primary amino acid sequence of "MRLIFIAKLQYSFLPFSPFNLLNFDNSISVSWFITYSVIVSIWGFAVIEWGAYRNKINLQLPRCTKIKCSRYNTRIKSPKWFNCKNWMHFFLLYLFLTASNLIVQLAYFSKEMCSQGINVPGTKKPGNRVYLSVIILMGNG" . The protein is classified as a membrane protein based on sequence analysis, suggesting it likely contains transmembrane domains . Despite being uncharacterized, its conservation in the yeast genome indicates functional importance that has been maintained through evolutionary pressure. Structural prediction methods suggest it may have alpha-helical transmembrane regions, but no crystal or NMR structure has been determined yet for this protein.
YIL029C is located on chromosome IX of the Saccharomyces cerevisiae genome, as indicated by the "YIL" prefix in its systematic name, where "Y" stands for yeast, "I" refers to chromosome IX, and "L" indicates it is on the left arm of the chromosome . It has a paralog, YPR071W, located on chromosome XVI, with which it shares limited sequence similarity, suggesting they arose from an ancient duplication event but have subsequently diverged significantly . Comparative genomic analyses reveal that YIL029C/YPR071W pair has a synonymous divergence value of 0.2154, classifying them as a chimeric duplication . These two genes have diverged to such an extent that standard BLAST nucleotide searches fail to detect their homology, indicating substantial sequence evolution since duplication .
Recombinant expression of YIL029C can be achieved using standard yeast expression systems with appropriate tags for detection and purification, such as FLAG, His, or GST tags . For membrane proteins like YIL029C, expression optimization may require testing different promoter strengths, growth temperatures, and induction conditions to prevent protein aggregation and maintain proper folding. Purification typically involves cell lysis under gentle conditions (to preserve membrane protein structure), followed by differential centrifugation to isolate membrane fractions containing the target protein . Subsequent solubilization with appropriate detergents (such as n-dodecyl-β-D-maltoside or digitonin) is critical before affinity chromatography using the protein's tag. Final purification steps may include size exclusion chromatography to ensure homogeneity, with the protein stored in a stabilizing buffer containing 50% glycerol at -20°C for short-term storage or -80°C for long-term storage .
Genetic interaction studies for YIL029C can be approached through systematic screens utilizing the yeast deletion collection combined with YIL029C overexpression or deletion . One effective method is synthetic genetic array (SGA) analysis, where a query strain containing a YIL029C deletion is crossed with the entire yeast deletion collection to identify double mutants with enhanced (synthetic sick/lethal) or suppressed (epistatic) phenotypes. Alternatively, high-throughput suppressor screens can identify genes that, when overexpressed or deleted, rescue phenotypes associated with YIL029C mutation . For targeted studies, researchers can employ the UPR-based genetic analysis method described in the literature, where the unfolded protein response is used as a reporter for genetic interactions . Flow cytometry-based screens can identify changes in cellular phenotypes such as cell size, DNA content, or quiescent fraction formation when YIL029C is deleted in combination with other mutations . Analysis should include careful statistical evaluation to distinguish between random fluctuation and true genetic interactions.
To analyze YIL029C's function in acid stress response, researchers should design experiments that systematically vary both pH and weak acid concentrations while monitoring cellular growth and survival . Growth assays should include multiple time points to capture both immediate and adaptive responses, with measurements taken using standard optical density methods or more precise counting techniques. Serial dilution spot assays on solid media containing different concentrations of propionic acid at controlled pH values provide visual confirmation of growth phenotypes and are particularly useful for comparing wild-type and mutant strains . Intracellular pH measurements using pH-sensitive fluorescent probes can directly assess the protein's role in pH homeostasis under acid stress conditions. Transcriptomic analysis using RNA-seq or microarrays can identify changes in gene expression patterns between wild-type and YIL029C deletion strains exposed to weak acid stress, helping to place YIL029C within specific response pathways . Metabolomic approaches can complement these studies by detecting changes in cellular metabolites, particularly changes in amino acid concentrations that might indicate altered metabolic functioning.
YIL029C has been identified as a component of the extended RIM101 signaling pathway network, which was previously known primarily for its role in alkaline pH response but has now been shown to function in weak acid stress responses as well . Network analysis of genetic interactions places YIL029C within this pathway, with enriched interactions around the RIM101 gene itself, suggesting functional relevance to the pathway's core activities. Transcriptomic data indicates that YIL029C expression is regulated as part of the Rim101p-dependent alterations of the yeast transcriptome following exposure to propionic acid stress at pH 4.0 . Mechanistically, YIL029C appears to be required for proper RIM101 pathway functioning under weak acid stress conditions, likely by affecting either signal transduction or the execution of downstream responses. The protein may participate in counteracting propionic acid-induced cytosolic acidification and maintaining proper vacuolar acidification, which are key aspects of the cellular adaptation to acid stress that involve the RIM101 pathway .
Beyond its involvement in the RIM101 pathway, YIL029C has been implicated in several other genetic networks through systematic screening approaches. Clustering analysis of genes that provide resistance to propionic acid reveals YIL029C associations with genes involved in protein catabolism through the multivesicular body pathway and in the homeostasis of internal pH and vacuolar function . Studies investigating cell wall integrity pathways have identified genetic interactions between YIL029C and genes such as DCW1, HOC1, MNN10, and MNN11, which are involved in cell wall organization and biogenesis . Metabolic profiling of deletion strains indicates that YIL029C affects amino acid homeostasis, suggesting potential interactions with genes involved in amino acid metabolism or transport . The genetic relationship with its paralog YPR071W remains unexplored but could provide insights into functional divergence or complementation between these duplicated genes .
YIL029C and its paralog YPR071W represent an interesting case of gene duplication and divergence in the Saccharomyces cerevisiae genome. With a synonymous divergence value of 0.2154, these genes are classified as having undergone chimeric duplication, indicating substantial sequence evolution since their origination . The sequence divergence between these paralogs is so extensive that they are not detected by standard BLASTN searches, suggesting they have evolved distinct functions over time . Comparative genomic analyses across different yeast species could provide insights into the evolutionary trajectory of YIL029C, but such comprehensive studies are currently lacking in the literature. Analysis using tools like HybridMine has failed to detect the YIL029C/YPR071W pair due to their extensive sequence divergence, highlighting the challenges in tracking evolutionary relationships for rapidly evolving genes . The functional significance of this divergence remains to be determined, but the retention of both genes in the genome suggests they may have undergone subfunctionalization or neofunctionalization to serve distinct cellular roles.
Determining the membrane topology of YIL029C requires a combination of computational prediction and experimental validation approaches. Initially, researchers should use multiple topology prediction algorithms (TMHMM, TopPred, HMMTOP) to generate consensus models of transmembrane domains and orientation . For experimental validation, systematic cysteine scanning mutagenesis coupled with accessibility assays can map exposed regions of the protein. This involves replacing individual amino acids with cysteine residues and then testing their accessibility to membrane-impermeable thiol-reactive reagents. Epitope tagging at different positions followed by protease protection assays can also reveal which portions of the protein are exposed to different cellular compartments. For structural studies, researchers should optimize expression and purification protocols specifically for membrane proteins, possibly using fusion partners that enhance stability . Detergent screening is critical to identify conditions that maintain protein folding and function during purification. For high-resolution structural determination, researchers can pursue X-ray crystallography (requiring well-diffracting crystals), cryo-electron microscopy (suitable for larger membrane protein complexes), or NMR spectroscopy (best for smaller membrane proteins or domains).
Identifying functional domains within YIL029C requires systematic mutagenesis approaches combined with functional assays tied to the protein's known phenotypes. A truncation series should be created to determine which regions of the protein are necessary and sufficient for complementing the acid sensitivity phenotype of YIL029C deletion strains . Site-directed mutagenesis targeting conserved amino acid residues or predicted functional motifs can pinpoint specific residues essential for function. Chimeric constructs swapping regions between YIL029C and its paralog YPR071W can help identify domains responsible for their functional divergence . Domain-specific functions can be assessed using acid stress survival assays, vacuolar pH measurements, and cell wall integrity tests, all of which have been linked to YIL029C function . Yeast two-hybrid or co-immunoprecipitation experiments with systematically truncated versions of the protein can map interaction domains with other proteins in the RIM101 pathway or related cellular processes. Advanced techniques like hydrogen-deuterium exchange mass spectrometry can identify regions with differential solvent accessibility under various conditions, potentially revealing domains that undergo conformational changes during activation.
Investigating protein complex formation by YIL029C under stress conditions requires approaches that can detect dynamic, condition-specific interactions. Researchers should first generate epitope-tagged versions of YIL029C (e.g., FLAG-tagged) that maintain functionality and can be used for native immunoprecipitation experiments under various stress conditions . Comparing the interactome of YIL029C under normal growth versus weak acid stress can reveal stress-specific interaction partners. Proximity-dependent biotin labeling methods (BioID or TurboID) can capture transient or weak interactions in living cells by tagging proteins that come into close proximity with YIL029C during stress responses. Cross-linking mass spectrometry (XL-MS) can provide structural information about the architecture of protein complexes involving YIL029C, with the cross-linking performed under specific stress conditions to capture stress-relevant interactions. Blue native PAGE or size exclusion chromatography coupled with multi-angle light scattering can identify the native complex size and potential oligomeric states of YIL029C under different conditions. For targeted analysis, split-reporter systems (like split-GFP) can verify specific predicted interactions and determine their subcellular localization during stress responses.
Phenotypic screening data for YIL029C should be organized in structured data tables with clear identification of independent and dependent variables410. For acid stress experiments, independent variables (acid concentration, pH) should be arranged in columns or rows with gradient formatting to visualize concentration effects, while dependent variables (growth rate, survival percentage) should include both mean values and measures of variation (standard deviation or standard error)10. Experimental data should always include appropriate controls (wild-type strain, empty vector control) and statistical analyses to determine significance of observed differences. A comprehensive data table example is shown below:
| Treatment | pH | Wild-type Survival (%) | YIL029C Deletion Survival (%) | p-value |
|---|---|---|---|---|
| Control | 6.0 | 100.0 ± 2.3 | 98.5 ± 3.1 | 0.542 |
| Propionic acid 2 mM | 4.0 | 89.3 ± 4.2 | 62.1 ± 5.7 | 0.003 |
| Propionic acid 5 mM | 4.0 | 75.6 ± 3.8 | 41.2 ± 4.3 | <0.001 |
| Propionic acid 10 mM | 4.0 | 58.9 ± 5.1 | 22.7 ± 3.9 | <0.001 |
| HCl to pH 4.0 | 4.0 | 96.2 ± 3.7 | 94.8 ± 4.2 | 0.720 |
| HCl to pH 3.0 | 3.0 | 92.3 ± 4.5 | 90.5 ± 5.1 | 0.675 |
Analysis should include both univariate statistics for individual conditions and multivariate approaches to identify patterns across multiple experimental variables . For large-scale phenotypic screens, clustering analyses can group genes with similar phenotypic profiles to YIL029C, potentially revealing functional relationships.
Transcriptomic analysis of YIL029C requires careful experimental design and robust statistical approaches to identify genes that are differentially expressed between wild-type and YIL029C deletion strains under various conditions. Experimental design should include multiple biological replicates (minimum three) and appropriate controls for batch effects that might influence gene expression measurements . Normalization methods should be selected based on the specific platform used (RNA-seq or microarray), with appropriate corrections for library size, gene length, and other technical factors. Differential expression analysis should employ statistical methods that account for the multiple testing problem, such as the Benjamini-Hochberg procedure to control false discovery rate . Gene set enrichment analysis (GSEA) or Gene Ontology (GO) term enrichment can identify biological processes, molecular functions, or cellular components that are overrepresented among differentially expressed genes. Network analysis approaches can place YIL029C within broader regulatory networks by identifying co-regulated genes and potential upstream regulators. Comparison with existing datasets, such as those examining the RIM101 pathway or acid stress responses, can contextualize YIL029C-specific transcriptional changes within broader cellular response programs .
The most promising approaches for definitively characterizing YIL029C function combine classical genetic methods with cutting-edge technologies to build a comprehensive understanding of this protein's role. CRISPR-based approaches offer precise genome editing capabilities to introduce targeted mutations or regulatory elements at the endogenous YIL029C locus, allowing study of the protein under physiological expression levels. Single-cell technologies can reveal cell-to-cell variability in YIL029C expression and function, potentially uncovering condition-specific roles that might be masked in population-level studies . Synthetic biology approaches, such as rewiring regulatory networks or designing minimal yeast genomes, can test the essentiality of YIL029C under defined conditions and reveal emergent properties. Evolutionary approaches comparing YIL029C function across different yeast species could reveal selective pressures and functional constraints that have shaped this protein over time. Systems biology integration of multiple data types (genomic, transcriptomic, proteomic, metabolomic, and phenomic) can place YIL029C within a comprehensive cellular network context, revealing its position in cellular response hierarchies . Structural biology coupled with molecular dynamics simulations can provide insights into the molecular mechanisms through which YIL029C functions, particularly if it proves to have enzymatic activity or undergoes conformational changes during stress responses.
The study of YIL029C in yeast stress responses offers insights into fundamental mechanisms that may be conserved across eukaryotes. By elucidating the role of YIL029C in the RIM101 pathway's response to weak acid stress, researchers can identify general principles of how cells sense and respond to environmental pH changes, which has relevance to many biological systems from fungi to higher eukaryotes . The apparent role of YIL029C in both weak acid stress response and cell wall integrity points to interconnected stress response networks that may reveal how cells prioritize and coordinate responses to multiple simultaneous stressors. The evolutionary divergence between YIL029C and its paralog YPR071W provides a model system for studying how duplicated genes evolve new functions while potentially retaining aspects of their original role, a process central to the expansion of eukaryotic stress response networks . Mechanistic studies of how YIL029C affects cellular processes like vacuolar acidification and cytosolic pH homeostasis may reveal conserved cellular strategies for maintaining pH gradients across membranes, a fundamental aspect of eukaryotic cell biology . Understanding the transcriptional and metabolic changes associated with YIL029C function can illuminate general principles of how stress responses are integrated with cellular metabolism, a relationship that is critical for cellular survival and adaptation in all eukaryotes.
Future research on YIL029C would benefit substantially from several technological advances that could overcome current limitations in studying uncharacterized membrane proteins. Improved cryo-electron microscopy techniques with enhanced resolution for smaller membrane proteins would enable structural determination of proteins like YIL029C without requiring crystallization, potentially revealing functional domains and interaction interfaces . Advanced protein-protein interaction detection methods with increased sensitivity for membrane proteins and transient interactions would help identify the protein complexes in which YIL029C participates under various conditions. Single-molecule imaging technologies capable of tracking protein dynamics in living cells would allow researchers to observe YIL029C localization and movement during stress responses in real time. Metabolic labeling techniques with higher temporal resolution could capture rapid changes in protein synthesis, degradation, and modification following stress exposure, potentially revealing regulatory mechanisms controlling YIL029C function. Massively parallel reporter assays could systematically test the effect of thousands of YIL029C variants on function, identifying critical residues and domains. Improved computational methods for predicting protein function from sequence alone would benefit not only YIL029C research but also the study of the approximately 20% of yeast genes that remain uncharacterized. Advances in synthetic genetic interaction mapping at higher throughput would enable more comprehensive placement of YIL029C within cellular genetic networks.