Recombinant Saccharomyces cerevisiae Putative uncharacterized protein YBR126W-A (YBR126W-A)

What is YBR126W-A (Meo1) and why was it previously uncharacterized?

YBR126W-A, now named Meo1 (Mini ER ORF1), belongs to the category of small Open Reading Frame (smORF) proteins in Saccharomyces cerevisiae, containing fewer than 110 amino acids. These smORFs were initially omitted from the first yeast genome annotation efforts and were only added in later annotation versions, explaining their historically uncharacterized status . The systematic visualization of this protein was achieved through the SWAT-GFP library approach, which enabled researchers to assign localization to numerous previously unvisualized proteins, including 80% of the smORFs in the library . The protein's small size made it technically challenging to detect and characterize using traditional methods, contributing to its previous obscurity in the research literature.

What is the subcellular localization of YBR126W-A (Meo1) and how was this determined?

YBR126W-A (Meo1) has been definitively localized to the endoplasmic reticulum (ER) using multiple complementary approaches. The primary localization was determined using the SWAT-GFP library, where the protein was tagged at its N-terminus with GFP . This localization was subsequently verified through subcellular fractionation experiments using C-terminal epitope tagging (HA or ProtA tags) to ensure that the observed localization was not an artifact of the N-terminal GFP tag . The protein was among 36 previously uncharacterized smORFs found to localize to the ER using this systematic approach, leading to its designation as Meo1 (Mini ER ORF1) .

How does the amino acid sequence of YBR126W-A (Meo1) provide clues about its potential function?

The amino acid sequence analysis of YBR126W-A (Meo1) requires careful interpretation due to its small size (under 110 amino acids). Researchers should conduct the following sequence-based analyses:

• Motif identification for ER retention/retrieval signals (such as KDEL-like sequences)

• Transmembrane domain prediction to determine membrane topology

• Signal peptide analysis to assess secretory pathway targeting

• Conservation analysis across fungal species to identify functionally important residues

• Structural prediction to identify potential interaction domains

The protein's ER localization suggests it may function in processes such as protein folding, quality control, lipid metabolism, or ER stress responses. The presence of any conserved domains shared with other ER proteins would provide valuable clues regarding its specific functional role within the ER network.

How is YBR126W-A (Meo1) expression regulated in different growth conditions?

Research into YBR126W-A (Meo1) expression patterns reveals condition-dependent regulation. The SWAT system studies demonstrated that proteins placed under control of their native promoters versus a constitutive promoter (SpNOP1) showed dramatically different expression levels spanning two orders of magnitude . When investigating YBR126W-A expression specifically, researchers should:

• Utilize quantitative PCR to measure transcript levels across various growth conditions

• Employ reporter constructs with the native promoter to identify regulatory elements

• Analyze the promoter region for transcription factor binding sites

• Compare expression patterns with other ER-localized smORFs to identify co-regulated genes

• Investigate expression changes under ER stress conditions (tunicamycin, DTT treatment)

The high-throughput data from the SWAT-GFP library suggests that some proteins may only be expressed under specific conditions, making comprehensive condition testing essential for understanding YBR126W-A regulation .

How can researchers effectively identify interaction partners of YBR126W-A (Meo1)?

Identifying interaction partners for small proteins like YBR126W-A (Meo1) presents unique challenges that require specialized approaches. Based on its ER localization, researchers should consider the following strategies:

• Proximity-dependent labeling approaches:

  • BioID or TurboID fusions to label proximal proteins in the ER environment

  • APEX2 tagging for electron microscopy visualization of the precise ER subdomain

• Co-immunoprecipitation strategies:

  • Gentle solubilization conditions optimized for ER membrane proteins

  • Crosslinking prior to lysis to capture transient interactions

  • Sequential co-IP from differentially tagged strains to verify interactions

• Genetic interaction mapping:

  • Synthetic genetic array (SGA) analysis with YBR126W-A deletion

  • Quantitative analysis of genetic interactions under ER stress conditions

  • Suppressor screens in YBR126W-A mutant backgrounds

• Split-reporter systems:

  • Yeast two-hybrid adaptations for membrane proteins

  • Split-ubiquitin assays specifically designed for ER-localized proteins

  • Bimolecular fluorescence complementation (BiFC) with candidate partners

The SWAT library system offers a powerful platform for these interaction studies, as demonstrated by the co-immunoprecipitation experiments that successfully identified interactions between other tagged proteins in the endomembrane system . The co-localization approach used to identify peroxisomal proteins could be adapted to systematically screen for proteins that precisely co-localize with YBR126W-A within ER subdomains .

How can the SWAT library system be optimized for further studies of YBR126W-A (Meo1)?

The SWAp-Tag (SWAT) library system provides a powerful platform for studying YBR126W-A (Meo1) through multiple tag swapping approaches. The system's versatility allows researchers to efficiently create numerous derivatives using a single parental strain . For optimizing this system specifically for YBR126W-A studies:

• Tag selection optimization:

  • Swap the original GFP tag with specialized tags for different applications

  • Implement proximity labeling tags (BioID, TurboID) for interaction studies

  • Utilize split tags for complementation assays

  • Incorporate degron tags for controlled protein depletion studies

• Promoter swapping strategies:

  • Replace the constitutive SpNOP1 promoter with condition-specific promoters

  • Implement titratable promoter systems for dose-response studies

  • Create a series of truncated native promoters to map regulatory elements

  • Develop reporters with the native promoter for regulation studies

• Genetic background modifications:

  • Cross SWAT strains into deletion collection backgrounds

  • Implement the system in different yeast strain backgrounds

  • Combine with genome-wide CRISPRi libraries for genetic interaction studies

  • Create double-tagged strains for co-localization studies

• Technical optimization for small proteins:

  • Optimize linker length and composition for minimal functional interference

  • Develop specialized immunoprecipitation protocols for small membrane proteins

  • Fine-tune expression levels to prevent aggregation or mislocalization

  • Implement sample preparation techniques optimized for small protein detection

The SWAT system is particularly valuable because the homologous recombination efficiency is extremely high (>98% recovery rate with 96% accuracy), enabling rapid creation of strain collections with different tags and regulatory elements . The proven ability to swap tags seamlessly while preserving native regulation makes this an ideal platform for YBR126W-A functional studies.

What approaches are most effective for detecting and quantifying native levels of YBR126W-A (Meo1)?

Detecting and quantifying native levels of small proteins like YBR126W-A (Meo1) presents technical challenges that require specialized approaches. Based on the challenges observed in systematic studies, researchers should consider:

• Transcriptional quantification methods:

  • RNA-seq with optimized library preparation for small transcripts

  • qRT-PCR with primers specifically validated for small ORFs

  • Single-molecule FISH for transcript visualization and quantification

  • Nascent transcript analysis to measure transcription rates

• Protein-level detection methods:

  • Targeted proteomics (SRM/MRM) optimized for small proteins

  • Custom antibody development against unique epitopes

  • Epitope tagging at endogenous loci with verification of functionality

  • Specialized extraction protocols to preserve small membrane proteins

• Translational activity measurement:

  • Ribosome profiling with optimized footprint collection

  • Polysome profiling to assess translation efficiency

  • Pulse labeling with amino acid analogs for synthesis rate measurement

  • In vitro translation assays to confirm coding potential

• Single-cell analysis approaches:

  • Flow cytometry of tagged variants for population heterogeneity assessment

  • Single-cell RNA-seq for expression heterogeneity analysis

  • Live-cell imaging of tagged variants for dynamic expression studies

  • Correlative light and electron microscopy for precise localization

The high-content microscopy approach used in the SWAT library studies provides a valuable model, demonstrating that even with constitutive expression, proteins maintain unique abundance levels spanning two orders of magnitude . This suggests that post-transcriptional mechanisms play a critical role in regulating YBR126W-A levels and should be a focus of quantification studies.

What statistical approaches are most appropriate for analyzing YBR126W-A (Meo1) expression data across different conditions?

Analyzing expression data for YBR126W-A (Meo1) requires statistical approaches tailored to the challenges of small proteins and condition-specific expression patterns. Based on the observations from the SWAT library studies, researchers should consider:

• Normalization strategies:

  • Size-factor normalization to account for library size differences

  • Spike-in controls for absolute quantification

  • Specialized normalization for small proteins often missed in global methods

  • Considering both fluorescence intensity and cell-to-cell variability metrics

• Differential expression analysis:

  • Employ methods robust to low count numbers (e.g., DESeq2, edgeR)

  • Account for technical variation specific to small transcripts/proteins

  • Implement statistical tests appropriate for potentially non-normal distributions

  • Consider specialized methods for detecting condition-specific expression

• Variance analysis:

  • Quantify both technical and biological sources of variance

  • Apply generalized linear mixed models to account for batch effects

  • Implement variance stabilizing transformations appropriate for low-abundance transcripts

  • Consider Bayesian approaches for improved estimation of small effect sizes

• Multivariate approaches:

  • Cluster analysis to identify co-regulated genes

  • Principal component analysis to identify major sources of variation

  • Time-series analysis for temporal expression patterns

  • Network analysis to place expression in pathway context

The SWAT library studies demonstrated that even with standardized expression from a constitutive promoter, protein abundance varied significantly between different proteins, spanning two orders of magnitude . This suggests post-transcriptional and post-translational regulation significantly impact YBR126W-A levels and should be accounted for in statistical analyses.

How can researchers determine if YBR126W-A (Meo1) functions in specialized ER subdomains?

Investigating YBR126W-A (Meo1) localization to specific ER subdomains requires specialized approaches that go beyond standard fluorescence microscopy. Given its confirmed ER localization, researchers should implement the following strategies:

• Super-resolution microscopy approaches:

  • Structured illumination microscopy (SIM) to resolve ER tubule structures

  • Stimulated emission depletion (STED) microscopy for nanoscale resolution

  • Single-molecule localization microscopy (PALM/STORM) for precise protein clustering analysis

  • Expansion microscopy to physically magnify subcellular structures

• ER subdomain markers co-localization:

  • Co-localization with ER-mitochondria encounter structure (ERMES) components

  • Association with ER-plasma membrane contact sites (cortical ER)

  • Co-distribution with specialized domains like ER exit sites or lipid droplet formation sites

  • Analysis with ER stress granule markers under stress conditions

• Biochemical fractionation approaches:

  • Density gradient separation of ER subdomains

  • Detergent resistance membrane fractionation

  • Immunoisolation of specialized ER regions

  • Protease protection assays to determine membrane topology

• Dynamic analysis methods:

  • Fluorescence recovery after photobleaching (FRAP) to measure mobility within the ER

  • Single-particle tracking to analyze diffusion patterns and constraints

  • Optogenetic approaches to induce relocalization and assess functional consequences

  • Correlative light and electron microscopy for ultrastructural context

The SWAT-GFP approach used to identify YBR126W-A's ER localization provides a foundation for these more detailed studies . The systematic co-localization approach used for peroxisomal proteins could be adapted to systematically test co-localization with markers of different ER subdomains, potentially revealing more specific localization patterns .

What evolutionary insights can be gained from studying YBR126W-A (Meo1) orthologs across fungal species?

Evolutionary analysis of YBR126W-A (Meo1) across fungal species can provide valuable insights into its function and importance. Given its classification as a small ORF protein that was only later annotated in the yeast genome, investigating its evolutionary conservation is particularly important .

Key evolutionary analyses should include:

• Ortholog identification approaches:

  • Sensitive sequence similarity searches optimized for small proteins

  • Synteny analysis to identify positionally conserved genes

  • Profile-based searches using position-specific scoring matrices

  • Structure-based homology detection for distant relationships

• Evolutionary rate analyses:

  • Calculation of dN/dS ratios to assess selection pressure

  • Identification of conserved residues suggesting functional importance

  • Analysis of insertion/deletion patterns across species

  • Relative evolutionary rate comparison with other ER proteins

• Comparative genomics approaches:

  • Presence/absence patterns across fungal phylogeny

  • Co-evolution with interacting partners

  • Association with specific metabolic or stress response pathways

  • Correlation with lifestyle or environmental adaptations

• Functional evolution assessment:

  • Expression pattern conservation across species

  • Localization conservation in model organisms

  • Complementation assays with orthologs from other species

  • Analysis of species-specific adaptations in sequence or regulation

The identification of YBR126W-A as part of a previously understudied group of smORFs suggests that similar proteins may exist across fungal species but might have been overlooked in genome annotations . Systematic analysis of these evolutionarily related proteins could reveal conserved functions and establish YBR126W-A as part of an ancient functional group of small ER proteins.