Recombinant Saccharomyces cerevisiae Phosphatidylinositol N-acetylglucosaminyltransferase ERI1 subunit (ERI1)

What is the basic structure and genomic context of Saccharomyces cerevisiae ERI1?

ERI1 (Endoplasmic reticulum-associated Ras Inhibitor 1) is encoded by a previously nonannotated ORF (YPL096C-A) in the S. cerevisiae genome. It is a small protein consisting of only 68 amino acids with the sequence: MRPRDQGFLVLGFTYSVLLISLATFYWLRNNDSFLHYWCVLLLCPATLWLWALIAWCDSEMFASSKDE . The ERI1 gene is located in the 498-bp intergenic region between YPL096w and YPL097w (MSY1) . Expression analysis using SAGE-tag identification has confirmed that a polyadenylated mRNA of approximately 300 nucleotides is expressed from this locus at relatively low levels in both log-phase and stationary-phase cells, confirming ERI1 as a bona fide gene despite its small size . The gene was relatively recently added to the Saccharomyces Genome Database, highlighting ongoing refinements in our understanding of the yeast genome .

What are the primary cellular functions attributed to ERI1 in yeast?

ERI1 functions primarily as an inhibitor of Ras signaling in S. cerevisiae. The protein associates in vivo with GTP-bound Ras in a manner that requires an intact Ras-effector loop, suggesting that ERI1 competes for the same binding site as Ras target proteins . This competition likely explains its inhibitory effect on Ras signaling pathways. ERI1 localizes predominantly to the membrane of the endoplasmic reticulum (ER), where it engages with Ras proteins . This localization is particularly significant because mammalian Ras signaling is not restricted to the cell surface but can also proceed from the cytoplasmic face of the ER, suggesting ERI1 may have an important regulatory function at that membrane . Phenotypically, deletion of ERI1 results in a growth defect at elevated temperatures (37°C), indicating its role in stress response mechanisms .

How does ERI1 differ functionally across species?

While the Saccharomyces cerevisiae ERI1 functions primarily as a Ras signaling inhibitor, homologs in other organisms often demonstrate different functions. For instance, in mice and humans, ERI1 acts as a 3′-to-5′ exoribonuclease that associates with ribosomes and ribosomal RNA (rRNA) . This exoribonuclease activity is essential for processing the 3′ end of 5.8S rRNA, as evidenced by the presence of aberrantly extended 5.8S rRNA in Eri1-deficient mice .

The functional divergence between yeast and mammalian ERI1 proteins is reflected in their subcellular localization patterns. While yeast ERI1 localizes primarily to the ER membrane, the mouse and human ERI1 proteins localize to both the cytoplasm and nucleus, with particular enrichment in the nucleolus, which is the site of preribosome biogenesis . Homologs of ERI1 have been identified in various fungi, including Candida albicans, Schizosaccharomyces pombe, Aspergillus fumigatus, and Neurospora crassa, but database searches have not revealed any metazoan homologs of the yeast ERI1 protein .

What are the recommended protocols for generating recombinant ERI1 for research applications?

To generate recombinant S. cerevisiae ERI1 for research applications, researchers typically express the protein with appropriate tags to facilitate purification and detection. Based on established methodologies, the following protocol is recommended:

• Cloning Strategy: The 204-bp ERI1 open reading frame should be amplified by PCR from S. cerevisiae genomic DNA (strain ATCC 204508/S288c recommended). For optimal expression, include appropriate restriction sites in the primers for subsequent cloning into an expression vector .

• Vector Selection: For detection purposes, construct an epitope-tagged version (typically HA-tagged) under an appropriate promoter. Options include:

  • The native ERI1 promoter (using ~680 bp of sequence 5′ to the ERI1 start codon)

  • The inducible GAL1 promoter for controlled expression

  • The constitutive MET25 promoter for stable expression

• Expression and Purification: Express the recombinant protein in an appropriate host system. For storage, maintain the purified protein in a Tris-based buffer with 50% glycerol at -20°C or -80°C for extended storage. Avoid repeated freeze-thaw cycles and prepare working aliquots for storage at 4°C for up to one week .

• Validation: Confirm protein identity by Western blotting and mass spectrometry. Functional validation can be performed through complementation assays in eri1Δ yeast strains, which should rescue the temperature-sensitive growth defect at 37°C .

What methods are most effective for studying ERI1's interactions with the Ras signaling pathway?

To investigate ERI1's interactions with the Ras signaling pathway, several complementary approaches have proven effective:

• Genetic Interaction Assays:

  • Create double mutants between eri1Δ and mutations in Ras pathway components (such as RAS2, IRA1, or IRA2)

  • Assess synthetic phenotypes, particularly under stress conditions such as heat shock

  • Quantify cell viability after heat shock by calculating colony-forming units (CFU) relative to non-heat-shocked controls

• Reporter Gene Assays:

  • Utilize Ras-responsive reporter constructs such as FRE::lacZ

  • Measure how ERI1 overexpression affects transcriptional activity driven by constitutively active Ras (Ras2-V19)

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation of epitope-tagged ERI1 with Ras proteins

  • Special attention should be paid to interactions with GTP-bound Ras forms

  • Mutational analysis of the Ras-effector loop to determine specificity of interaction

• Subcellular Localization:

  • Fluorescence microscopy using tagged ERI1 to confirm ER localization

  • Co-localization studies with Ras proteins and ER markers

These methods collectively provide a comprehensive approach to understanding ERI1's function as a Ras pathway inhibitor, particularly its mechanism of competing with Ras effectors at the ER membrane.

What experimental design considerations are important when investigating ERI1 deletion phenotypes?

When investigating phenotypes associated with ERI1 deletion, several methodological considerations are critical:

• Strain Background Effects:

  • The genetic background significantly influences ERI1 deletion phenotypes

  • Neonatal lethality in mice with ERI1 deficiency is partially rescued by outbred strain backgrounds

  • For yeast studies, phenotypes should be assessed in multiple strain backgrounds to ensure reproducibility

• Temperature Sensitivity Assessment:

  • Growth assays should be performed at both permissive (30°C) and restrictive (37°C) temperatures

  • Quantitative growth measurements are preferred over qualitative assessments

  • Serial dilution spot assays provide a visual representation of growth defects

• Cellular Growth Analysis:

  • For yeast, measure growth rates in liquid culture using spectrophotometric methods

  • For mammalian cells, automated microscopy to measure doubling times provides objective quantification

  • ERI1-deficient mouse embryonic fibroblasts show significantly slower growth (p=0.006) compared to wild-type cells

• Rescue Experiments:

  • Complementation with wild-type ERI1 should rescue the deletion phenotype

  • Include controls with catalytically inactive mutants (for exonuclease studies) or localization-defective variants

  • Expression levels should be controlled using appropriate promoters (native, constitutive, or inducible)

• Molecular Phenotype Analysis:

  • In addition to growth phenotypes, investigate molecular consequences of ERI1 deletion

  • For rRNA processing studies, analyze the 3′ end of 5.8S rRNA for extensions

  • For Ras signaling studies, examine downstream pathway activation

How can researchers effectively study ERI1's role in ribosomal RNA processing?

To investigate ERI1's role in ribosomal RNA processing, researchers should implement a multi-faceted approach:

• Ribosome Association Analysis:

  • Isolate ribosomes through sucrose gradient centrifugation

  • Detect ERI1 in ribosomal fractions via Western blotting

  • Analyze ribosomal RNA content to identify processing defects

• In Vitro Processing Assays:

  • Purify ribosomes from ERI1-deficient cells

  • Incubate with recombinant wild-type ERI1 or catalytically inactive mutants

  • Monitor 5.8S rRNA processing via high-resolution gel electrophoresis

  • Compare processing efficiency between intact ribosomes and reconstituted 5.8S-28S rRNA duplexes

• RNA-Protein Interaction Studies:

  • Evaluate the contribution of ERI1's SAP and linker domains to stable rRNA association

  • Mutate key RNA-binding residues and assess impact on processing activity

  • Quantify binding affinities for different rRNA substrates

• Substrate Specificity Determination:

  • Design synthetic RNA substrates mimicking the 3′-extended 5.8S rRNA

  • Test processing efficiency with varying substrate concentrations

  • Note that the 5.8S-28S rRNA duplex requires approximately 10-fold higher ERI1 concentration compared to intact ribosomes, suggesting contribution of other ribosomal elements to specificity

• Subcellular Localization Studies:

  • Track ERI1 localization in relation to ribosome biogenesis

  • Focus on nucleolar enrichment in mammalian cells

  • Correlate localization patterns with processing activity

What approaches can be used to investigate ERI1's potential role in viral infections?

ERI1 has been implicated in viral infection processes, particularly with Influenza A virus. To investigate this connection, researchers should consider:

• Viral Ribonucleoprotein (vRNP) Association Studies:

  • Isolate vRNPs from infected cells

  • Detect co-purification of ERI1 with vRNPs via immunoblotting

  • Analyze association with viral RNA using RNA immunoprecipitation

• Histone mRNA Processing Complex Analysis:

  • Investigate the relationship between ERI1, SLBP protein, and histone mRNAs during infection

  • Determine if viral factors recruit ERI1 when it is present in the histone pre-mRNA processing complex

• Nuclear-Cytoplasmic Trafficking:

  • Track changes in ERI1 localization during different stages of viral infection

  • Examine potential recruitment mechanisms to nuclear compartments

• Viral Replication Impact Assessment:

  • Generate ERI1 knockdown or knockout cell lines

  • Infect with influenza A virus and quantify viral replication efficiency

  • Measure viral RNA and protein synthesis in the presence/absence of ERI1

• Mechanistic Studies:

  • Determine whether ERI1's exonuclease activity is relevant to its role in viral infection

  • Test catalytically inactive ERI1 mutants for their ability to associate with vRNPs

How does ERI1 impact cellular growth regulation, and what methods best capture these effects?

ERI1's impact on cellular growth can be investigated through several complementary approaches:

• Growth Curve Analysis:

  • For yeast: Compare growth rates of wild-type and eri1Δ strains in liquid culture

  • For mammalian cells: Use automated microscopy to measure doubling times of ERI1-deficient vs. wild-type cells

  • ERI1-deficient mouse embryonic fibroblasts show significantly slower growth (p=0.006)

• Body Size Phenotyping in Animal Models:

  • ERI1-deficient mice exhibit reduced body size observable as early as embryonic day 15.5

  • The phenotype persists in surviving adult mice

  • Quantitative measurements should include weight and length parameters

• Stress Response Assessment:

  • Heat shock survival: Measure percentage of survivors after heat shock treatment

  • In yeast, test growth at elevated temperatures (37°C)

  • Calculate colony-forming units relative to non-stressed controls

• Viability Studies:

  • Neonatal lethality in ERI1-deficient mice suggests essential developmental functions

  • Genetic background influences survival rates (partial rescue observed in outbred strains)

  • Careful breeding strategies and viability tracking are essential

• Molecular Mechanism Investigation:

  • For yeast ERI1: Focus on Ras signaling inhibition at the ER membrane

  • For mammalian ERI1: Examine ribosome biogenesis and 5.8S rRNA processing

  • The connection between these molecular functions and growth phenotypes requires investigation of downstream effectors

What are the key differences between yeast ERI1 and its homologs in other species?

The ERI1 protein family exhibits significant functional divergence across species, with important implications for evolutionary biology and comparative research:

The most striking difference is the apparent functional shift from a Ras signaling regulator in yeast to an RNA processing enzyme in metazoans. This functional divergence is reflected in different domain architectures, with metazoan ERI1 proteins containing RNA-binding SAP domains and exonuclease domains not found in the yeast protein .

Despite these differences, some functional conservation exists in the role of ERI1 in growth regulation across species, with deficiencies resulting in growth phenotypes in both yeast and mice .

How can researchers use cross-species complementation to understand ERI1 function?

Cross-species complementation represents a powerful approach to dissect the functional conservation and divergence of ERI1:

• Experimental Design Framework:

  • Express mammalian ERI1 in eri1Δ yeast and assess rescue of temperature sensitivity

  • Express yeast ERI1 in Eri1-deficient mammalian cells and assess rescue of rRNA processing

  • Create chimeric proteins with domains from different species to map functional regions

• Domain Swap Experiments:

  • Create fusion proteins containing the yeast ERI1 membrane localization regions with mammalian ERI1 catalytic domains

  • Test functionality in both yeast and mammalian systems

  • Map minimal functional units required for different activities

• Evolution-Guided Mutagenesis:

  • Identify conserved residues across species

  • Generate point mutations at these positions

  • Assess impact on respective functions (Ras inhibition vs. rRNA processing)

• Subcellular Targeting Studies:

  • Redirect yeast ERI1 to the nucleolus using targeting sequences

  • Target mammalian ERI1 to the ER membrane

  • Determine if localization dictates function

• Pathway Integration Analysis:

  • Examine if mammalian ERI1 can interact with yeast Ras proteins

  • Test if yeast ERI1 affects mammalian RNA processing

  • Identify shared binding partners across species

These approaches can help uncover the evolutionary path by which ERI1 diversified from a Ras regulator in lower eukaryotes to an RNA processing enzyme in higher eukaryotes, while potentially maintaining some overlapping functions.

What are the current hypotheses regarding the evolution of ERI1's diverse functions?

Several hypotheses have emerged to explain the functional divergence of ERI1 across evolution:

• Functional Repurposing Hypothesis:

  • The small yeast ERI1 protein may have been incorporated into larger protein architectures in metazoans

  • Additional domains (SAP, exonuclease) were acquired that conferred new RNA processing functions

  • The original Ras regulatory function was either lost or became secondary in higher eukaryotes

• Dual Function Conservation Hypothesis:

  • Both Ras signaling and RNA processing functions might coexist in some species

  • Different cellular compartments (ER membrane vs. nucleolus) could host the different activities

  • Regulatory mechanisms might determine which function predominates in different contexts

• Convergent Evolution Hypothesis:

  • Despite the shared name, yeast and metazoan ERI1 proteins might represent convergent evolution

  • The lack of detectable sequence homology between yeast ERI1 and metazoan Eri1 supports this idea

  • Functional studies are needed to definitively resolve this question

• Connector Hypothesis:

  • ERI1 might represent an evolutionary link between fundamental cellular processes

  • The connection between Ras signaling (growth control) and ribosome biogenesis (translation machinery) suggests a potential regulatory nexus

  • This is supported by the fact that ERI1 deficiency causes growth defects across species

Research addressing these hypotheses would benefit from comprehensive phylogenetic analysis of ERI1-like proteins across diverse taxa, coupled with structural studies to identify potentially conserved structural motifs despite sequence divergence.

What are the most promising unexplored aspects of ERI1 biology for future research?

Several underexplored areas of ERI1 biology present significant opportunities for future research:

• Potential Role in Disease Processes:

  • Investigation of ERI1's association with Ras-driven cancers, given its role as a Ras inhibitor in yeast

  • Exploration of connections between ribosome biogenesis defects and disease phenotypes in ERI1-deficient models

  • Examination of ERI1's role in viral pathogenesis beyond influenza A

• Systems Biology Integration:

  • Comprehensive mapping of ERI1 interaction networks across species

  • Integration of transcriptomic, proteomic, and metabolomic data from ERI1-deficient models

  • Mathematical modeling of ERI1's impact on cellular growth regulation

• Structural Biology:

  • Determination of the three-dimensional structure of S. cerevisiae ERI1

  • Comparative structural analysis with mammalian ERI1

  • Structure-guided design of modulators for functional studies

• Evolutionary Biology:

  • Detailed phylogenetic analysis of ERI1-like proteins across diverse taxa

  • Reconstruction of the evolutionary history of ERI1 functional divergence

  • Identification of potential selective pressures driving functional specialization

• Translational Applications:

  • Exploration of ERI1 as a potential target for antiviral therapies

  • Investigation of ERI1 modulation as an approach to regulate Ras signaling in cancer

  • Development of ERI1-based tools for manipulating ribosome biogenesis

What advanced methodologies would benefit ERI1 research but remain underutilized?

Several cutting-edge methodologies could significantly advance ERI1 research:

• Cryo-Electron Microscopy:

  • Visualization of ERI1 in complex with ribosomes or Ras proteins

  • Structural determination at near-atomic resolution

  • Mapping of conformational changes during substrate engagement

• CRISPR-Based Genetic Screens:

  • Genome-wide identification of genetic interactions with ERI1

  • Creation of sophisticated conditional knockout models

  • Generation of precise point mutations to dissect domain functions

• Single-Molecule Techniques:

  • Real-time visualization of ERI1's exonuclease activity

  • Analysis of kinetics and processivity on different RNA substrates

  • Direct observation of ERI1-Ras interactions

• Proteomics Approaches:

  • Proximity labeling to identify the complete ERI1 interactome

  • Quantitative phosphoproteomics to map signaling changes in ERI1-deficient cells

  • Cross-linking mass spectrometry to identify direct binding partners

• Advanced Imaging Methods:

  • Super-resolution microscopy to visualize ERI1 localization with nanometer precision

  • Live-cell imaging to track ERI1 dynamics during cellular processes

  • Correlative light and electron microscopy to connect function with ultrastructure

How might the study of ERI1 contribute to broader understanding of cellular regulatory networks?

ERI1 research has the potential to illuminate fundamental aspects of cellular regulation:

• Integration of Growth Signaling and Protein Synthesis:

  • ERI1's dual roles in Ras signaling and ribosome biogenesis suggest coordination between growth signals and protein synthesis machinery

  • This connection could reveal new regulatory principles governing cellular growth and proliferation

• Evolutionary Plasticity of Regulatory Proteins:

  • The functional divergence of ERI1 across species provides a model for studying how proteins acquire new functions during evolution

  • Understanding this process could shed light on the emergence of complex regulatory networks

• Subcellular Compartmentalization of Signaling:

  • ERI1's localization to specific cellular compartments (ER membrane, nucleolus) highlights the importance of spatial regulation

  • This aspect of ERI1 biology could inform broader principles of compartmentalized signaling

• Host-Pathogen Interactions:

  • ERI1's association with viral ribonucleoproteins suggests potential roles in host defense or viral subversion

  • Further investigation could reveal novel aspects of the cellular response to infection

• Systems-Level Growth Regulation:

  • The consistent growth phenotypes associated with ERI1 deficiency across species point to fundamental roles in cellular homeostasis

  • Understanding how ERI1 contributes to growth regulation could provide insights into developmental disorders and diseases of cellular proliferation

By addressing these broader questions, ERI1 research transcends the study of a single protein and contributes to our understanding of complex cellular regulatory networks and their evolution.