Recombinant Saccharomyces cerevisiae Inactive deaminase YBR284W (YBR284W)

What is YBR284W and what is its known functional status in Saccharomyces cerevisiae?

YBR284W is classified as a putative metallo-dependent hydrolase superfamily protein in Saccharomyces cerevisiae. Despite its structural similarity to AMP deaminases, it lacks key catalytic residues essential for deaminase activity . The protein is considered "inactive" because it cannot rescue the purine nucleotide metabolic defect observed in quadruple aah1 ade8 amd1 his1 mutants .

YBR284W is not an essential gene in S. cerevisiae, meaning that null mutants remain viable under standard laboratory conditions . Its UniProt accession number is P38150, and it is sometimes referred to as yAMPD2 (yeast AMP deaminase 2) in the literature, though this nomenclature can be misleading given its lack of demonstrable deaminase activity .

Methodologically, when investigating its potential function, researchers should note that overexpression studies have confirmed that YBR284W cannot contribute to AMPD activity nor to adenosine or adenine deaminase activity, distinguishing it from true functional deaminases .

What is the evolutionary relationship between YBR284W and its paralog YJL070C?

YBR284W has a paralog, YJL070C, that arose from the whole genome duplication event that occurred approximately 100 million years ago in the Saccharomyces lineage . Both proteins belong to the metallo-dependent hydrolase superfamily and share significant sequence similarity, particularly in their C-terminal regions where they are >30% identical to the active AMP deaminase (Amd1p) .

Evolutionary analysis suggests that these paralogs have been maintained in the genome despite losing their ancestral deaminase activity. Interestingly, while YBR284W and YJL070C have similar structures, they appear to have diverged in certain functions:

• Overexpression of YJL070C, but not YBR284W, results in a strong decrease of both GDP and GTP intracellular concentration when cells are grown in the presence of adenine

• This phenocopies the effect of amd1 deletion, suggesting that YJL070C may play a regulatory role in purine nucleotide homeostasis, whereas YBR284W does not demonstrate this property

For researchers studying gene duplication and functional divergence, these paralogs provide an excellent model system for investigating how duplicated genes can evolve distinct functions or regulatory roles.

What are the optimal storage and handling conditions for recombinant YBR284W protein?

For optimal maintenance of recombinant YBR284W protein integrity, follow these evidence-based protocols:

Storage conditions:

• Store at -20°C for standard use

• For extended storage, conserve at -20°C or -80°C

• Working aliquots can be maintained at 4°C for up to one week

Buffer composition:

• Use Tris-based buffer with 50% glycerol, optimized for this protein

• The specific buffer composition should be adjusted based on downstream applications

Handling precautions:

• Repeated freezing and thawing is not recommended as it may lead to protein degradation or aggregation

• When designing experiments, prepare small aliquots of the protein to avoid multiple freeze-thaw cycles

Quality control:

• Before use in critical experiments, verify protein integrity by SDS-PAGE

• For functional studies, even though YBR284W lacks deaminase activity, control experiments using known active deaminases (such as AMD1) are recommended as comparators

These handling recommendations are particularly important for structural and interaction studies where protein conformation is critical to experimental outcomes.

How should researchers design experiments to evaluate potential regulatory functions of YBR284W?

Since YBR284W lacks deaminase activity but may have regulatory functions in nucleotide metabolism, consider these methodological approaches:

1. Nucleotide pool analysis:

• Implement LC-MS/MS methods to quantify changes in purine nucleotide pools (AMP, ADP, ATP, GMP, GDP, GTP) in wild-type, YBR284W deletion, and YBR284W overexpression strains

• Compare results under different growth conditions, particularly in media supplemented with adenine, as previous studies revealed that YJL070C overexpression affects GTP/GDP levels only when adenine is present

2. Transcriptomic analysis:

• Perform RNA-Seq comparing wild-type, ΔybR284W, and overexpression strains

• Analyze for expression changes in genes involved in purine metabolism

• Previous studies showed that YJL070C overexpression and AMD1 deletion resulted in overlapping transcriptional responses (246 commonly affected genes), suggesting regulatory roles

3. Protein-protein interaction studies:

• Use BioID or proximity labeling approaches to identify proteins that physically interact with YBR284W

• Focus on predicted functional partners identified in STRING database, including YJL070C (paralog), AMD1 (AMP deaminase), YHR140W, YHR202W, and ESC8

4. Reporter gene assays:

• Utilize IMD2-lacZ reporter constructs, as IMD2 is strongly induced by guanylic nucleotide limitation

• This approach successfully demonstrated that YJL070C overexpression, like AMD1 deletion, induces IMD2-lacZ expression in adenine-containing media

Experimental design considerations:

• Include appropriate controls (wild-type, known deaminase-deficient strains)

• Test under various stress conditions (oxidative stress, nutrient limitation)

• Consider combinatorial gene deletions (ybr284w/yjl070c double mutants, or combinations with other purine metabolism genes)

These approaches allow for distinguishing direct from indirect effects and can reveal regulatory roles beyond catalytic function.

What phenotypes are associated with YBR284W deletion in yeast?

YBR284W null mutants exhibit several distinct phenotypes that provide clues to its biological role:

Telomere-related phenotypes:

• YBR284W deletion results in elongated telomeres

• This suggests a potential role in telomere length regulation, which could be investigated using Southern blot analysis of terminal restriction fragments

Transposon mobility:

• Altered Ty mobility is observed in ΔybR284W strains

• Researchers can quantify this using transposition assays that measure the frequency of Ty element movement in the genome

Drug sensitivity:

• Decreased resistance to rapamycin and wortmannin

• These are inhibitors of the TOR and PI3K pathways, respectively, suggesting YBR284W may function in nutrient-sensing or growth control pathways

Stress response:

• The gene is induced in response to hydrostatic pressure

• This indicates potential involvement in stress response mechanisms

Nucleotide metabolism:

• Unlike its paralog YJL070C, deletion of YBR284W alone does not significantly affect guanylic nucleotide pools

• Single ybr284w mutants, as well as ybr284w/amd1 double mutants, do not show additional phenotypes beyond those of amd1 single mutants

For comprehensive phenotypic analysis, researchers should employ:

• Growth curve analysis under various conditions (temperature, pH, osmotic stress)

• Metabolomic profiling focusing on purine nucleotide intermediates

• Chemical genomic screening with diverse inhibitors to identify condition-specific sensitivities

• Epistasis analysis with genes in related pathways to establish genetic interactions

How does YBR284W interact with the cellular response to DNA damage?

While YBR284W itself has not been directly implicated in DNA damage responses in the search results, S. cerevisiae has well-characterized DNA damage checkpoint pathways that could interact with YBR284W functions:

Potential interactions with checkpoint proteins:

• The RAD9, RAD17, and RAD24 genes regulate DNA damage checkpoints in S. cerevisiae

• Research has shown that defects in S-phase checkpoint regulation lead to increased reliance on mutagenic translesion synthesis

• Given YBR284W's effects on telomere length and Ty mobility (both related to genome stability), potential interactions with these pathways warrant investigation

Experimental approach for investigating YBR284W in DNA damage responses:

• Assess sensitivity of ybr284w deletion strains to DNA damaging agents (MMS, UV, hydroxyurea)

• Create double mutants with key DNA repair genes (rad9Δ ybr284wΔ, etc.) to detect genetic interactions

• Monitor checkpoint activation (Rad53 phosphorylation) in wild-type versus ybr284wΔ strains after DNA damage

• Measure mutation rates using fluctuation analysis in wild-type versus mutant backgrounds

• Analyze replication fork progression using DNA combing or 2D gel electrophoresis

Methodological considerations:

• When testing DNA damage sensitivity, use both acute high-dose and chronic low-dose treatments, as different phenotypes may emerge under different treatment regimens

• Include cell cycle analysis, as checkpoint defects often manifest as cell cycle-specific vulnerabilities

• Consider the relationship between nucleotide pools (potentially affected by YBR284W) and DNA repair efficiency

How can YBR284W and its paralog YJL070C be used to study functional divergence after gene duplication?

YBR284W and YJL070C represent an excellent model for studying subfunctionalization and neofunctionalization following gene duplication:

Research framework:

• Comparative sequence analysis:

  • Align YBR284W and YJL070C sequences to identify conserved versus divergent regions

  • Map these differences to structural models to predict functional implications

  • Focus particularly on the C-terminal regions that share >30% identity with the active AMP deaminase

• Domain swapping experiments:

  • Create chimeric proteins exchanging domains between YBR284W and YJL070C

  • Test whether specific domains from YJL070C can confer its ability to affect guanylic nucleotide pools when overexpressed

  • This approach can identify the structural basis for their functional differences

• Transcriptional response analysis:

  • Previous transcriptomic analysis showed that YJL070C overexpression and AMD1 deletion affect 246 common genes

  • Perform similar analysis with YBR284W overexpression to identify unique and shared transcriptional effects

  • This table summarizes key transcriptional differences:

  Condition	Number of affected genes	Overlap with AMD1 deletion	Most affected pathways
  YJL070C overexpression	358	246	Purine metabolism, stress response
  AMD1 deletion	407	(reference)	Purine metabolism
  YBR284W overexpression	To be determined	To be determined	To be determined

• Evolutionary rate analysis:

  • Compare substitution rates in YBR284W and YJL070C across different yeast species

  • Identify positions under purifying versus relaxed selection

  • This can reveal whether functional constraints differ between paralogs

Interpretation framework:

• Functional divergence can occur through:

  • Changes in protein-protein interactions

  • Alterations in regulatory mechanisms

  • Subcellular localization differences

  • Temporal expression pattern shifts

Researchers should combine these approaches to build a comprehensive model of how these paralogs have diverged since the whole genome duplication event approximately 100 million years ago .

What are the most effective methods for measuring nucleotide pool changes in relation to YBR284W function?

To accurately assess how YBR284W affects nucleotide metabolism, researchers should implement these advanced methodological approaches:

1. LC-MS/MS quantification of nucleotide pools:

• Extract nucleotides using cold trichloroacetic acid precipitation followed by neutralization

• Separate nucleotides by reversed-phase HPLC with ion-pairing agents

• Quantify using tandem mass spectrometry with appropriate internal standards

• This method can detect changes in the concentrations of AMP, ADP, ATP, IMP, GMP, GDP, and GTP simultaneously

2. Genetic reporter systems for nucleotide imbalance:

• Utilize the IMD2-lacZ reporter system, which is strongly induced under guanylic nucleotide limitation

• This approach previously demonstrated that YJL070C overexpression induces IMD2-lacZ expression when cells are grown in adenine-supplemented media

3. Metabolic flux analysis:

• Use 15N or 13C labeled precursors to trace the flow of atoms through the purine biosynthetic and salvage pathways

• Combine with computational modeling to identify rate-limiting steps affected by YBR284W

4. Nucleotide stress response monitoring:

• Measure responses to nucleotide pool imbalance using appropriate stress reporters

• Previous research showed that both YJL070C overexpression and AMD1 deletion result in strong upregulation of IMD2 (>10-fold)

5. Single-cell analysis techniques:

• Implement microfluidics-based approaches to assess cell-to-cell variation in response to nucleotide stress

• This can reveal whether YBR284W affects the heterogeneity of cellular responses

Data analysis considerations:

• Account for cell cycle-dependent fluctuations in nucleotide pools

• Normalize to appropriate cellular parameters (cell number, protein content)

• Compare results under different nutrient conditions, especially varying adenine concentrations

• Include appropriate controls (amd1Δ, yjl070cΔ) for comparative analysis

These methodologies provide complementary data that can distinguish direct from indirect effects of YBR284W on nucleotide metabolism.

How can YBR284W research contribute to understanding purine metabolism regulation?

YBR284W and its paralog YJL070C offer unique insights into the evolution and regulation of purine metabolism networks:

Regulatory mechanisms in purine homeostasis:

• Despite lacking deaminase activity, YJL070C overexpression affects guanylic nucleotide pools

• This suggests non-catalytic regulatory mechanisms may exist in purine metabolism

• YBR284W may represent an evolutionary intermediate in this regulatory system

Experimental framework for investigation:

• Comparative systems biology approach:

  • Create a comprehensive model of purine metabolism in S. cerevisiae

  • Map effects of YBR284W, YJL070C, and AMD1 perturbations on this network

  • Identify feedback loops and regulatory nodes

• Protein interaction mapping:

  • Identify proteins that interact with YBR284W using protein-fragment complementation assays or co-immunoprecipitation

  • Focus on interactions that differ between YBR284W and YJL070C to explain their different effects on nucleotide pools

• Cross-species comparative analysis:

  • Determine whether YBR284W homologs exist in other fungal species

  • Compare deaminase activity and regulatory functions across evolutionary distance

  • Previous research showed differences in AMP deaminase between S. cerevisiae and Schizosaccharomyces pombe at both DNA sequence and immunoreactivity levels, despite similar catalytic properties

Translational implications:

• Understanding non-catalytic regulation of purine metabolism may have implications for:

  • Cancer metabolism (rapidly dividing cells have high nucleotide demands)

  • Metabolic disorders affecting purine homeostasis

  • Drug development targeting metabolic vulnerabilities

Research on YBR284W contributes to understanding how metabolic networks maintain homeostasis through both catalytic and non-catalytic mechanisms, potentially revealing new regulatory principles applicable across species.

What role might YBR284W play in cellular stress responses and adaptation?

Several lines of evidence suggest YBR284W may function in stress response pathways:

Stress-related phenotypes:

• YBR284W is induced in response to hydrostatic pressure

• YBR284W deletion results in decreased resistance to rapamycin and wortmannin, inhibitors of nutrient-sensing pathways

• These phenotypes suggest a role in cellular adaptation to environmental stress

Connection to oxidative stress response:

• The oxidative stress response in S. cerevisiae involves complex transcriptional regulation and mRNA stability changes

• Given YBR284W's connection to purine metabolism and the importance of nucleotide balance in stress conditions, potential roles in oxidative stress warrant investigation

Methodological approach for investigation:

• Transcriptional profiling under stress conditions:

  • Compare transcriptional responses to various stressors (oxidative, osmotic, temperature) in wild-type versus ybr284wΔ strains

  • Analyze for defects in specific stress response pathways

• Protein localization during stress:

  • Use fluorescently tagged YBR284W to track its localization under normal versus stress conditions

  • Determine whether stress induces changes in subcellular distribution

• Metabolic adaptation assessment:

  • Measure changes in nucleotide pools, energy charge, and redox status during stress adaptation

  • Compare wild-type, ybr284wΔ, and overexpression strains

• Genetic interaction mapping under stress:

  • Perform synthetic genetic array analysis under stress conditions

  • Identify genetic interactions that become essential specifically during stress adaptation

Interconnection with other cellular pathways:

• YBR284W's effects on telomere length and Ty mobility suggest connections to genome stability

• Its relationship to rapamycin resistance indicates potential connections to TOR signaling

• The Rpd3 histone deacetylase complex is essential for heat stress adaptation in yeast , and potential interactions with YBR284W could be investigated

Understanding YBR284W's role in stress responses may reveal how cells integrate metabolic status with stress adaptation mechanisms, a fundamental aspect of cellular homeostasis.

What are the most promising approaches for identifying the true biological function of YBR284W?

Given that YBR284W lacks deaminase activity despite structural similarity to AMP deaminases, novel approaches are needed to uncover its actual biological role:

Multi-omics integration strategy:

• Condition-specific expression profiling:

  • Identify conditions where YBR284W is strongly induced or repressed

  • The gene is known to be induced under hydrostatic pressure , suggesting other stress conditions may be relevant

  • Systematically test growth conditions, developmental stages, and stress responses

• Protein-protein interaction network mapping:

  • Implement BioID or proximity labeling approaches to identify interaction partners

  • Cross-reference with predicted functional partners from STRING database

  • Special focus on interactions that differ between YBR284W and its paralog YJL070C

• Metabolomics beyond nucleotides:

  • Expand metabolomic analysis beyond purine metabolism

  • Identify unexpected metabolic changes in ybr284wΔ strains

  • Use untargeted approaches to discover novel connections

• Evolutionary and comparative genomics:

  • Analyze patterns of conservation and co-evolution with other genes

  • Identify species where YBR284W homologs have maintained or lost function

  • Use phylogenetic profiling to predict functional relationships

Novel experimental techniques:

• CRISPR-based screens: Perform genome-wide CRISPR screens in ybr284wΔ background to identify synthetic interactions

• Deaminase domain engineering: Introduce mutations to restore potential catalytic activity and test functional consequences

• Structural biology approaches: Determine high-resolution structure to identify potential binding sites for metabolites or proteins

Research design considerations:

• Implement reciprocal hemizygosity analysis to validate candidate interacting genes

• Use chemical genetic profiling to identify conditions where YBR284W becomes essential

• Consider potential moonlighting functions unrelated to nucleotide metabolism

Uncovering YBR284W's true function will likely require integrating these diverse approaches to build a comprehensive model of its role in cellular physiology.

How can cytidine deaminase research inform our understanding of inactive deaminases like YBR284W?

Recent advances in cytidine deaminase research provide valuable frameworks for understanding inactive deaminases like YBR284W:

Structural and functional insights from active deaminases:

• Research on cytidine deaminase structures has identified key catalytic residues and mechanisms

• Structure-guided discovery approaches have yielded highly efficient cytidine deaminases with diverse properties

• These studies provide templates for analyzing which specific residues YBR284W lacks and their functional consequences

Experimental application of deaminase research to YBR284W:

• Structure-based analysis:

  • Use AlphaFold2 or similar approaches to predict YBR284W structure

  • Compare with structures of active deaminases to identify key differences

  • Recent studies used AlphaFold2 to predict structures of 1,483 cytidine deaminases , providing a methodological framework

• Residue-level investigation:

  • Identify specific residues in YBR284W that differ from conserved catalytic residues in active deaminases

  • Perform site-directed mutagenesis to restore these residues and test for gain of function

  • Analyze how these residues affect protein stability and interactions

• Functional evolution studies:

  • Compare residue conservation patterns between active and inactive deaminases across species

  • Determine whether inactive deaminases like YBR284W show different selective pressures

  • Investigate whether alternative functions have evolved in these proteins

Table: Key differences between active and inactive deaminases

Translational implications:

• Understanding how inactive deaminases evolved may provide insights into protein evolution and neofunctionalization

• The study of inactive enzymes can reveal regulatory mechanisms in metabolic networks

• Engineering approaches based on cytidine deaminase research could potentially restore activity to YBR284W