Recombinant Saccharomyces cerevisiae UPF0495 protein YPR010C-A (YPR010C-A)

What is the amino acid sequence of YPR010C-A and what structural features does it possess?

YPR010C-A is a small protein with the following amino acid sequence: MRPAQLLLNTAKKTSGGYKIPVELTPLFLAVGVALCSGTYFTYKKLRTDETLRLTGNPELSSLDEVLAKDKD . The protein belongs to the UPF0495 family and consists of 72 amino acids in its full expression region .

Structurally, YPR010C-A contains hydrophobic regions that likely facilitate membrane association, particularly with mitochondrial membranes. The protein's structure supports its dual functionality in both nuclear processes (RNA binding) and mitochondrial processes (respiratory chain complex assembly). Secondary structure predictions suggest a combination of alpha-helical segments and beta-sheets that contribute to its binding interfaces with partner proteins and RNA molecules.

How is YPR010C-A classified in protein databases and what are its known orthologs?

YPR010C-A is classified in the UPF0495 protein family, with database identifiers including UniProt accession number A5Z2X5 . In functional databases, it is referenced as:

• KEGG: sce:YPR010C-A

• STRING: 4932.YPR010C-A

The protein has orthologs in other fungal species, including pathogenic fungi such as Histoplasma capsulatum, with which it shares 56.76% sequence identity. This conservation across species suggests evolutionary importance of its functions. The protein has been observed in the comprehensive Saccharomyces cerevisiae PeptideAtlas, which validates its expression at the protein level through mass spectrometry data .

What is the subcellular localization of YPR010C-A and how does this relate to its function?

YPR010C-A demonstrates dual localization patterns within yeast cells, correlating with its multiple functional roles. The protein associates with both nuclear components (interacting with nuclear polyadenylated RNA-binding proteins) and mitochondrial structures (particularly respiratory chain complexes).

In the nucleus, YPR010C-A interacts with proteins like Nab2p to modulate mRNA export and poly(A) tail length control. Within mitochondria, it associates with respiratory chain components including Cor1p and Cox13p, supporting oxidative phosphorylation (OXPHOS) complex assembly. This dual localization explains its diverse functions in both RNA processing and mitochondrial energy metabolism.

For research methodologies, immunofluorescence microscopy using GFP-tagged constructs or specific antibodies provides the most direct visualization of this dual localization pattern. Subcellular fractionation followed by Western blotting offers quantitative assessment of the protein's distribution between nuclear and mitochondrial compartments.

What are the primary functional roles of YPR010C-A in cellular processes?

YPR010C-A participates in multiple cellular pathways, with three primary functional roles:

• RNA Processing and Export: YPR010C-A interacts with nuclear polyadenylated RNA-binding proteins (particularly Nab2p) to modulate mRNA export and poly(A) tail length control. This function directly impacts post-transcriptional gene regulation.

• Mitochondrial Respiratory Chain Assembly: The protein associates with respiratory chain components (Cor1p, Cox13p) and supports mitochondrial oxidative phosphorylation (OXPHOS) complex assembly, contributing to cellular energy metabolism.

• Genome Stability Maintenance: YPR010C-A interacts with Aim46p, a mitochondrial protein linked to genome integrity, suggesting a role in maintaining genomic stability, potentially through mitochondrial DNA maintenance mechanisms.

These functions highlight YPR010C-A as a multifunctional protein that bridges nuclear gene expression processes and mitochondrial energy production, potentially serving as a coordinator between these cellular compartments.

What proteins does YPR010C-A interact with and what are their interaction scores?

YPR010C-A engages in specific protein-protein interactions that support its various cellular functions. The key interactions and their respective scores are summarized in the following table:

These interaction scores are derived from experimental evidence and computational predictions. The highest confidence interaction is with COR1 (score 0.709), which supports YPR010C-A's role in mitochondrial respiratory chain assembly. The validated interaction with AIM46 lacks a numerical score but has been experimentally confirmed through methods such as co-immunoprecipitation or yeast two-hybrid assays.

How does YPR010C-A contribute to mitochondrial supercomplex assembly?

YPR010C-A plays a crucial role in mitochondrial supercomplex assembly by associating with key respiratory chain components. The protein particularly interacts with Cor1p (a component of ubiquinol-cytochrome c reductase) and Cox13p (a subunit of cytochrome c oxidase), functioning as a potential assembly factor or stabilizing element for these respiratory complexes.

For experimental verification of this function, researchers should consider:

• Blue native PAGE analysis of respiratory chain complexes in wild-type versus YPR010C-A deletion strains

• Co-immunoprecipitation assays with tagged respiratory chain components

• Respiration measurements to assess functional consequences of YPR010C-A absence

What are the optimal storage conditions for recombinant YPR010C-A protein?

Recombinant YPR010C-A requires specific storage conditions to maintain its structural integrity and functional activity:

• Short-term storage: Working aliquots should be maintained at 4°C for up to one week .

• Standard storage: Store the protein at -20°C in a Tris-based buffer containing 50% glycerol, which has been optimized for this specific protein .

• Long-term storage: For extended preservation, conserve the protein at -20°C or -80°C, with -80°C being preferable for maintaining activity beyond 6 months .

Critical considerations include:

• Repeated freezing and thawing should be avoided as this can lead to protein denaturation and activity loss .

• The protein should be aliquoted into single-use volumes before freezing to prevent repeated freeze-thaw cycles.

• When thawing, samples should be placed on ice and used immediately for optimal results.

What expression systems are most effective for producing recombinant YPR010C-A?

When producing recombinant YPR010C-A, several expression systems can be employed, each with specific advantages:

• E. coli-based expression: Commonly used for its simplicity and high yield. For YPR010C-A, codon optimization may be necessary due to differences between yeast and bacterial codon usage preferences. BL21(DE3) or Rosetta strains are recommended for better expression of eukaryotic proteins.

• Yeast expression systems: Using S. cerevisiae or Pichia pastoris provides a eukaryotic environment with appropriate post-translational modifications. For homologous expression in S. cerevisiae, the GAL1 promoter allows for inducible expression with galactose.

• Insect cell/baculovirus systems: Offer advantages for proteins that require eukaryotic processing machinery while providing higher yields than yeast systems.

Expression of YPR010C-A with fusion tags (His, GST, or MBP) facilitates purification and can enhance solubility. When designing constructs, researchers should note that the tag type will be determined during the production process for optimal results .

What methods are most effective for studying YPR010C-A interactions with RNA?

To investigate YPR010C-A's RNA-binding properties and interactions with nuclear polyadenylated RNA-binding proteins, several methodological approaches are recommended:

• RNA Immunoprecipitation (RIP): This technique allows for the identification of RNAs that directly associate with YPR010C-A. Using tagged versions of the protein (such as HA-tag or FLAG-tag), followed by immunoprecipitation and RNA isolation, researchers can identify bound RNAs through sequencing or RT-PCR.

• Electrophoretic Mobility Shift Assay (EMSA): This approach can determine direct RNA binding by recombinant YPR010C-A. Various RNA sequences can be tested to identify binding preferences and affinities.

• Crosslinking and Immunoprecipitation (CLIP): More advanced than standard RIP, CLIP techniques (such as PAR-CLIP or iCLIP) provide single-nucleotide resolution of RNA-protein interaction sites.

• Yeast Three-Hybrid System: This genetic approach can screen for RNA sequences that interact with YPR010C-A in vivo.

• Co-immunoprecipitation with RNA-binding proteins: This method can reveal whether YPR010C-A forms complexes with proteins like Nab2p in an RNA-dependent manner. Treatment with RNases before immunoprecipitation can determine if interactions are RNA-dependent or direct protein-protein interactions.

How can the interaction between YPR010C-A and Aim46p be leveraged to study mitochondrial genome stability?

The validated interaction between YPR010C-A and Aim46p presents a valuable opportunity to investigate mechanisms of mitochondrial genome stability. Researchers can leverage this interaction through several experimental approaches:

• Double knockout studies: Creating strains with deletions of both YPR010C-A and AIM46 genes would allow assessment of potential synthetic phenotypes that might reveal redundant or complementary roles in genome stability.

• Mitochondrial DNA (mtDNA) stability assays: Measuring mtDNA mutation rates and copy number in wild-type versus YPR010C-A deletion strains can quantify the protein's contribution to genome stability.

• Fluorescent tagging and live-cell imaging: Visualizing the co-localization of YPR010C-A and Aim46p during various cellular stresses can provide insights into their functional relationship during conditions that challenge mitochondrial genome integrity.

• ChIP-seq of mitochondrial DNA: Chromatin immunoprecipitation followed by sequencing can map binding sites of YPR010C-A on mtDNA, potentially revealing direct interactions with the mitochondrial genome.

• Protein domain mapping: Creating truncated versions of both proteins can identify the specific domains required for their interaction and for mitochondrial genome maintenance functions.

What approaches can be used to study YPR010C-A's role in mRNA export and poly(A) tail length control?

Investigating YPR010C-A's function in mRNA processing requires specialized methodologies focusing on nuclear export and polyadenylation:

• Poly(A) tail length analysis: Using methods like ePAT (extension Poly(A) Test) or TAIL-seq to measure poly(A) tail lengths in wild-type versus YPR010C-A deletion strains can directly assess its impact on polyadenylation.

• mRNA export assays: Fluorescence in situ hybridization (FISH) with oligo(dT) probes can visualize poly(A) RNA distribution, identifying potential nuclear accumulation in YPR010C-A mutants that would indicate export defects.

• Interaction studies with mRNA export factors: Co-immunoprecipitation and mass spectrometry can identify components of the mRNA export machinery that associate with YPR010C-A beyond the known Nab2p interaction.

• Transcriptome-wide analyses: RNA-seq comparing nuclear and cytoplasmic fractions in wild-type versus YPR010C-A deletion strains can identify specific transcripts whose export depends on this protein.

• Single-molecule tracking: Using MS2-GFP systems to visualize individual mRNAs can provide real-time data on export kinetics and how they are affected by YPR010C-A deletion.

These approaches would help delineate whether YPR010C-A functions directly in the mRNA export process or indirectly through its interactions with established export factors like Nab2p.

How can researchers utilize YPR010C-A orthologs in pathogenic fungi for antifungal drug discovery?

The structural similarity (56.76% identity) between YPR010C-A and its orthologs in pathogenic fungi such as Histoplasma capsulatum presents opportunities for antifungal drug discovery. Researchers can pursue this avenue through several approaches:

• Comparative structural biology: Solving crystal structures of both S. cerevisiae YPR010C-A and its pathogenic orthologs would reveal conserved pockets that could serve as drug targets.

• Essentiality testing: Determining whether the YPR010C-A ortholog is essential in pathogenic fungi through CRISPR-Cas9 or RNAi approaches would validate it as a potential drug target.

• High-throughput screening: Developing assays based on the protein's function (RNA binding or mitochondrial assembly) would enable screening of compound libraries for molecules that specifically inhibit the pathogenic ortholog.

• Structure-based drug design: Using in silico docking studies with the protein's structure to design molecules that could disrupt its function in pathogenic fungi while minimizing effects on human proteins.

• Heterologous complementation studies: Testing whether the pathogenic ortholog can complement YPR010C-A deletion in S. cerevisiae would reveal functional conservation and provide a simple model system for drug screening.

This research direction could leverage the non-pathogenic S. cerevisiae as a model system while developing therapeutics targeting clinically relevant fungal pathogens.

How reliable are the protein-protein interaction scores for YPR010C-A, and what might cause false positives or negatives?

The protein-protein interaction scores reported for YPR010C-A (ranging from 0.631 to 0.709) represent confidence values derived from various experimental and computational approaches. When interpreting these scores, researchers should consider several factors:

• Score reliability factors:

  • Higher scores (>0.7) like the COR1 interaction (0.709) indicate high confidence interactions likely to be biologically relevant.

  • Medium scores (0.5-0.7) such as those with NAB2 (0.635) and RCF3 (0.631) suggest probable interactions that benefit from additional validation.

  • The "Validated" designation for AIM46 indicates experimental confirmation through methods like co-immunoprecipitation, regardless of numerical score.

• Potential sources of false positives:

  • Artificial associations during cell lysis

  • Overexpression artifacts in two-hybrid studies

  • Indirect interactions through intermediate proteins

  • Structural similarities causing cross-reactivity in antibody-based detection

• Potential sources of false negatives:

  • Transient or weak interactions that escape detection

  • Context-dependent interactions requiring specific conditions

  • Technical limitations in detecting membrane-associated complexes

  • Poor expression or solubility of tagged protein constructs

For validation, researchers should employ orthogonal methods such as co-immunoprecipitation, proximity ligation assays, and functional studies to confirm the biological relevance of reported interactions.

What considerations should be made when interpreting proteomics data for YPR010C-A from the Saccharomyces PeptideAtlas?

The Saccharomyces cerevisiae PeptideAtlas represents a comprehensive proteomics resource composed from 47 diverse experiments and 4.9 million tandem mass spectra . When interpreting YPR010C-A data from this resource, researchers should consider:

• Statistical confidence levels: Different probability thresholds yield varying expected error rates as shown in this table:

• Peptide observation frequency: Peptides observed multiple times have higher reliability. For "verified" ORFs like YPR010C-A, 74% are represented in the PeptideAtlas, but this decreases to 56% when removing peptides observed only once .

• Sample preparation variations: Different extraction methods may bias against membrane-associated proteins like YPR010C-A, potentially affecting its representation in certain experiments.

• Post-translational modifications: Modified peptides may not be identified using standard search parameters, leading to underrepresentation of modified forms of the protein.

To maximize confidence in proteomics data, researchers should focus on peptides observed multiple times and identified with high probability scores (P>0.95 or P>0.99).

What troubleshooting approaches are recommended when recombinant YPR010C-A shows reduced activity?

When recombinant YPR010C-A exhibits diminished functional activity, systematic troubleshooting should address potential causes:

• Protein integrity assessment:

  • SDS-PAGE and western blotting to verify protein size and integrity

  • Mass spectrometry to confirm sequence and detect potential modifications

  • Circular dichroism to assess secondary structure integrity

• Storage condition optimization:

  • Verify storage at recommended conditions (-20°C in Tris-based buffer with 50% glycerol)

  • Check for evidence of freeze-thaw cycles which should be avoided

  • Test alternative buffer compositions if activity issues persist

• Functional assay validation:

  • Include positive and negative controls in all activity assays

  • Titrate protein concentration to identify optimal working range

  • Verify assay components (buffer conditions, cofactors, interaction partners)

• Recombinant production strategies:

  • Test expression with different fusion tags

  • Evaluate expression in alternative systems (bacterial vs. yeast)

  • Consider co-expression with interaction partners (e.g., Nab2p) to stabilize structure

For persistent issues, researchers might consider developing a native purification protocol from S. cerevisiae to compare activity with recombinant versions, potentially identifying missing cofactors or post-translational modifications critical for function.

What are the most promising avenues for future research on YPR010C-A function and applications?

Future research on YPR010C-A should focus on several promising directions that build upon current knowledge while addressing significant gaps:

• Structural biology: Determining the three-dimensional structure of YPR010C-A through X-ray crystallography or cryo-EM would provide critical insights into its function. Particular attention should be given to structural changes upon RNA binding or protein partner interactions.

• Systems biology approach: Integrating transcriptomics, proteomics, and metabolomics data from YPR010C-A deletion strains would provide a comprehensive understanding of its cellular impact, particularly focusing on the intersection of RNA processing and mitochondrial function.

• Conditional regulation studies: Investigating how YPR010C-A function is regulated under various cellular stresses could reveal its role in adaptive responses, particularly during mitochondrial dysfunction or RNA processing challenges.

• Therapeutic applications: Further exploring the differences between YPR010C-A and its pathogenic fungal orthologs could yield selective antifungal approaches with minimal cross-reactivity to human proteins.

• Evolutionary analysis: Comparative studies across fungal species could reveal how YPR010C-A's dual function in nuclear and mitochondrial processes evolved and provide insights into the co-evolution of these cellular compartments.

These research directions would address fundamental questions about YPR010C-A while potentially yielding practical applications in biotechnology and medicine.

How might CRISPR-Cas9 technology be applied to study YPR010C-A function in vivo?

CRISPR-Cas9 technology offers powerful approaches for investigating YPR010C-A function through precise genomic modifications:

• Gene knockout studies: Complete deletion of YPR010C-A to assess phenotypic consequences, particularly focusing on:

  • Growth rates under various carbon sources to evaluate mitochondrial function

  • mRNA export efficiency through nuclear/cytoplasmic fractionation

  • mtDNA stability through mutation rate analysis

• Domain-specific mutations: Creating precise mutations in functional domains to dissect their specific contributions:

  • RNA-binding regions to affect nuclear functions while preserving mitochondrial roles

  • Mitochondrial targeting sequences to redirect localization

  • Interface regions mediating specific protein-protein interactions

• Endogenous tagging: Adding fluorescent or affinity tags to the endogenous gene to:

  • Visualize real-time localization changes under various conditions

  • Perform chromatin immunoprecipitation to identify DNA binding sites

  • Conduct co-immunoprecipitation under native expression levels

• Promoter modifications: Replacing the endogenous promoter with inducible systems to:

  • Control expression levels for dosage studies

  • Create conditional knockouts for essential function analysis

  • Study temporal aspects of YPR010C-A function during the cell cycle

• Ortholog replacement: Swapping the S. cerevisiae gene with orthologs from pathogenic fungi to:

  • Assess functional conservation

  • Identify species-specific activities

  • Develop platforms for antifungal screening

These CRISPR-based approaches would provide unprecedented insights into YPR010C-A's multifaceted functions while maintaining physiologically relevant expression contexts.

What integrative approaches can link YPR010C-A's dual roles in RNA processing and mitochondrial function?

YPR010C-A's involvement in both nuclear RNA processing and mitochondrial function presents an intriguing case of potential coordination between these cellular processes. Integrative approaches to investigate this connection include:

• Mitochondrial transcriptome analysis: Comparing mitochondrial transcript processing in wild-type versus YPR010C-A mutant strains could reveal whether its RNA processing activity extends to mitochondrial transcripts, potentially explaining the dual localization.

• Metabolic flux analysis: Tracing carbon metabolism through techniques like 13C-glucose labeling could quantify how YPR010C-A deletion affects the coordination between nuclear-encoded mitochondrial proteins and mitochondrial function.

• Interactome mapping under stress conditions: Performing systematic protein-protein interaction studies under conditions that stress either RNA processing (e.g., heat shock) or mitochondrial function (e.g., oxidative stress) could reveal condition-specific interaction networks.

• Synchronized cell cycle analysis: Examining YPR010C-A localization and interaction partners throughout the cell cycle could identify temporal patterns in its functional switching between nuclear and mitochondrial roles.

• Retrograde signaling investigation: Determining whether YPR010C-A participates in mitochondria-to-nucleus signaling pathways could explain how it coordinates nuclear gene expression with mitochondrial status.