Recombinant Saccharomyces cerevisiae Uncharacterized protein YDR282C (YDR282C)

What is YDR282C in Saccharomyces cerevisiae?

YDR282C (also known as RMND1 ortholog) is an uncharacterized protein in Saccharomyces cerevisiae localized to the inner mitochondrial membrane with its C-terminus facing the intermembrane space . It is the yeast ortholog of the human RMND1 gene, which has been implicated in mitochondrial protein synthesis . Immunoblot analysis has detected YDR282C as a protein of approximately 50 kDa in size when tagged with an HA epitope . Despite being classified as "uncharacterized," preliminary studies suggest its involvement in mitochondrial functions, particularly related to mitochondrial translation machinery.

What is the cellular localization pattern of YDR282C?

YDR282C has been experimentally determined to localize to the inner mitochondrial membrane . This localization was established through mitochondrial fractionation and intramitochondrial localization studies using antibodies against epitope tags. Specifically, the C-terminus of the protein faces the intermembrane space, suggesting a potential role in communication between mitochondrial compartments or in protein transport . This orientation may be critical for understanding its function within the context of mitochondrial protein synthesis and organelle biogenesis.

What phenotype is associated with YDR282C deletion?

Interestingly, yeast strains containing the ydr282c-null allele demonstrate a normal phenotype, including normal growth in respiration-dependent glycerol medium and unimpaired mitochondrial protein synthesis . This lack of obvious phenotype suggests possible functional redundancy with other proteins or that its role may become apparent only under specific stress conditions not typically examined in standard laboratory assays. The absence of a clear phenotype presents a challenge for functional characterization but also suggests that comparative studies under various stress conditions might reveal conditional phenotypes.

What are effective approaches for generating recombinant YDR282C protein?

For recombinant expression of YDR282C, researchers can adapt methodologies similar to those used for other yeast proteins. Based on successful expression of yeast proteins in E. coli, the following approach can be effective:

• Optimize the ribosome-binding site through site-specific mutagenesis to enhance expression in bacterial systems

• Clone the gene into an expression vector with an inducible promoter (e.g., IPTG-inducible tac promoter)

• Transform the construct into an appropriate E. coli strain (e.g., TG2 or BL21)

• Induce expression with IPTG and purify using a combination of chromatography techniques, such as anion-exchange and hydroxyapatite gel chromatography

This approach has yielded approximately 10 mg of purified recombinant yeast protein per liter of cell culture for other yeast proteins .

What methods are recommended for studying YDR282C subcellular localization?

To verify and characterize the subcellular localization of YDR282C, researchers should consider:

• Epitope tagging: Adding HA, YFP, or similar tags to facilitate detection with commercial antibodies

• Mitochondrial fractionation: Separating different mitochondrial compartments through gradient centrifugation

• Immunodetection: Using epitope antibodies for Western blotting and immunocytochemistry

• Fluorescence microscopy: For YFP-tagged constructs to visualize localization in living cells

When using YFP-RMND1 fusion proteins, researchers should note that immunoblot analysis typically reveals two bands: the expected long isoform (approximately 52 kDa) and a cleaved mitochondrial protein (approximately 28 kDa) .

How can researchers generate and validate YDR282C knockout strains?

For generating YDR282C knockout strains:

• Use homologous recombination-based gene replacement with a selectable marker

• Verify gene deletion through PCR analysis of genomic DNA

• Confirm protein absence through Western blotting if antibodies are available

• Validate the knockout by testing for phenotypes under various growth conditions, particularly those requiring respiratory function

For phenotypic characterization, examine growth in both fermentable (glucose) and non-fermentable (glycerol) carbon sources to assess respiratory capacity .

What is the relationship between YDR282C and mitochondrial protein synthesis?

• Ribosome profiling of wild-type versus knockout strains

• Analysis of mitochondrial translation products using 35S-methionine labeling

• Examination of potential genetic interactions through synthetic genetic arrays

What structural features characterize YDR282C protein?

Based on available data from orthologous proteins:

The detection of both 52 kDa and 28 kDa forms of the protein suggests post-translational processing, potentially through proteolytic cleavage of a precursor form after mitochondrial import .

How does YDR282C compare to its orthologs in other species?

The human ortholog of YDR282C, RMND1, has at least three isoforms produced by alternative splicing :

• Isoform 1: 449 amino acids (longest form), localized to mitochondria

• Isoform 2: Lacking amino acids 1-211

• Isoform 3: Containing alternative amino acids 205-208 (DAAN>GTSS) and missing amino acids 209-449

While mutations in human RMND1 are associated with infantile encephaloneuromyopathy and defective mitochondrial translation, the yeast ortholog knockout shows no obvious phenotype . This difference suggests either functional divergence or the presence of compensatory mechanisms in yeast. Comparative analysis across species could reveal conserved domains critical for function and species-specific adaptations.

What yeast ghost preparation methods can be applied to YDR282C studies?

Yeast ghost preparation provides a unique approach for studying membrane proteins like YDR282C. The protocol involves:

• Determining Minimum Inhibitory Concentration (MIC) for chemical agents (NaOH, SDS, NaHCO₃, H₂O₂)

• Applying these agents at critical concentrations to create gentle pores in the yeast cell walls

• Allowing cytoplasmic content evacuation while maintaining the 3D structure and membrane integrity

• Verifying ghost formation through light and scanning electron microscopy

This approach preserves the membrane structure where YDR282C resides, potentially allowing for isolation of membrane protein complexes in their native orientation . The evacuation of cytoplasmic contents can be verified by measuring released DNA and protein using spectrophotometry at 260 nm and 280 nm, respectively .

What heterologous expression systems are suitable for YDR282C characterization?

For heterologous expression of YDR282C, researchers can adapt approaches used for other yeast proteins:

• E. coli expression system:

  • Modify the ribosome-binding site to match E. coli consensus sequences

  • Use an inducible promoter system like the tac promoter with IPTG induction

  • Create fusion proteins with purification tags (His, GST, MBP) for easier purification

  • Expression yields of approximately 10 mg/L of culture have been achieved for other yeast proteins

• Alternative host systems:

  • Other yeast species (Pichia pastoris for higher expression)

  • Mammalian cell lines (for studying interaction with mammalian orthologs)

  • Cell-free expression systems (for difficult-to-express proteins)

How can researchers investigate protein-protein interactions involving YDR282C?

To identify and characterize protein-protein interactions:

• Affinity purification coupled with mass spectrometry:

  • Express epitope-tagged YDR282C in yeast

  • Isolate mitochondria and solubilize membrane proteins

  • Perform pull-down experiments followed by mass spectrometry analysis

• Yeast two-hybrid screening:

  • Use the mature form of YDR282C (post-processing) as bait

  • Screen against a mitochondrial protein library

  • Validate interactions through co-immunoprecipitation

• Proximity labeling approaches:

  • Fuse YDR282C with BioID or APEX2 enzymes

  • Identify proteins in close proximity through biotinylation

  • Analyze biotinylated proteins by mass spectrometry

Why might YDR282C knockout strains show no obvious phenotype?

The absence of an obvious phenotype in ydr282c-null strains could be attributed to:

• Functional redundancy: Other proteins may compensate for YDR282C loss

• Condition-specific requirements: The protein may be important only under specific stress conditions not typically tested

• Subtle phenotypes: The effects may be quantitatively small or affect processes not examined in standard assays

• Evolutionary differences: The function may have diverged between yeast and higher eukaryotes

Researchers should consider examining the knockout strain under various stress conditions (oxidative stress, temperature stress, nutrient limitation) and utilizing more sensitive analytical methods (transcriptomics, metabolomics) to detect subtle phenotypic changes.

What challenges might arise in purifying recombinant YDR282C?

Common challenges and solutions include:

When working with membrane proteins like YDR282C, consider using specialized approaches such as amphipols or nanodiscs to maintain protein structure and function during purification.

How might CRISPR-Cas9 technology enhance YDR282C research?

CRISPR-Cas9 technology offers several advantages for YDR282C research:

• Precise genome editing:

  • Introduction of point mutations to study specific domains

  • Creation of conditional alleles through insertion of degradation tags

  • Integration of reporter genes for live-cell imaging

• Transcriptional modulation:

  • CRISPRi for partial knockdown to study dosage effects

  • CRISPRa for overexpression studies

  • Timed modulation using inducible CRISPR systems

• High-throughput screening:

  • CRISPR libraries targeting genetic interactors

  • Systematic mutagenesis of YDR282C domains

  • Parallel phenotypic analysis using pooled screens

What systems biology approaches can reveal YDR282C function?

Integrated systems biology approaches for YDR282C characterization include:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics of wild-type vs. knockout strains

  • Map changes onto biochemical pathways to identify affected processes

  • Identify potential biomarkers for functional assays

• Network analysis:

  • Construct protein-protein interaction networks

  • Perform genetic interaction mapping through synthetic genetic arrays

  • Identify functional modules through co-expression analysis

• Comparative genomics:

  • Analyze evolutionary conservation patterns

  • Identify co-evolved gene clusters

  • Leverage phenotypic data from orthologous genes in model organisms