Recombinant Saccharomyces cerevisiae DUP240 protein YAR023C

What is the structural composition of YAR023C DUP240 protein?

YAR023C is a member of the DUP240 protein family in Saccharomyces cerevisiae. The full-length protein consists of 179 amino acids with the following sequence: MINFLLFVLTILATLTNIWFSGVLSPAMVIRICLGGSMVVLQIWSFSRPISNETFRTKLL LEVITHRPSIAGKEWKTITYNMNQYLFKAGLWKTPYHFFCEHQCYEFFKDLIKGKYPDVQ WDTANTQPFISVPENQAATQNSDVEPTVKWCLFKAAEIQAHAVREYWQSQYPDVGIPAI .

Like other DUP240 family proteins, YAR023C likely has two predicted transmembrane domains separated by a short inter-helix linker, as well as three conserved domains . The transmembrane topology is critical for its localization and function in the cell membrane.

How does YAR023C relate to other DUP240 family proteins?

YAR023C belongs to the DUP240 gene family, which includes other members such as YAR028W (also known as KTD1 or killer toxin defense) and UIP3. These proteins share structural similarities but have evolved distinct functions. Recent research has identified YAR028W as playing a critical role in defense against killer toxin K28, while the specific function of YAR023C remains under investigation .

Evolutionary analysis suggests that DUP240 proteins may have diversified through gene duplication events, allowing for specialization of different family members. The presence of conserved domains across family members indicates shared ancestral functions, while variations in the inter-helix linker and C-terminal regions likely contribute to their functional divergence.

What expression systems are suitable for recombinant production of YAR023C?

YAR023C can be successfully expressed in multiple heterologous systems:

For structural studies, E. coli expression with an N-terminal His-tag has been demonstrated to be effective, yielding protein with greater than 90% purity as determined by SDS-PAGE . For functional studies requiring proper protein folding and activity, yeast expression systems may provide advantages due to more native-like post-translational modifications .

How can researchers optimize YAR023C protein stability during purification and storage?

Optimizing YAR023C stability requires careful consideration of buffer composition and storage conditions. Recombinant YAR023C is typically provided in a Tris/PBS-based buffer with 6% trehalose at pH 8.0 to enhance stability . The addition of trehalose is critical as it acts as a cryoprotectant that prevents protein denaturation during freeze-thaw cycles.

For reconstitution, it is recommended to:

• Briefly centrifuge the vial containing lyophilized protein

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (optimally 50%)

• Aliquot into smaller volumes to avoid repeated freeze-thaw cycles

• Store at -20°C/-80°C for long-term storage

Working aliquots may be stored at 4°C for up to one week, but repeated freezing and thawing should be avoided as it significantly reduces protein activity and integrity .

What are the critical considerations when designing experiments to investigate YAR023C function?

When designing experiments to investigate YAR023C function, researchers should consider:

• Appropriate controls: Include both positive and negative controls. For deletion studies, complementation with the wild-type gene should be performed to confirm phenotypic changes are due to the absence of YAR023C.

• Strain background considerations: Different yeast strain backgrounds (e.g., BY vs. RM) may have significant genetic differences that affect experimental outcomes. Researchers should characterize their strain background thoroughly and consider how genetic background may influence results .

• Variable isolation: Clearly define and control variables in the experimental design:

  • Independent variable: The factor being manipulated (e.g., YAR023C expression levels)

  • Dependent variable: The outcome being measured (e.g., cell growth, toxin resistance)

  • Controlled variables: Factors kept constant (e.g., temperature, media composition)

  • Constants: Unchanging elements throughout the experiment

• Replication: Include at least three technical replicates for each experimental condition and conduct biological replicates as well to ensure reliability and reproducibility of results .

• Chimeric protein analysis: When investigating functional domains, design chimeras that transition between YAR023C and related proteins at the boundaries of transmembrane helices or conserved domains to identify regions important for specific functions .

How can researchers effectively characterize the membrane topology of YAR023C?

Characterizing membrane topology of YAR023C requires a combination of computational prediction and experimental validation approaches:

• Computational prediction:

  • Use topology prediction algorithms (e.g., TMHMM, Phobius) to identify potential transmembrane regions

  • Analyze hydrophobicity plots to confirm transmembrane domain boundaries

  • Compare with known DUP240 family proteins like YAR028W/KTD1

• Experimental validation:

  • Protease protection assays: Treating intact cells or membrane fractions with proteases to determine which regions are accessible

  • Glycosylation mapping: Introducing glycosylation sites at various positions to determine luminal/cytoplasmic orientation

  • GFP-fusion analysis: Creating fusions with GFP at N- and C-termini to visualize localization

  • Cysteine accessibility methods: Introducing cysteine residues and testing their accessibility to membrane-impermeable thiol-reactive reagents

• Structural analysis:

  • Circular dichroism (CD) spectroscopy to determine secondary structure content

  • NMR or X-ray crystallography for high-resolution structural information

Comparative analysis with the characterized transmembrane topology of related DUP240 proteins provides additional insights, particularly the two predicted transmembrane domains separated by the inter-helix linker that has been shown to be functionally important in YAR028W .

What evidence suggests a role for YAR023C in killer toxin defense mechanisms?

While direct evidence for YAR023C in killer toxin defense is limited, strong evidence exists for its family member YAR028W (KTD1). Comprehensive genetic analysis has revealed that:

• Linkage analysis of BY × RM cross segregants identified a QTL on chromosome I where genetic differences caused variation in K28 toxin resistance .

• The YAR028W gene from the BY strain provides strong protection against K28 toxin when expressed in sensitive strains, while a frameshift mutation in the RM allele abolishes this protection .

• Deletion of YAR028W (yar028wΔ) in the BY background results in hypersensitivity to K28 toxin .

How do genetic variations in YAR023C affect protein function across different yeast strains?

Analysis of genetic variations in YAR023C across different Saccharomyces cerevisiae strains can provide insights into its functional evolution. Similar to what has been observed with YAR028W, where the RM strain contains a frameshift mutation that abolishes its protective function against K28 toxin , variations in YAR023C may lead to functional differences.

To systematically analyze such variations:

• Sequence alignment: Compare YAR023C sequences from different yeast strains to identify polymorphisms.

• Functional complementation: Express YAR023C variants in a deletion background and assess phenotypic rescue.

• Domain swapping: Create chimeric proteins between variants to identify which regions are responsible for functional differences.

• Evolutionary analysis: Calculate the ratio of non-synonymous to synonymous substitutions (dN/dS) to determine if the gene is under positive selection, which would suggest a role in adaptation to different environments or in host-pathogen interactions.

• Expression analysis: Use ribosome footprinting data to confirm translation and identify potential differences in expression levels or translation efficiency between strains .

What are the implications of the inter-helix linker and C-terminus regions for YAR023C function?

Studies of the related DUP240 protein YAR028W (KTD1) have identified the inter-helix linker and C-terminus as critical for its function in killer toxin defense . By extension, these regions may also be important for YAR023C function:

• Inter-helix linker: In YAR028W, chimeras with different inter-helix linkers showed dramatic differences in K28 protection ability, suggesting this region may be involved in toxin recognition or interaction with cell wall components .

• C-terminus: Replacement of the C-terminal 88 amino acids of YAR028W with the corresponding region from another DUP240 family member (UIP3) completely abolished K28 resistance, indicating this region is essential for functional specificity .

For YAR023C, targeted mutagenesis of these regions coupled with functional assays would help determine:

• Whether similar functional domains exist

• If these regions confer specific interactions with particular molecules

• How structural differences in these regions contribute to potentially distinct functions compared to other DUP240 family members

What are the optimal conditions for expressing and purifying recombinant YAR023C?

Optimal expression and purification of recombinant YAR023C involves several key considerations:

When expressing in E. coli, codon optimization for the host organism may improve expression levels, particularly for eukaryotic proteins. For functional studies, expression in S. cerevisiae may provide advantages due to native post-translational modifications and proper folding environment .

How can researchers design chimeric constructs to identify functional domains of YAR023C?

Creating chimeric constructs between YAR023C and other DUP240 family members provides a powerful approach to identify functional domains. Based on successful strategies with YAR028W , researchers should:

• Identify domain boundaries: Use computational analysis to identify:

  • Transmembrane helices

  • Inter-helix linker

  • Conserved domains

  • C-terminal region

• Design chimera construction strategy:

  • Create transitions at natural domain boundaries

  • Use overlap extension PCR or Gibson assembly for seamless fusion

  • Maintain reading frame across fusion junctions

  • Include epitope tags for detection if necessary

• Functional validation:

  • Express chimeras in appropriate deletion backgrounds

  • Test for complementation of phenotypes

  • Compare activity levels quantitatively

• Sequential domain swapping: Follow a systematic approach:

  • Swap entire N-terminal or C-terminal halves first

  • Narrow down to individual domains

  • Focus on fine mapping of regions showing functional differences

For example, with YAR028W, chimeras transitioning between Ktd1p and Uip3p at the boundaries of transmembrane helices or conserved domains revealed that the inter-helix linker and C-terminus were critical for K28 toxin protection . A similar approach with YAR023C could reveal domains important for its specific functions.

What quantitative methods are most appropriate for analyzing YAR023C-mediated phenotypes?

Several quantitative methods can be employed to analyze YAR023C-mediated phenotypes:

Each of these methods should include appropriate controls and statistical analysis to ensure reliable and reproducible results.

How might YAR023C be utilized in biotechnological applications?

Understanding YAR023C's structure and function could lead to several biotechnological applications:

• Stress resistance engineering: If YAR023C contributes to stress or toxin resistance like its family member YAR028W, it could be used to engineer yeast strains with enhanced tolerance to industrial conditions.

• Biosensor development: The specificity of DUP240 proteins for certain environmental factors or toxins could be exploited to develop biosensors that detect specific compounds.

• Protein scaffold engineering: The membrane topology and domain structure of YAR023C could serve as a scaffold for designing proteins with novel functions, particularly for membrane-associated applications.

• Yeast surface display: YAR023C could potentially be utilized in yeast surface display systems for protein engineering or antibody development.

• Drug discovery platform: If YAR023C interacts with specific molecules, it could serve as a target or tool in drug discovery efforts focused on antifungal compounds.

These applications would build upon fundamental research into YAR023C's natural function and would require detailed structural and functional characterization.

What are the most promising approaches for elucidating the natural biological function of YAR023C?

To elucidate the natural biological function of YAR023C, researchers should consider a multi-faceted approach:

• Comprehensive phenotypic analysis:

  • Create precise deletion and overexpression strains

  • Subject these strains to various stress conditions (chemical, temperature, pH)

  • Test resistance to different yeast killer toxins beyond K28

  • Analyze cellular physiology and morphology under different conditions

• Evolutionary and comparative genomics:

  • Compare YAR023C sequences across Saccharomyces species and strains

  • Analyze patterns of selection and conservation

  • Correlate genetic variations with phenotypic differences

  • Build upon findings from related DUP240 proteins like YAR028W

• Protein interaction studies:

  • Identify binding partners through co-immunoprecipitation coupled with mass spectrometry

  • Validate interactions using orthogonal methods (yeast two-hybrid, FRET)

  • Determine subcellular localization under different conditions

• Structural biology approaches:

  • Obtain high-resolution structure through X-ray crystallography or cryo-EM

  • Use structure to guide functional hypotheses and mutagenesis

  • Compare with structures of related proteins with known functions

• Systems biology integration:

  • Analyze transcriptomics and proteomics data in response to YAR023C deletion or overexpression

  • Integrate with existing datasets on DUP240 family proteins

  • Model potential functions based on system-wide effects

By combining these approaches, researchers can develop and test specific hypotheses about YAR023C function, potentially revealing its role in yeast biology and evolution.