Recombinant Saccharomyces cerevisiae Uncharacterized protein YIL102C-A (YIL102C-A)
Description
Introduction to Recombinant Saccharomyces cerevisiae Uncharacterized Protein YIL102C-A
YIL102C-A is a recombinant protein derived from Saccharomyces cerevisiae, originally annotated as an uncharacterized protein in genomic databases . Recent functional studies have redefined its role as a regulatory subunit of dolichyl phosphate mannose (DPM) synthase, a critical enzyme in glycosylation pathways . Despite its historical classification as uncharacterized, YIL102C-A exhibits functional homology to DPM2 in humans and DPMII in Trichoderma reesei .
Gene and Protein Information
*Proposed renaming to DPM2 based on functional similarity .
Protein Structure and Recombinant Production
YIL102C-A lacks defined domains in early annotations but shares functional motifs with DPM2/DPMII proteins . Recombinant versions are produced in E. coli with His-tagged purification systems, yielding >85% purity . The truncated form (1–75 aa) is commonly used in research .
Role in DPM Synthase Activity
YIL102C-A interacts directly with Dpm1 (the catalytic subunit of DPM synthase) in S. cerevisiae, modulating enzymatic activity . Deletion of YIL102C-A is lethal, but this phenotype is rescued by expressing dpm2 from T. reesei . This confirms its conserved role in glycosylation.
Interaction with GPI-GnT
YIL102C-A binds glucosylphosphatidylinositol-N-acetylglucosaminyl transferase (GPI-GnT), mirroring DPM2’s interaction in human cells . This highlights its dual role in GPI anchor biosynthesis and protein glycosylation.
Essentiality and Functional Homology
| Organism | DPM Synthase Subunit | YIL102C-A Homology |
|---|---|---|
| S. cerevisiae | YIL102C-A/DPM2 | Regulatory subunit |
| Homo sapiens | DPM2 | Functional analog |
| T. reesei | DPMII | Structural analog |
Nomenclature and Research Implications
Recent proteomic studies propose renaming YIL102C-A to DPM2 to reflect its conserved function . This update aligns with its essential role in yeast glycosylation and its interaction with Dpm1 and GPI-GnT .
Limitations in Early Annotations
Product Specs
Note: While we prioritize shipping the format we have in stock, we are happy to accommodate your specific requirements. Please indicate your preferred format in your order notes, and we will fulfill your request whenever possible.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance as additional fees may apply.
Generally, the shelf life of liquid forms is 6 months at -20°C/-80°C. Lyophilized forms typically have a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: sce:YIL102C-A
STRING: 4932.YIL102C-A
Q&A
What is the basic function of YIL102C-A in Saccharomyces cerevisiae?
YIL102C-A functions as a regulatory subunit of dolichyl phosphate mannose synthase in S. cerevisiae. While previously labeled as uncharacterized in the Saccharomyces Genome Database (SGD), more recent investigations have identified it as a functional homologue of the DPMII subunit involved in dolichyl phosphate mannose (DPM) synthesis . This pathway is crucial for protein glycosylation, which affects numerous cellular processes including cell wall integrity, protein folding, and cellular recognition.
How was YIL102C-A initially identified as an essential gene?
YIL102C-A was not originally identified as essential in the Yeast Knockout (YKO) collection but was later discovered to be essential through complementary approaches. Recent studies using the C' AID-GFP library (combining the Auxin-Inducible Degron system with GFP tagging) demonstrated that depletion of YIL102C-A protein results in lethality . This finding was validated using both AID induction and orthogonal approaches such as replacing the endogenous promoter with the repressible GAL1 promoter, confirming its essential nature through multiple experimental methodologies .
Why might YIL102C-A have been misannotated in early genomic studies?
The misannotation of YIL102C-A as non-essential may be attributed to several factors common in genome-wide studies:
| Factor | Explanation |
|---|---|
| Expression level | YIL102C-A may be expressed at low levels under standard laboratory conditions, making it difficult to detect in early studies |
| Genetic redundancy | Partial functional overlap with other genes may have masked its essential nature in knockout studies |
| Conditional essentiality | The protein may be essential only under specific growth or stress conditions not tested in initial screens |
| Technical limitations | Limitations in early genome annotation techniques may have led to its misidentification |
High-throughput validation with newer technologies like the AID2 system has been instrumental in correctly identifying its essential function .
What are the most effective methods for studying YIL102C-A protein depletion phenotypes?
The Auxin-Inducible Degron (AID2) system has proven particularly effective for studying YIL102C-A. This approach involves:
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Fusing the small AID* tag (amino acids 71-114 from AtIAA17) followed by eGFP to the C-terminus of YIL102C-A
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Expressing the adaptor OsTIR1(F74G) constitutively in the same strain
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Inducing protein degradation by adding the modified auxin, 5-Ph-IAA (5-Phenyl 1H-indole-3-acetic acid)
This system allows for rapid, reversible depletion with minimal baseline degradation in the absence of inducer. For validation, orthogonal approaches such as replacing the endogenous promoter with the galactose-inducible/glucose-repressible GAL1 promoter can be used, where protein depletion is achieved by growing cells in glucose-containing media .
How can researchers effectively visualize and quantify YIL102C-A localization and abundance?
For visualization and quantification of YIL102C-A:
| Method | Application | Advantages |
|---|---|---|
| C-terminal GFP tagging | Live-cell visualization | Allows real-time tracking with minimal disruption |
| High-throughput fluorescence microscopy | Systematic analysis of localization changes | Enables screening across different conditions |
| Western blotting | Protein abundance quantification | Provides precise quantitative measurements |
| Flow cytometry | Population-level protein abundance | Offers statistical power through large sample sizes |
When implementing the AID2 system with GFP fusion, researchers can simultaneously monitor protein degradation kinetics and localization changes before complete depletion . This approach has shown that approximately 90% of AID-tagged proteins, including YIL102C-A, respond effectively to the degradation system .
What considerations should be made when designing experiments to study YIL102C-A interactions?
When studying protein-protein interactions involving YIL102C-A:
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Consider that the C-terminal tagging approach used in the AID-GFP library may affect some protein interactions
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Test multiple induction timepoints (e.g., 30 minutes vs. 24 hours) as proteins show variation in depletion dynamics
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Include appropriate controls to account for potential effects of the AID tag or 5-Ph-IAA on cellular processes
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Combine approaches such as co-immunoprecipitation, proximity labeling, or yeast two-hybrid assays to validate interactions
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Focus on potential interactions with other components of the dolichyl phosphate mannose synthesis pathway
How does YIL102C-A contribute to dolichyl phosphate mannose synthesis?
As a functional homologue of the DPMII subunit, YIL102C-A likely plays a regulatory role in dolichyl phosphate mannose (DPM) synthesis . The DPM synthesis complex typically consists of a catalytic subunit and regulatory subunits that stabilize the complex and potentially modulate its activity. In this pathway:
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Dolichol phosphate serves as a lipid carrier
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A mannose residue from GDP-mannose is transferred to dolichol phosphate by the DPM synthase complex
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The resulting DPM serves as a mannose donor for various glycosylation reactions
YIL102C-A's essentiality suggests it may be critical for maintaining the structural integrity or catalytic efficiency of this complex, though detailed biochemical characterization is still needed.
What cellular processes are affected by YIL102C-A depletion?
Based on its role in DPM synthesis, YIL102C-A depletion would likely affect:
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N-linked glycosylation of proteins
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O-linked glycosylation of proteins
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GPI anchor synthesis
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Cell wall integrity
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Protein folding and quality control in the endoplasmic reticulum
The essentiality of YIL102C-A demonstrated through conditional depletion systems suggests that disruption of these processes leads to cellular lethality . Future studies using partial depletion or temperature-sensitive alleles could help identify the most critical processes and potential compensatory mechanisms.
How does YIL102C-A relate to iron homeostasis or aluminum sensitivity?
Interestingly, the search results mention that YIL102C (a different but possibly related gene) was reported to be sensitive to Al(III) . The connection between YIL102C-A and metal sensitivity requires further investigation, but it raises several research questions:
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Whether YIL102C-A has a similar sensitivity to metals like aluminum
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If glycosylation defects resulting from YIL102C-A dysfunction affect metal transport or toxicity
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Potential regulatory connections between dolichyl phosphate mannose synthesis and metal homeostasis pathways
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If metal ions directly affect YIL102C-A function or stability
This represents an unexplored area that might reveal novel connections between glycosylation and metal homeostasis.
What structural features characterize YIL102C-A and how do they relate to its function?
While detailed structural information about YIL102C-A is not provided in the search results, we can outline approaches to characterize its structure:
| Approach | Information Gained | Experimental Considerations |
|---|---|---|
| Sequence analysis | Domain prediction, conserved motifs | Requires homologous sequences for comparison |
| Secondary structure prediction | α-helices, β-sheets, disordered regions | Computational predictions need experimental validation |
| Membrane topology analysis | Transmembrane regions, orientation | Important if associated with ER membrane like other DPM components |
| Crystallography/Cryo-EM | Atomic-level structure | May require co-crystallization with binding partners |
| Cross-linking studies | Protein-protein interaction interfaces | Can identify contact points with other DPM synthase components |
As a DPMII homologue, YIL102C-A likely contains domains involved in protein-protein interactions within the DPM synthase complex and potentially membrane association domains if it resembles other eukaryotic DPM components.
How can researchers distinguish between direct and indirect effects of YIL102C-A manipulation?
Distinguishing direct from indirect effects in YIL102C-A studies requires:
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Time-course experiments following rapid protein depletion to identify primary versus secondary effects
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Rescue experiments using catalytically inactive mutants to separate structural from enzymatic roles
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Complementation studies with homologous proteins from other species
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Targeted analysis of immediate biochemical consequences (e.g., measuring DPM levels directly after YIL102C-A depletion)
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Comparison with phenotypes resulting from depletion of other DPM synthase components
The AID2 system is particularly valuable for such analyses as it enables rapid depletion with minimal side effects, allowing researchers to observe immediate consequences before secondary effects manifest .
What can comparative genomics tell us about YIL102C-A conservation and evolution?
Comparative genomic analysis of YIL102C-A would investigate:
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Conservation across fungal species and beyond
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Presence of functional homologues in other eukaryotes
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Domain conservation patterns
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Evolutionary rate compared to other components of the DPM synthase complex
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Evidence of co-evolution with interacting partners
Such analysis could reveal whether YIL102C-A represents a fungal-specific adaptation or a conserved component of eukaryotic glycosylation machinery. The identification of YIL102C-A as a DPMII homologue suggests some degree of conservation in dolichyl phosphate mannose synthesis across species .
How do functional homologues of YIL102C-A in other organisms differ from the S. cerevisiae protein?
Understanding differences between YIL102C-A and its homologues requires:
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Sequence alignment to identify conserved and variable regions
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Functional complementation assays to test interchangeability
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Examination of species-specific protein interactions
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Analysis of different regulatory mechanisms across species
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Investigation of structural variations that might reflect adaptations to different cellular environments
This comparative approach could identify critical functional domains and provide insights into evolutionary constraints on the DPM synthesis pathway.
How can synthetic genetic array (SGA) approaches be applied to study YIL102C-A genetic interactions?
For studying YIL102C-A genetic interactions:
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Generate conditional alleles (using AID2 or temperature-sensitive mutations) to overcome the essentiality barrier
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Perform systematic genetic interaction screens by crossing with genome-wide deletion or hypomorphic collections
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Analyze genetic interaction profiles for functional clustering
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Focus on genetic interactions with other glycosylation pathway components
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Compare interaction profiles with those of known DPMII homologues or other DPM synthase components
The combination of the AID2 system with SGA methodology is particularly powerful, as it allows for conditional depletion in combination with thousands of genetic backgrounds .
What are the challenges and opportunities in developing YIL102C-A as a potential antifungal target?
YIL102C-A presents several characteristics relevant to antifungal development:
Research challenges include developing specific inhibitors that distinguish between fungal and human homologues, and designing appropriate assays to monitor DPM synthase activity in vitro and in vivo.
