Recombinant Saccharomyces cerevisiae ULP1-interacting protein 3 (UIP3)

What is ULP1 and what is its significance in studying S. cerevisiae protein interactions?

ULP1 (Ubiquitin-like protease 1) in Saccharomyces cerevisiae is a SUMO-specific protease that catalyzes two essential functions in the SUMO pathway: processing of full-length SUMO (Smt3 in yeast) to its mature form and deconjugation of SUMO from targeted proteins . The significance of ULP1 lies in its critical role in nuclear processes and cell cycle progression. Genetic analysis guided by structural studies has revealed regulatory elements N-terminal to the proteolytic domain that are required for cell growth in yeast . Understanding ULP1 and its interacting proteins provides insights into fundamental cellular processes including DNA repair, recombination, and cell cycle regulation.

How is UIP3 identified and isolated from S. cerevisiae?

Methodological approach: UIP3, as a ULP1-interacting protein, can be identified through several complementary approaches:

• Yeast two-hybrid screening: Using ULP1 as bait to identify potential interaction partners from a S. cerevisiae cDNA library.

• Co-immunoprecipitation followed by mass spectrometry: ULP1 can be tagged (e.g., with FLAG or HA epitopes) and used to pull down interacting proteins.

• Genetic screening: Similar to the approach used with Srs2, synthetic lethality screens can identify genes that, when mutated alongside ULP1, cause severe growth defects or lethality .

Once identified, the UIP3 gene can be amplified via PCR from genomic DNA, cloned into expression vectors, and recombinantly expressed, similar to the methods described for CPY where ORFs are isolated via PCR from genomic DNA, purified, and cloned into appropriate expression vectors .

What experimental systems are most effective for studying ULP1-UIP3 interactions?

For studying ULP1-UIP3 interactions, several experimental systems have proven effective:

• The YESS-PSSC (Yeast Endoplasmic Reticulum Sequestration Screening-Protease Substrate Specificity Characterization) system allows for in vivo characterization of protein interactions . This system can be adapted by creating fusion constructs (such as Aga2-FLAG-UIP3-HA) that can be co-expressed with ULP1.

• Crystal structure analysis: Similar to the approach used for the Ulp1-Smt3 complex, X-ray crystallography can elucidate the structural basis of ULP1-UIP3 interactions .

• Genetic complementation assays: These can determine whether UIP3 affects ULP1's essential functions by expressing wild-type or mutant UIP3 in strains with compromised ULP1 function.

• Fluorescence microscopy with tagged proteins to visualize co-localization and potential interaction dynamics in living cells.

What are the key parameters to control when expressing recombinant UIP3 in yeast?

When expressing recombinant UIP3 in yeast, researchers should control the following key parameters:

• Expression vector selection: Similar to the protein production strategy described in search result , appropriate selection between low copy (centromeric pRS413 vector) and high copy number vectors (2μ pRS423 vector) is crucial for controlling expression levels.

• Promoter choice: The GAL promoter system allows for inducible expression, providing temporal control over UIP3 production .

• Growth conditions: Temperature (typically 30°C), media composition, and induction timing significantly affect protein expression.

• Protein folding considerations: As demonstrated with CPY and CPY* expression, protein misfolding can trigger cellular stress responses . Monitoring unfolded protein response (UPR) activation using biosensors can help assess whether UIP3 expression causes cellular stress.

• Genetic background: Using appropriate strain backgrounds, such as wild-type or specific deletion strains (e.g., Δulp1, Δsrs2), to evaluate genetic interactions.

How does UIP3 influence the substrate specificity of ULP1 in the SUMO pathway?

Understanding how UIP3 might influence ULP1's substrate specificity requires examination of the structural and biochemical basis of ULP1's SUMO processing activity. Research demonstrates that ULP1 recognizes SUMO (Smt3) through an extensive interface involving six structural motifs that interact with the exposed β sheet and C-terminal strand of Smt3 .

To investigate UIP3's influence on this process, researchers should:

• Perform in vitro deconjugation assays with purified ULP1, with and without UIP3, using various SUMO-conjugated substrates.

• Analyze whether UIP3 alters ULP1's ability to cleave substrates with mutations at the Gly-Gly motif. Current research shows that ULP1 can cleave Gly-Gly↓ motif-mutated substrates, indicating flexibility in recognition .

• Assess changes in the kinetic parameters of ULP1-mediated SUMO processing in the presence of UIP3.

• Employ structural modeling to predict how UIP3 might interact with the ULP1-SUMO complex, particularly focusing on the "tapered active pocket" of ULP1 that confers selectivity for small residues at the P1-P2 positions of Smt3 .

The analysis of substrate specificity can be quantified using approaches similar to those in the following table derived from research on ULP1-Smt3 interactions:

What role does UIP3 play in the DNA repair and recombination pathways associated with ULP1?

Investigating UIP3's role in DNA repair and recombination requires understanding the established connections between ULP1 and these pathways. Research has identified synthetic lethality between srs2 deletion and a mutated allele of ULP1 (ulp1-I615N), which causes accumulation of Smt3-conjugated proteins .

Methodological approaches to investigate UIP3's function in these pathways include:

• Genetic interaction studies: Create double mutants (Δuip3 with mutations in known DNA repair genes, including Δsrs2) and assess phenotypes related to DNA damage sensitivity, recombination rates, and cell cycle progression.

• Analysis of recombination rates: Determine whether UIP3 deletion or overexpression affects the hyperrecombination phenotype observed in ulp1 mutants .

• Protein SUMOylation profiling: Compare the profile of SUMOylated proteins in wild-type, Δulp1, and Δuip3 strains under normal conditions and after DNA damage induction.

• Chromatin immunoprecipitation (ChIP) to determine whether UIP3 localizes to sites of DNA damage along with ULP1 and other repair factors.

• Assessing interactions with the RAD51 pathway, which has shown genetic interactions with ulp1 mutations .

How can site-directed mutagenesis be used to map the critical interaction domains between ULP1 and UIP3?

Site-directed mutagenesis provides a powerful approach to mapping interaction domains between ULP1 and UIP3. Based on the structural understanding of ULP1-Smt3 interactions, a systematic mutagenesis strategy should:

• Target conserved residues in the catalytic domain of ULP1, particularly focusing on the six structural motifs known to interact with Smt3 .

• Create mutations in the regulatory element N-terminal to the proteolytic domain that has been shown to be required for cell growth .

• Generate a series of UIP3 mutants targeting predicted interaction surfaces.

Experimental validation of these mutants can employ:

• The YESS-PSSC system for in vivo characterization of interaction efficiency .

• Quantitative binding assays, such as isothermal titration calorimetry or surface plasmon resonance.

• Functional complementation assays to determine which mutations disrupt the biological functions mediated by ULP1-UIP3 interaction.

• Flow cytometry analysis to quantify interaction efficiencies, similar to the approach used for analyzing ULP1-Smt3 interactions .

The structural approach should be guided by the extensive interface model revealed in the Ulp1-Smt3 crystal structure, where recognition depends on multiple residues, making single mutations sometimes insufficient to disrupt interactions .

What high-throughput screening methods can identify small molecules that modulate ULP1-UIP3 interactions?

High-throughput screening (HTS) for modulators of ULP1-UIP3 interactions can be approached through several methodologies:

• Fluorescence-based interaction assays: Develop a system similar to the fluorescence-based detection used in the YESS-PSSC approach . By tagging ULP1 and UIP3 with appropriate fluorophores, Förster resonance energy transfer (FRET) can quantify their interaction in the presence of test compounds.

• Split-reporter complementation assays: Engineer ULP1 and UIP3 fusions with fragments of a reporter protein (e.g., luciferase or GFP) that becomes functional only when the proteins interact.

• Yeast-based screens: Adapt the synthetic minimal biosensor approach described for UPR monitoring to develop a reporter system where ULP1-UIP3 interaction drives the expression of a selectable marker or fluorescent protein.

• In silico screening: Use the structural data from ULP1-Smt3 interactions to model the ULP1-UIP3 interface and perform virtual screening of chemical libraries for compounds predicted to disrupt or enhance the interaction.

• Phenotypic screens: Screen for compounds that rescue growth defects in strains with compromised ULP1-UIP3 function or that mimic these defects in wild-type strains.

Validation of hits should include:

• Dose-response analysis

• Specificity testing against other protein-protein interactions

• Evaluation of effects on ULP1 catalytic activity

• Assessment of cellular phenotypes related to known ULP1 functions

How does temperature stress affect the dynamics of ULP1-UIP3 interactions and associated cellular processes?

The investigation of temperature stress effects on ULP1-UIP3 interactions is particularly relevant given that the ulp1-I615N mutant is unable to grow at 37°C but shows phenotypes at permissive temperatures . Methodological approaches to study this include:

• Temperature-shift experiments: Monitor ULP1-UIP3 interactions at various temperatures (e.g., 25°C, 30°C, 37°C) using co-immunoprecipitation or live-cell imaging with fluorescently tagged proteins.

• Analysis of SUMO conjugation patterns: Compare SUMOylated protein profiles at different temperatures in wild-type, Δuip3, and ulp1 mutant strains.

• Gene expression analysis: Perform RNA sequencing to identify genes differentially expressed in response to temperature stress in the presence and absence of UIP3.

• Protein stability assays: Determine whether UIP3 affects the thermal stability of ULP1 or vice versa, using techniques such as thermal shift assays or limited proteolysis at different temperatures.

• Microscopy: Analyze the subcellular localization of ULP1 and UIP3 at different temperatures to determine if stress induces changes in their distribution.

• Growth phenotype analysis: Compare growth curves of wild-type and mutant strains at different temperatures to correlate ULP1-UIP3 interaction dynamics with cellular fitness.

What are the optimal conditions for purifying recombinant UIP3 for structural studies?

The purification of recombinant UIP3 for structural studies requires careful optimization:

• Expression system selection:

  • For high-yield expression, consider using the yeast GAL-inducible system with a 2μ high-copy vector similar to the pRS423-GAL-CYC1 system described for CPY expression .

  • For structural studies requiring isotopic labeling, bacterial or insect cell expression systems may be preferable.

• Purification tags and strategies:

  • N-terminal or C-terminal His6 tags facilitate initial purification by immobilized metal affinity chromatography.

  • For complex structural studies, consider a cleavable tag system to remove artificial elements after purification.

  • For co-crystallization with ULP1, the approach used for the Ulp1-Smt3 complex formation may be adapted, potentially using a covalent thiohemiacetal transition state complex strategy .

• Buffer optimization:

  • Initial screening of buffer conditions (pH, salt concentration, additives) is essential for maintaining protein stability.

  • Given ULP1's role in SUMO processing, consider including reducing agents (DTT or TCEP) to maintain cysteine residues in the reduced state.

• Quality control:

  • Size-exclusion chromatography to ensure monodispersity

  • Thermal shift assays to identify stabilizing buffer conditions

  • Dynamic light scattering to assess aggregation propensity

  • Limited proteolysis to identify stable domains for crystallization

How can computational methods improve our understanding of ULP1-UIP3 functional relationships?

Computational approaches offer powerful tools for understanding ULP1-UIP3 interactions:

• Homology modeling and molecular docking:

  • Using the Ulp1-Smt3 crystal structure as a template, predict the UIP3 structure and model its interaction with ULP1.

  • Molecular dynamics simulations can assess the stability of predicted interaction interfaces.

• Network analysis:

  • Integrate protein-protein interaction data, genetic interaction profiles, and expression correlations to place UIP3 in the broader context of ULP1-associated pathways.

  • Identify potential functional redundancies or compensatory mechanisms.

• Evolutionary analysis:

  • Compare UIP3 sequences across fungal species to identify conserved regions that might be critical for ULP1 interaction.

  • Correlate evolutionary conservation patterns between ULP1 and UIP3.

• Machine learning approaches:

  • Train models on known ULP1 substrates to predict whether UIP3 affects substrate recognition.

  • Use text mining of scientific literature to identify unreported connections between UIP3 and other cellular processes.

• Structural bioinformatics:

  • Analyze the "tapered active pocket" of ULP1 to predict how UIP3 might influence substrate access or specificity.

  • Identify potential allosteric sites where UIP3 binding might affect ULP1 activity.

What controls are essential when analyzing the effects of UIP3 on ULP1-mediated SUMO processing?

Rigorous controls are critical when studying UIP3's effects on ULP1-mediated SUMO processing:

• Protein-level controls:

  • Catalytically inactive ULP1 mutant (e.g., C580S mutation in the catalytic triad)

  • ULP1 without its regulatory domain

  • Non-interacting UIP3 mutant (once interaction domains are identified)

  • Unrelated protein of similar size to UIP3 as a negative control

• Substrate controls:

  • Wild-type Smt3-substrate conjugates

  • Smt3 mutants affecting the Gly-Gly motif (G98A shows ~95% cleavage efficiency)

  • Smt3 interface mutants (R64G, R68G, etc.)

  • Non-cleavable substrate (Smt3-P at P1' position)

• Assay-specific controls:

  • For in vivo assays, include Δhac1 strains as controls for UPR activation

  • For in vitro assays, include time-course analyses to distinguish between effects on reaction rate versus reaction extent

  • Include both processing (Smt3 maturation) and deconjugation assays to determine if UIP3 differentially affects these two ULP1 functions

• Genetic controls:

  • Compare effects in wild-type versus ulp1-I615N mutant backgrounds

  • Include srs2 deletion strains to assess connections to recombination pathways

How can proteomics approaches be optimized to identify the full range of proteins affected by ULP1-UIP3 interactions?

Optimizing proteomics approaches for studying ULP1-UIP3 effects requires:

• Enrichment strategies:

  • Develop a system for efficiently purifying SUMOylated proteins, potentially using tandem affinity purification (TAP) tags on Smt3.

  • Consider using SUMO mutants that are resistant to ULP1 cleavage to stabilize conjugates.

  • Employ antibodies specific to the branched peptides formed after tryptic digestion of SUMOylated proteins.

• Quantitative approaches:

  • Stable isotope labeling with amino acids in cell culture (SILAC) to compare SUMOylated proteomes between wild-type, Δuip3, and ulp1 mutant strains.

  • Tandem mass tag (TMT) labeling for multiplexed analysis of multiple conditions.

  • Parallel reaction monitoring (PRM) for targeted quantification of specific SUMOylated proteins.

• Data analysis optimization:

  • Develop specialized search algorithms that account for the complex branched peptides resulting from SUMO conjugation.

  • Implement machine learning approaches to improve identification of SUMOylation sites.

  • Create databases of known SUMOylated proteins in yeast to improve identification rates.

• Validation strategies:

  • Western blotting for key SUMOylated proteins

  • Targeted mutagenesis of identified SUMOylation sites

  • Functional assays to verify the biological relevance of SUMOylation changes

How can the ULP1-UIP3 interaction system be exploited as a research tool in synthetic biology?

The ULP1-UIP3 interaction system offers several applications in synthetic biology:

• Protein expression control systems:

  • Develop a SUMO-based protein destabilization system where UIP3 regulates ULP1 activity, thereby controlling the stability of SUMO-tagged proteins.

  • This could function similar to degron systems but with potentially more temporal and spatial control.

• Biosensor development:

  • Engineer split-reporter systems based on ULP1-UIP3 interactions to detect cellular conditions that affect SUMO pathway activity.

  • Similar to the synthetic minimal UPR sensors , but specific for SUMO pathway perturbations.

• Protein localization tools:

  • Create synthetic systems for conditionally targeting proteins to specific subcellular compartments based on ULP1-UIP3 interaction dynamics.

  • Potential applications in studying spatially restricted processes.

• SUMO processing regulation:

  • Develop synthetic regulatory circuits where UIP3 expression or activity is controlled by cellular inputs, thereby modulating ULP1-dependent SUMO processing.

  • This could allow for conditional control of SUMOylation-dependent processes.

• Model system for studying protein-protein interactions:

  • The extensive interface characterized for ULP1-Smt3 provides a framework for engineering synthetic protein interaction systems with tunable affinity and specificity.

What emerging technologies might provide new insights into the spatial and temporal dynamics of ULP1-UIP3 interactions?

Several emerging technologies show promise for studying ULP1-UIP3 dynamics:

• Advanced imaging approaches:

  • Super-resolution microscopy (e.g., PALM, STORM) to visualize ULP1-UIP3 interactions at nanometer resolution.

  • Lattice light-sheet microscopy for long-term imaging with minimal phototoxicity.

  • Single-molecule tracking to follow individual ULP1 and UIP3 molecules in living cells.

• Proximity labeling techniques:

  • BioID or TurboID fusions with ULP1 or UIP3 to identify proteins in their vicinity under different conditions.

  • APEX2-based proximity labeling for temporal control of the labeling reaction.

• Optogenetic tools:

  • Light-inducible protein interaction systems to control ULP1-UIP3 association with temporal and spatial precision.

  • Optogenetic control of UIP3 localization to study the effects of its recruitment to specific subcellular structures.

• Microfluidics and single-cell analysis:

  • Microfluidic systems for precise control of yeast growth conditions and rapid media changes.

  • Single-cell RNA-seq to capture cell-to-cell variability in responses to ULP1-UIP3 perturbations.

• CRISPR-based technologies:

  • CRISPRi for tunable repression of UIP3 expression.

  • CRISPR activation systems for controlled upregulation.

  • Base editing or prime editing for precise introduction of mutations without double-strand breaks.

How can findings from ULP1-UIP3 studies be integrated with broader knowledge of SUMO pathway regulation in eukaryotes?

Integration of ULP1-UIP3 findings with broader SUMO pathway knowledge requires:

• Comparative analysis across species:

  • Identify homologs or functional equivalents of UIP3 in other eukaryotes.

  • Compare the regulatory mechanisms of SUMO proteases across evolutionary distance.

  • Determine whether the ULP1-UIP3 interaction represents a conserved regulatory mechanism.

• Pathway interconnections:

  • Map how UIP3-mediated regulation of ULP1 interfaces with other SUMO pathway components.

  • Investigate potential crosstalk between UIP3 and other post-translational modification systems (ubiquitination, phosphorylation).

  • Determine whether UIP3 affects the balance between SUMO conjugation and deconjugation activities.

• Systems biology approaches:

  • Develop mathematical models of the SUMO cycle that incorporate UIP3's influence on ULP1 activity.

  • Use these models to predict system-level responses to perturbations.

  • Validate model predictions with targeted experiments.

• Translation to complex eukaryotes:

  • Assess whether findings in S. cerevisiae can inform understanding of mammalian SUMO proteases and their regulators.

  • Consider therapeutic implications for diseases involving dysregulation of the SUMO pathway.

What are the most promising future research directions for understanding ULP1-UIP3 function in cellular homeostasis?

The most promising future research directions include:

• Single-molecule studies of ULP1-UIP3-Smt3 interactions to understand the dynamic process of substrate recognition and processing.

• Investigation of UIP3's potential role in ULP1 localization and how this affects substrate accessibility.

• Exploration of condition-specific regulation of UIP3 (stress conditions, cell cycle stages) and how this translates to changes in global SUMOylation patterns.

• Development of mathematical models that can predict how perturbations to ULP1-UIP3 interactions propagate through cellular networks.

• Integration of genetic, biochemical, and structural approaches to build a comprehensive understanding of how UIP3 fits into the complex regulatory network controlling SUMO homeostasis.

• Investigation of potential roles in stress responses, given that ulp1 mutations show temperature sensitivity and protein folding stress activates cellular response pathways .

• Assessment of how UIP3 might influence the recently characterized substrate specificity of ULP1, particularly regarding the finding that the conserved Gly-Gly motif is not strictly required for Ulp1 cleavage .