Recombinant Arabidopsis thaliana E3 ubiquitin-protein ligase SIS3 (SIS3)

What is the expression pattern of SIS3 in Arabidopsis?

Quantitative real-time RT-PCR experiments have shown that SIS3 is expressed at consistent levels across different Arabidopsis organs, including roots, stems, leaves, siliques, and flowers . According to Genevestigator analyses, SIS3 is ubiquitously expressed in all major organ and tissue types except in pollen, where mRNA levels are very low. The developmental expression profile reveals slightly higher expression in germinated seeds (1,140) compared to seedlings (879) . Interestingly, SIS3 transcript levels are not significantly regulated by high glucose concentrations in germinating seeds, suggesting that any effects of glucose on SIS3 activity occur at the posttranscriptional level .

How does SIS3 function as an E3 ligase at the molecular level?

SIS3 functions as a RING-type E3 ubiquitin ligase, facilitating the transfer of ubiquitin from an E2 enzyme to specific target proteins. In vitro biochemical assays have demonstrated that both full-length GST-SIS3 (GST-SIS3 FL) and the RING domain alone (GST-SIS3 RING) can stimulate E2-dependent ubiquitination . The process requires the presence of both E1 (ubiquitin-activating enzyme) and E2 (ubiquitin-conjugating enzyme, specifically UBC8 in the reported experiments). The RING domain of SIS3 (amino acids 235-275) is sufficient for this ubiquitination activity, as shown by in vitro assays where the RING domain alone could facilitate ubiquitin transfer . This indicates that the RING motif is the functional catalytic core of SIS3's E3 ligase activity.

What are the biochemical requirements for SIS3 E3 ligase activity in vitro?

The in vitro E3 ligase activity of SIS3 requires specific components:

• Arabidopsis E1 (ubiquitin-activating enzyme)

• Arabidopsis E2 (UBC8, ubiquitin-conjugating enzyme)

• The RING domain of SIS3 (sufficient for activity)

• ATP

• Ubiquitin

In experimental setups, recombinant GST-SIS3 FL (full-length) and GST-SIS3 RING (containing only the RING domain) proteins are produced in Escherichia coli and affinity purified using glutathione agarose . In the presence of Arabidopsis E1 and E2 (UBC8), both protein forms stimulate E2-dependent ubiquitination. Without either E1 or E2, no ubiquitination activity is observed, confirming SIS3's role as an E3 ligase in the ubiquitination cascade .

How can researchers design effective experiments to study SIS3 function in sugar signaling?

Designing effective experiments to study SIS3 function requires careful consideration of several factors:

• Genetic materials: Use multiple alleles of sis3 mutants (e.g., sis3-1, sis3-2, sis3-3) alongside wild-type controls and complementation lines. Include other sugar-response mutants (e.g., hxk1, rgs1, prl1) for comparative analyses.

• Growth conditions: Standardize growth conditions with defined media:

  • Minimal Arabidopsis medium supplemented with varying concentrations of sugars (typically 270-300 mM sucrose or glucose)

  • Include osmotic controls (e.g., 300 mM sorbitol + 10 mM sucrose)

  • Surface sterilize seeds and stratify at 4°C for 3 days

  • Grow under continuous white fluorescent light at 22°C

• Phenotypic analysis: Assess developmental parameters at 12-14 days post-germination:

  • Cotyledon expansion and greening

  • True leaf development

  • Root growth

  • Quantitative measurements of seedling fresh weight and chlorophyll content

• Molecular analysis: Examine SIS3 expression and activity:

  • Quantitative real-time RT-PCR to measure transcript levels

  • Western blotting to detect protein levels

  • In vitro ubiquitination assays to assess E3 ligase activity

• Integration with other pathways: Test interactions with known sugar signaling components and other hormonal pathways (e.g., ABA, GA) .

What experimental controls are essential when investigating SIS3 function?

When investigating SIS3 function, several crucial controls must be included:

• Osmotic controls: Since high sugar concentrations create osmotic stress, include equivalent osmotic controls (e.g., 300 mM sorbitol + 10 mM sucrose) to distinguish sugar-specific responses from osmotic effects. For example, while sis3 mutants show resistance to 300 mM glucose or sucrose, they exhibit similar phenotypes to wild-type on media with equivalent osmolality but non-metabolizable sugars .

• Multiple allele testing: Use multiple independent sis3 alleles (e.g., sis3-1, sis3-2, sis3-3) to confirm phenotypes are due to SIS3 disruption rather than background mutations.

• Complementation lines: Generate transgenic lines expressing SIS3 in the sis3 mutant background to confirm gene identity and function. Restoration of wild-type sensitivity to sugars in these lines confirms SIS3's role .

• Different ecotype backgrounds: Test phenotypes in different Arabidopsis ecotypes (e.g., Col-0, Ws-2) to ensure the observations are not ecotype-specific.

• Hormone response controls: Include tests for responses to plant hormones (e.g., ABA, GA) to distinguish sugar-specific pathways from hormone-mediated responses .

How can researchers identify potential target proteins of SIS3 E3 ligase activity?

Identifying SIS3 targets requires a multi-faceted approach:

• Yeast two-hybrid screening: Use SIS3 as bait to screen Arabidopsis cDNA libraries for interacting proteins. This can identify direct binding partners, some of which may be ubiquitination targets.

• Co-immunoprecipitation (Co-IP) coupled with mass spectrometry: Express tagged versions of SIS3 (e.g., GFP-SIS3 or HA-SIS3) in Arabidopsis, immunoprecipitate the protein complexes, and identify associated proteins through mass spectrometry.

• Ubiquitinome analysis: Compare the ubiquitinated proteome of wild-type and sis3 mutant plants using antibodies against ubiquitin or ubiquitin remnant motifs, followed by mass spectrometry to identify differentially ubiquitinated proteins.

• in vitro ubiquitination assays: Test candidate proteins as substrates for SIS3-mediated ubiquitination in reconstituted systems containing E1, E2, SIS3, ubiquitin, and the potential target protein.

• Protein stability assays: Compare the stability of candidate target proteins in wild-type versus sis3 mutant backgrounds using cycloheximide chase experiments to identify proteins whose degradation depends on SIS3 activity.

• Genetic suppressor screens: Identify mutations that suppress the sis3 phenotype, which may reveal downstream components of the SIS3 pathway.

What are the most effective strategies for producing recombinant SIS3 protein for biochemical studies?

Based on published methodologies, the most effective strategies for producing recombinant SIS3 include:

• E. coli expression systems:

  • Express SIS3 as a GST fusion protein (GST-SIS3) in E. coli

  • Use BL21(DE3) or similar strains optimized for protein expression

  • Induce expression with IPTG at lower temperatures (16-18°C) to enhance solubility

  • Affinity purify using glutathione agarose

• Protein domain considerations:

  • Express full-length SIS3 (GST-SIS3 FL) for complete functional studies

  • Express the RING domain alone (GST-SIS3 RING, amino acids 235-275) for E3 ligase activity studies

  • Consider removing transmembrane domains for improved solubility

• Purification optimizations:

  • Include protease inhibitors throughout purification

  • Add reducing agents (e.g., DTT) to maintain cysteine residues in the RING domain

  • Use size exclusion chromatography as a final purification step

• Activity preservation:

  • Store purified protein in small aliquots at -80°C with glycerol

  • Avoid repeated freeze-thaw cycles

  • Verify protein activity using in vitro ubiquitination assays before use in experiments

In successful studies, this approach has yielded functional recombinant SIS3 protein capable of demonstrating E3 ligase activity in vitro .

How does SIS3 integrate with other known sugar signaling pathways in Arabidopsis?

SIS3 functions within a complex network of sugar signaling components in Arabidopsis:

• Relationship with HXK1-dependent pathway: Hexokinase 1 (HXK1) is a well-established glucose sensor in plants. Research suggests that SIS3 may act in parallel to or downstream of the HXK1-dependent pathway, as both affect early seedling development responses to high sugar concentrations .

• Connection to RGS1/GPA1 pathway: The heterotrimeric G-protein components RGS1 and GPA1 are involved in sugar sensing. The relationship between SIS3 and this pathway requires further investigation, but they may converge on common downstream targets.

• Interaction with PRL1/SnRK1 pathway: PRL1 (PLEIOTROPIC REGULATORY LOCUS1) and SnRK1 (Snf1-RELATED PROTEIN KINASE1) are key regulators of sugar and energy signaling. SIS3 may function in relation to this pathway to coordinate growth responses to sugar availability .

• Cross-talk with hormonal pathways: Unlike some other sugar response mutants, sis3 exhibits wild-type responses to abscisic acid (ABA) and paclobutrazol (a gibberellic acid biosynthesis inhibitor) during seed germination, suggesting SIS3 functions specifically in sugar signaling rather than in general stress or hormone responses .

Understanding these relationships requires detailed genetic analysis using double mutant combinations and transcriptomic studies to map the regulatory networks.

What is known about transcriptional regulation downstream of SIS3?

While the complete transcriptional network downstream of SIS3 remains to be fully elucidated, several key points have emerged from research:

• Transcriptomic analyses comparing wild-type and sis3 mutant plants have revealed that SIS3 affects the expression of genes involved in various cellular processes:

  • Sugar-responsive gene expression

  • Stress-related gene expression

  • Growth and development genes

• According to Genevestigator analyses, SIS3 steady-state mRNA levels are affected by various stimuli:

  • Increased 2.5-fold by syringolin (a cell death-inducing chemical)

  • Increased 1.5-fold by cold and anoxia treatment

  • Decreased nearly 2-fold by heat

This suggests SIS3 may influence pathogen and abiotic stress responses in addition to sugar signaling .

• As an E3 ubiquitin ligase, SIS3 likely regulates protein abundance rather than directly affecting transcription. The transcriptional changes observed in sis3 mutants are likely consequences of altered stability or activity of transcription factors or other signaling components that are SIS3 ubiquitination targets.

How can researchers effectively characterize the phenotypes of sis3 mutants at different developmental stages?

Effective characterization of sis3 mutants requires systematic analysis at multiple developmental stages:

• Seed germination stage:

  • Measure germination rates on media with varying concentrations of sugars (0-300 mM)

  • Test germination in the presence of plant hormones (ABA, GA) to assess pathway specificity

  • Analyze the effect of stratification (cold treatment) on germination efficiency

• Early seedling development (1-14 days):

  • Document cotyledon expansion, greening, and true leaf development

  • Measure hypocotyl and root elongation

  • Quantify chlorophyll and anthocyanin content

  • Assess carbohydrate content (glucose, fructose, sucrose, starch)

• Vegetative growth stage:

  • Monitor rosette size and leaf morphology

  • Measure biomass accumulation

  • Analyze photosynthetic efficiency using chlorophyll fluorescence

  • Examine root system architecture

• Reproductive stage:

  • Record flowering time and inflorescence development

  • Assess silique development and seed production

  • Evaluate seed quality and dormancy characteristics

• Response to environmental stresses:

  • Test tolerance to drought, salt, cold, and heat stresses

  • Examine pathogen resistance or susceptibility

  • Analyze responses to nutrient limitation

For each stage, compare multiple sis3 alleles to wild-type controls under identical conditions, and include well-characterized sugar-signaling mutants for reference .

What genetic approaches can be used to identify suppressors or enhancers of the sis3 phenotype?

Several genetic approaches can be employed to identify genes that interact with SIS3:

• Suppressor screens:

  • Mutagenize sis3 mutant seeds (using EMS or other mutagens)

  • Screen for reversion to wild-type sugar sensitivity

  • Map and identify suppressor mutations using next-generation sequencing

  • This approach can identify downstream components required for the sis3 phenotype

• Enhancer screens:

  • Mutagenize sis3 mutant seeds

  • Screen for enhanced sugar insensitivity or new phenotypes

  • This can reveal genes with partially redundant functions to SIS3

• Double mutant analysis:

  • Cross sis3 with other sugar-response mutants (e.g., hxk1, gin1, rgs1)

  • Evaluate phenotypes of double mutants to establish genetic relationships

  • Epistasis analysis can place SIS3 in existing signaling pathways

• Transcription factor overexpression:

  • Transform sis3 mutants with an activation-tagging library

  • Screen for restoration of sugar sensitivity

  • Identify transcription factors that can bypass the requirement for SIS3

• CRISPR-based screens:

  • Use multiplexed CRISPR libraries to target multiple genes in the sis3 background

  • Screen for phenotypic modifiers

  • This approach allows systematic testing of candidate interactors

These approaches can provide valuable insights into the genetic network in which SIS3 functions .

In vitro analysis:

• Recombinant protein-based ubiquitination assay:

  • Express and purify GST-SIS3 (full-length or RING domain only)

  • Combine with purified E1, E2 (UBC8), ATP, and ubiquitin

  • Incubate at 30°C for 1-3 hours

  • Analyze ubiquitination by Western blotting using anti-ubiquitin antibodies

  • This approach has successfully demonstrated SIS3's E3 ligase activity

• Target protein ubiquitination:

  • Include purified candidate substrate proteins in the reaction

  • Detect substrate-specific ubiquitination by Western blotting

  • Analyze ubiquitination patterns (mono- vs. poly-ubiquitination)

  • Determine ubiquitin chain linkage types using linkage-specific antibodies

In vivo analysis:

• Cell-free degradation assays:

  • Prepare protein extracts from wild-type and sis3 mutant plants

  • Add recombinant candidate substrate proteins

  • Monitor their degradation over time

  • Add proteasome inhibitors to confirm ubiquitin-proteasome involvement

• In planta ubiquitination:

  • Express tagged versions of SIS3 and candidate substrates in plants

  • Immunoprecipitate substrates under denaturing conditions

  • Detect ubiquitination by Western blotting with anti-ubiquitin antibodies

  • Compare ubiquitination levels between wild-type and sis3 mutant backgrounds

• Fluorescent protein fusion degradation:

  • Create fusions between candidate substrates and fluorescent proteins

  • Express in wild-type and sis3 mutant plants

  • Monitor fluorescence levels as a proxy for protein stability

  • Use cycloheximide to block new protein synthesis and focus on degradation

These complementary approaches provide a comprehensive view of SIS3's E3 ligase activity and its biological targets.

How can researchers visualize SIS3 subcellular localization and dynamics during sugar responses?

Several techniques can be employed to visualize SIS3 localization and dynamics:

• Fluorescent protein fusion constructs:

  • Generate N- and C-terminal fusions of SIS3 with fluorescent proteins (GFP, YFP, mCherry)

  • Express in Arabidopsis under native or constitutive promoters

  • Observe localization using confocal microscopy

  • Consider the impact of the three predicted transmembrane domains on localization

• Co-localization studies:

  • Combine SIS3-FP with markers for different cellular compartments

  • Quantify co-localization using Pearson's correlation coefficient

  • Particularly focus on ER, Golgi, and plasma membrane markers given SIS3's predicted membrane association

• Dynamic imaging during sugar treatments:

  • Treat seedlings expressing SIS3-FP with different sugar concentrations

  • Perform time-lapse imaging to track changes in localization or abundance

  • Quantify fluorescence intensity changes over time

• Photoconvertible or photoactivatable tags:

  • Use tags like Dendra2 or PA-GFP fused to SIS3

  • Photoconvert/activate SIS3 in specific subcellular regions

  • Track protein movement to assess dynamics and turnover

• Immunogold electron microscopy:

  • Use anti-SIS3 antibodies or anti-tag antibodies for endogenous or tagged SIS3

  • Visualize at ultrastructural level to precisely define membrane associations

  • This provides higher resolution than light microscopy

• FRET/FLIM analysis:

  • Create donor-acceptor pairs with SIS3 and potential interactors

  • Measure FRET efficiency to detect protein-protein interactions in vivo

  • This approach can reveal dynamic interactions during sugar responses

These visualization techniques will provide crucial insights into SIS3's subcellular location and how this might change during sugar signaling events.

How should researchers analyze and interpret contradictory data regarding SIS3 function?

When confronted with contradictory data regarding SIS3 function, researchers should follow these systematic steps:

• Validate experimental conditions:

  • Verify that sugar concentrations, media composition, and growth conditions are comparable across experiments

  • Ensure genetic materials are correctly genotyped and free of contamination

  • Confirm that different sis3 alleles show consistent phenotypes

• Consider genetic background effects:

  • Determine if contradictions arise from different Arabidopsis ecotypes (Col-0 vs. Ws-2)

  • Backcross mutants to standardize genetic backgrounds

  • Include multiple independent alleles in analyses

• Assess experimental design differences:

  • Evaluate differences in developmental stages examined

  • Consider variations in treatment duration and intensity

  • Analyze differences in assay methods and endpoints

• Perform statistical analysis:

  • Apply appropriate statistical tests based on data distribution

  • Consider biological versus technical replication

  • Calculate effect sizes to determine biological significance

• Implement integrative approaches:

  • Combine multiple methodologies to address the same question

  • Use both in vitro and in vivo approaches when possible

  • Consider system-level analyses (transcriptomics, proteomics) alongside focused experiments

• Design decisive experiments:

  • Develop experiments specifically designed to resolve contradictions

  • Include appropriate positive and negative controls

  • Consider epistasis analysis with well-characterized pathway components

When publishing, transparently discuss contradictory findings and propose models that accommodate different observations or specify conditions under which different outcomes occur.

What statistical approaches are most appropriate for analyzing sugar sensitivity phenotypes in SIS3 research?

Several statistical approaches are suitable for analyzing sugar sensitivity phenotypes:

• For categorical data (e.g., germination vs. non-germination, green vs. pale seedlings):

  • Chi-square tests for comparing proportions across genotypes

  • Fisher's exact test for smaller sample sizes

  • Logistic regression for modeling binary outcomes with multiple factors

• For continuous measurements (e.g., cotyledon area, root length, chlorophyll content):

  • ANOVA followed by appropriate post-hoc tests (Tukey's HSD, Dunnett's test)

  • Linear mixed models when including random effects (e.g., experimental batch)

  • Non-parametric alternatives (Kruskal-Wallis, Mann-Whitney) when assumptions of normality are violated

• For dose-response relationships:

  • Fit sigmoidal dose-response curves to determine EC50 values

  • Compare curve parameters (slope, maximum response) across genotypes

  • Example application: analyzing growth inhibition across a range of sugar concentrations

• For time-course experiments:

  • Repeated measures ANOVA or mixed models with time as a factor

  • Growth curve analysis using non-linear models

  • Area under the curve (AUC) calculations followed by standard statistical tests

• For multivariate phenotypes:

  • Principal component analysis (PCA) to reduce dimensionality

  • MANOVA for simultaneous analysis of multiple response variables

  • Cluster analysis to identify patterns across multiple phenotypic traits

Sample size determination should be based on power analysis, with typical experiments requiring at least 30-50 seedlings per genotype per condition to detect biologically meaningful differences in sugar sensitivity.

What are common technical challenges in studying SIS3 and how can they be overcome?

Researchers commonly encounter several challenges when studying SIS3:

• Protein solubility issues:

  • Challenge: SIS3 contains predicted transmembrane domains, potentially causing solubility problems during recombinant expression.

  • Solution: Express only the soluble domains (e.g., RING domain) for biochemical studies, use detergents for full-length protein, or employ membrane-mimicking systems like nanodiscs.

• Maintaining E3 ligase activity:

  • Challenge: RING E3 ligases can lose activity due to oxidation of cysteine residues in the RING domain.

  • Solution: Include reducing agents (DTT, β-mercaptoethanol) in buffers, handle proteins under nitrogen atmosphere, and verify activity before each experiment.

• Identifying physiological substrates:

  • Challenge: Determining the authentic targets of SIS3 in vivo is difficult.

  • Solution: Combine proteomics approaches (e.g., comparing ubiquitinomes of wild-type and sis3 plants) with targeted validation of candidates through in vitro and in vivo assays.

• Variability in sugar sensitivity assays:

  • Challenge: Sugar sensitivity phenotypes can be affected by light conditions, media composition, and seed storage conditions.

  • Solution: Standardize growth conditions rigorously, include multiple controls, and use fresh seed batches harvested from plants grown under identical conditions.

• Genetic redundancy:

  • Challenge: Arabidopsis contains numerous RING E3 ligases that may have overlapping functions with SIS3.

  • Solution: Generate higher-order mutants by crossing sis3 with mutants of related E3 ligases, or use inducible artificial microRNA approaches to silence multiple genes simultaneously.

• Transient nature of ubiquitination:

  • Challenge: Ubiquitinated proteins are often rapidly degraded, making detection difficult.

  • Solution: Use proteasome inhibitors (MG132) in experiments, employ tandem-repeated ubiquitin-binding entities (TUBEs) to enrich ubiquitinated proteins, and use denaturing conditions during extraction.

How can researchers troubleshoot failed in vitro ubiquitination assays with recombinant SIS3?

When in vitro ubiquitination assays with recombinant SIS3 fail, consider the following troubleshooting approaches:

• Protein quality issues:

  • Verify protein integrity by SDS-PAGE and Western blotting

  • Check for degradation or aggregation

  • Ensure the RING domain is intact (Coomassie staining, mass spectrometry)

  • Use freshly purified protein or minimize freeze-thaw cycles

• Reaction component analysis:

  • Test each component separately with control reactions

  • Verify E1 and E2 activity using known functional E3 ligases

  • Try different E2 enzymes (UBC8 was successful in published studies )

  • Ensure ATP is fresh and at the correct concentration (typically 2-5 mM)

• Buffer optimization:

  • Test different pH ranges (typically pH 7.0-8.0)

  • Adjust salt concentration (50-150 mM NaCl)

  • Include zinc (10-50 μM ZnCl₂) to stabilize the RING domain

  • Add reducing agents (0.5-1 mM DTT) to maintain cysteine residues

• Detection methods:

  • Try different antibodies for ubiquitin detection

  • Consider using tagged ubiquitin (HA-Ub, FLAG-Ub) for enhanced detection

  • Increase sensitivity with chemiluminescent or fluorescent detection systems

  • Extend reaction time (1-3 hours) or increase enzyme concentrations

• Temperature and time variables:

  • Test different incubation temperatures (25°C, 30°C, 37°C)

  • Perform time-course experiments to determine optimal reaction time

  • Take aliquots at different time points to monitor reaction progression

• Substrate considerations:

  • If testing specific substrates, ensure they are properly folded

  • Try different substrate concentrations

  • Consider using known E3 substrates as positive controls

A systematic approach working through these variables will help identify and resolve issues with in vitro ubiquitination assays.

What are promising future research directions for understanding SIS3 function in plant development and stress responses?

Several promising research directions could advance our understanding of SIS3:

• Identification of physiological substrates:

  • Perform quantitative proteomics comparing ubiquitinomes of wild-type and sis3 mutants

  • Develop proximity labeling approaches to identify proteins in the vicinity of SIS3

  • Characterize how substrate ubiquitination changes under different sugar conditions

• Integration with energy homeostasis networks:

  • Investigate connections between SIS3 and energy sensors like SnRK1

  • Examine the role of SIS3 in balancing growth and stress responses

  • Study metabolic adaptations in sis3 mutants under energy limitation

• Cross-talk with other signaling pathways:

  • Explore connections between sugar signaling and hormone responses

  • Investigate SIS3's role in integrating environmental and developmental signals

  • Study potential roles in pathogen defense, as suggested by transcriptional responses

• Structural biology approaches:

  • Determine the three-dimensional structure of SIS3's RING domain

  • Characterize structural changes upon E2 binding

  • Develop structure-based approaches to identify chemical modulators of SIS3 activity

• Translational research:

  • Apply knowledge of SIS3 function to crop improvement

  • Identify and characterize SIS3 homologs in major crops

  • Develop strategies to modify sugar sensitivity for enhanced stress tolerance

• Systems biology integration:

  • Create computational models of sugar signaling incorporating SIS3

  • Apply network analysis to position SIS3 in global regulatory networks

  • Use multi-omics approaches to comprehensively characterize SIS3 function

• Evolutionary perspectives:

  • Compare SIS3 function across plant species

  • Investigate how sugar sensing mechanisms evolved in plants

  • Examine the co-evolution of E3 ligases and their substrates in sugar signaling

These directions would significantly advance our understanding of SIS3's biological roles and potentially lead to applications in agriculture.

How might advanced genetic technologies be applied to further elucidate SIS3 function?

Advanced genetic technologies offer powerful approaches to study SIS3:

• CRISPR/Cas9 genome editing:

  • Generate precise mutations in specific domains of SIS3

  • Create allelic series with varying degrees of function

  • Introduce tags at endogenous loci for visualization and purification

  • Develop inducible degradation systems to control SIS3 levels temporally

• Synthetic biology approaches:

  • Engineer synthetic ubiquitination circuits to study SIS3 specificity

  • Create chimeric E3 ligases to study domain functions

  • Develop optogenetic tools to control SIS3 activity with light

• Single-cell technologies:

  • Apply single-cell RNA-seq to understand cell-type specific responses to SIS3 disruption

  • Use single-cell proteomics to examine variation in protein levels

  • Develop single-cell reporters for ubiquitination activity

• In vivo structural studies:

  • Apply proximity labeling (BioID, TurboID) to map SIS3 interaction networks in living cells

  • Use in-cell NMR to study structural changes in SIS3 in response to sugar

  • Develop FRET-based sensors for monitoring SIS3 activity in real-time

• Multi-omics integration:

  • Combine transcriptomics, proteomics, metabolomics, and phenomics data

  • Apply machine learning to identify patterns and predict SIS3 functions

  • Create comprehensive models of SIS3's role in sugar signaling networks

• Tissue-specific and inducible approaches:

  • Generate tissue-specific knockout or overexpression lines

  • Develop chemically or environmentally inducible SIS3 expression systems

  • Study spatiotemporal aspects of SIS3 function during development