Recombinant HTH-type transcriptional regulator reg1 (reg1)

What is the primary molecular function of REG1 in yeast cells?

REG1 functions as a regulatory subunit of protein phosphatase type 1 (PP1), which is encoded by the essential gene GLC7 in Saccharomyces cerevisiae. The primary role of REG1 is to target PP1 activity specifically to proteins involved in the glucose repression regulatory pathway. This targeting specificity allows for precise control of metabolic responses to changing glucose conditions. REG1 physically associates with GLC7 (PP1), demonstrating strong and specific interactions in two-hybrid system analyses, and REG1-GLC7 fusion proteins can be co-immunoprecipitated from cell extracts, confirming their direct physical interaction .

Methodologically, researchers can study this interaction through:

• Yeast two-hybrid assays to detect protein-protein interactions

• Co-immunoprecipitation experiments with tagged versions of REG1 and GLC7

• Complementation studies where REG1 overexpression can suppress certain GLC7 mutant phenotypes

How does REG1 regulate the Snf1 kinase pathway?

REG1's major known function is to repress Snf1, the yeast ortholog of mammalian AMP-activated protein kinase (AMPK). This repression occurs through stimulating the GLC7-dependent dephosphorylation of Snf1 at a specific residue (Thr-210) . This regulatory relationship forms a critical control point in cellular energy metabolism.

When studying this regulatory relationship, researchers should:

• Monitor phosphorylation status of Snf1-Thr210 using phospho-specific antibodies

• Perform kinase activity assays in reg1Δ strains compared to wild-type cells

• Analyze expression of Snf1-regulated genes in the presence and absence of REG1

What evidence supports REG1's role in the glucose repression pathway?

Multiple lines of genetic and biochemical evidence indicate that REG1 functions with GLC7 in regulating glucose repression. A specific mutation in GLC7 (glc7-T152K) relieves glucose repression but does not affect other GLC7 functions like glycogen metabolism. Importantly, overexpression of REG1 fusion protein can suppress this mutant defect in glucose repression, indicating their cooperative function in this specific pathway .

Research methodologies to establish this relationship include:

• Glucose repression assays measuring expression of glucose-repressed genes

• Suppressor screens to identify functional relationships

• Epistasis analysis with different components of the glucose sensing pathway

How do stress conditions affect REG1 function and localization?

Under specific stress conditions, particularly arsenite exposure, REG1 mediates the relocalization of GLC7 (PP1) into cytoplasmic granules. This stress-induced relocalization is highly specific to arsenite and appears distinct from canonical stress granules. The process involves a regulatory loop where GLC7's localization is influenced by its own activity through a mechanism centered around Snf1 .

To investigate this phenomenon, researchers should:

• Use fluorescently tagged GLC7 to monitor subcellular localization

• Compare responses across multiple stress conditions

• Analyze localization patterns in reg1Δ and snf1Δ mutants

What molecular mechanisms allow REG1 to specifically target PP1 to glucose repression pathway proteins?

The specificity of REG1-GLC7 interactions with target proteins in the glucose repression pathway likely involves specific protein domains and recognition sequences. Research suggests that REG1 acts as a targeting subunit that brings PP1 in proximity to its substrates. The glc7-T152K mutation specifically disrupts the interaction with REG1 but not other PP1 regulatory subunits, indicating a specific binding interface .

Advanced experimental approaches include:

• Domain mapping through truncation and point mutation analysis

• Structural studies of REG1-GLC7 complexes

• Proteomic identification of the REG1-GLC7 interactome under different conditions

• CRISPR-mediated mutagenesis of potential interaction interfaces

How does the REG1-SNF1 regulatory circuit respond to different metabolic signals?

The REG1-SNF1 regulatory circuit functions as a metabolic sensor that responds to changes in cellular energy status. When glucose is abundant, REG1-GLC7 actively dephosphorylates and inactivates SNF1. Under glucose limitation or other stress conditions, this repression is relieved, allowing SNF1 activation. Intriguingly, this circuit also appears to regulate its own components, as suggested by the finding that Glc7 influences its own localization through a regulatory loop centered around Snf1 .

Methodological approaches for dissecting this circuit include:

• Time-course analyses of REG1-SNF1 interactions following metabolic shifts

• Quantitative phosphoproteomics to identify all targets affected by REG1-mediated dephosphorylation

• Real-time monitoring of SNF1 activity using FRET-based biosensors

• Metabolomic profiling in reg1Δ versus wild-type cells under different conditions

What is the relationship between REG1-mediated stress granule formation and translational control?

Arsenite induces potent translational inhibition, and translational recovery is strongly dependent on GLC7, but independent of GLC7's well-established role in regulating eIF2α. This suggests a novel form of stress-induced cytoplasmic granule and a new mode of translational control by GLC7 that is mediated by REG1 . The specific mechanisms linking these granules to translational control remain to be fully elucidated.

Research approaches should include:

• Polysome profiling in reg1Δ versus wild-type cells during stress and recovery

• Proximity labeling to identify proteins associated with REG1-GLC7 granules

• In vitro translation assays with components isolated from stressed cells

• Ribosome profiling to identify translational effects at nucleotide resolution

How do the different regulatory subunits of SNF1 influence REG1-mediated functions?

The Snf1 complex consists of the catalytic subunit (Snf1), one of three β-subunits (Sip1, Sip2, Gal83), and a single γ-subunit (Snf4). These different regulatory configurations may mediate distinct aspects of REG1 function. Research has shown that loss of the γ-subunit Snf4 phenocopies certain aspects of Snf1 regulation .

Systematic investigation requires:

• Comparison of reg1Δ phenotypes in strains lacking different Snf1 regulatory subunits

• Analysis of GLC7 granule formation in mutants lacking specific Snf1 complex components

• Co-immunoprecipitation studies to determine if REG1 preferentially interacts with specific Snf1 complex configurations

• Phosphoproteomics to identify differential substrate targeting

What are the most effective methods for analyzing REG1-dependent transcriptional regulation?

REG1 indirectly affects transcriptional regulation through its regulation of the glucose repression pathway. To effectively analyze these effects, researchers should employ multiple complementary approaches:

• Genome-wide expression analysis:

  • RNA-seq or microarray analysis comparing wild-type, reg1Δ, and glc7 mutant strains

  • ChIP-seq to identify binding sites of transcription factors affected by REG1-GLC7 activity

  • NET-seq to measure nascent transcription in response to REG1 perturbation

• Reporter gene assays:

  • Luciferase reporter constructs containing promoters of glucose-repressed genes

  • Flow cytometry with fluorescent reporters to measure cell-to-cell variability in response

  • Time-resolved reporter assays to capture dynamic responses to glucose shifts

• Single-cell analyses:

  • Single-cell RNA-seq to identify cell-to-cell variability in REG1-dependent regulation

  • Live-cell imaging with transcriptional reporters to monitor dynamic responses

What experimental approaches can distinguish between direct and indirect effects of REG1?

Distinguishing direct from indirect effects is a common challenge in studying regulatory proteins like REG1. Several experimental approaches can help address this issue:

• Temporal analysis:

  • Use rapidly inducible or repressible REG1 constructs (e.g., auxin-inducible degron tags)

  • Monitor immediate versus delayed responses to REG1 depletion or induction

  • Implement kinetic modeling to infer direct versus indirect relationships

• Biochemical approaches:

  • In vitro dephosphorylation assays with purified components

  • Substrate trapping using catalytically inactive GLC7 variants

  • Crosslinking mass spectrometry to identify direct interaction partners

• Genetic approaches:

  • Epistasis analysis with components of REG1-regulated pathways

  • Suppressor screens to identify direct functional relationships

  • Synthetic genetic array analysis to map genetic interaction networks

How can researchers optimize systems for recombinant REG1 expression and purification?

Obtaining pure, functional recombinant REG1 protein is essential for many biochemical studies:

• Expression systems:

  • Bacterial expression often yields inclusion bodies; consider using specialized strains (e.g., Rosetta for rare codon optimization)

  • Yeast expression systems maintain proper folding and post-translational modifications

  • Insect cell systems may provide a good balance of yield and proper folding

• Purification strategies:

  • Tandem affinity tags (e.g., His-MBP tag) to improve solubility and purity

  • Size-exclusion chromatography to isolate monomeric versus oligomeric forms

  • Consider co-expression with GLC7 to stabilize the complex

• Functional validation:

  • In vitro phosphatase assays with known REG1-GLC7 substrates

  • Circular dichroism to confirm proper folding

  • Limited proteolysis to assess structural integrity

How does REG1 contribute to arsenite-specific stress responses?

REG1 plays a specific role in arsenite stress response, mediating the relocalization of GLC7 into cytoplasmic granules that are distinct from canonical stress granules. This response appears highly specific to arsenite compared to other stressors .

When investigating this phenomenon, researchers should:

• Compare multiple stress conditions (oxidative, heat, osmotic) to identify arsenite-specific responses

• Use fluorescently tagged proteins to track co-localization with known stress granule markers

• Perform time-course experiments to determine the kinetics of granule formation and dissolution

• Analyze the proteome and phosphoproteome of isolated REG1-dependent granules

What is the relationship between REG1 and translational recovery after stress?

Arsenite induces potent translational inhibition, and translational recovery is strongly dependent on GLC7, the phosphatase regulated by REG1. This recovery mechanism appears to be independent of GLC7's established role in regulating eIF2α, suggesting a novel mode of translational control .

Experimental approaches should include:

• Polysome profiling in wild-type versus reg1Δ cells during stress and recovery phases

• Phosphorylation analysis of translation initiation factors in the presence/absence of REG1

• Ribosome profiling to identify transcripts most affected by REG1-dependent regulation

• Genetic separation-of-function studies using GLC7 mutants specifically defective in REG1 interaction

How do specific mutations in REG1 affect its stress response functions?

Understanding the structure-function relationships in REG1 is crucial for dissecting its diverse roles:

• Targeted mutagenesis approaches:

  • Create point mutations in putative GLC7-binding domains

  • Mutate potential phosphorylation sites that might regulate REG1 activity

  • Generate truncation variants to identify functional domains

• Phenotypic characterization:

  • Assess growth under various stress conditions

  • Monitor GLC7 localization in response to stress

  • Measure SNF1 phosphorylation status in mutant backgrounds

• Biochemical characterization:

  • Determine GLC7 binding affinity of mutant REG1 proteins

  • Assess ability to direct GLC7 phosphatase activity toward specific substrates

  • Evaluate protein stability and turnover rates of mutant proteins

How can contradictory results in REG1 studies be reconciled?

Contradictory results are common in complex regulatory systems like the REG1-GLC7-SNF1 pathway. Researchers should consider:

• Experimental context differences:

  • Different yeast strain backgrounds may have compensatory mutations

  • Growth conditions (media composition, temperature) can dramatically affect results

  • Acute versus chronic loss of REG1 may produce different phenotypes due to compensation

• Methodological considerations:

  • Different assay sensitivities and dynamic ranges

  • Temporal resolution of measurements (immediate versus steady-state effects)

  • Tags or fusion proteins may partially compromise function

• Systematic approaches to resolution:

  • Perform experiments in multiple strain backgrounds

  • Use complementary methodologies to measure the same biological outcome

  • Consider genetic interaction context (synthetic effects may reveal redundant pathways)

What controls are essential when studying REG1-mediated phosphorylation events?

Phosphorylation studies require rigorous controls:

• Essential controls for western blot analysis:

  • Phosphatase treatment controls to confirm specificity of phospho-antibodies

  • Total protein controls alongside phosphoprotein detection

  • Multiple time points to capture dynamic phosphorylation changes

  • Quantitative loading controls independent of the regulated pathway

• Controls for phosphoproteomic studies:

  • Comparison of reg1Δ, glc7 mutant, and snf1Δ phosphoproteomes

  • Inclusion of known REG1-regulated phosphosites as internal controls

  • Spike-in standards for accurate quantification across samples

  • Validation of key phosphosites using targeted approaches (PRM mass spectrometry)

• Genetic controls:

  • Phosphomimetic and non-phosphorylatable mutants of key substrates

  • Analog-sensitive kinase mutants to distinguish direct versus indirect effects

  • Catalytically inactive phosphatase controls

How might single-cell approaches advance our understanding of REG1 function?

Traditional population-based studies may mask important cell-to-cell variability in REG1-mediated responses. Single-cell approaches can provide new insights:

• Single-cell multi-omics:

  • scRNA-seq to identify cell-to-cell variability in transcriptional responses

  • Single-cell proteomics to detect protein-level heterogeneity

  • Combined with lineage tracing to identify heritable regulatory states

• Live-cell imaging:

  • Real-time monitoring of REG1-GLC7 localization during stress response

  • FRET-based sensors to detect SNF1 activity in individual cells

  • Microfluidics platforms for precise temporal control of environmental shifts

• Analysis approaches:

  • Trajectory inference to identify regulatory states and transitions

  • Information theory metrics to quantify signaling fidelity

  • Network modeling of single-cell data to infer causal relationships

What is the evolutionary conservation of REG1 function across species?

Understanding the evolutionary context of REG1 can provide insights into its fundamental functions:

• Comparative genomics approaches:

  • Identify REG1 orthologs across fungal species

  • Analyze conservation of key functional domains and regulatory sites

  • Correlate presence/absence of REG1 with metabolic capabilities

• Functional complementation studies:

  • Test if REG1 orthologs from different species can complement yeast reg1Δ

  • Identify species-specific regulatory features through domain swapping

  • Reconstruct ancestral REG1 sequences to test evolutionary hypotheses

• Systems-level analysis:

  • Compare glucose repression networks across species

  • Identify conserved versus species-specific REG1 targets

  • Model the evolution of the REG1-GLC7-SNF1 regulatory circuit