Recombinant Schizosaccharomyces pombe Vacuolar transporter chaperone 1 (nrf1)

What is the relationship between Nrf1 and vacuolar transport in S. pombe?

Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is primarily known as a transcription factor that regulates antioxidant response element (ARE)-driven genes. In S. pombe, protein sorting to the vacuole (analogous to the mammalian lysosome) involves specific targeting mechanisms where proteins transit the early stages of the secretory pathway before being sorted in a late Golgi compartment . While Nrf1 itself has not been directly characterized as a vacuolar transporter chaperone in the provided research, it contains an N-terminal domain (NTD) that directs it to the endoplasmic reticulum, suggesting potential involvement in protein trafficking pathways .

Recent studies indicate that the N-terminal targeting sequences in proteins like Nrf1 can play crucial roles in determining subcellular localization. In particular, research has shown that the NTD of Nrf1 (amino acids 1-124) functions as a negative regulator of its transcriptional activity by targeting it to the endoplasmic reticulum rather than allowing nuclear localization . This targeting mechanism could potentially influence vacuolar protein trafficking pathways, though direct evidence for Nrf1's role as a vacuolar transporter chaperone requires further investigation.

How is Nrf1 structurally organized in S. pombe?

Nrf1 is a modular protein that can be divided into distinct functional domains. Based on bioinformatic analysis, Nrf1 contains at least nine domains . Most distinctively, Nrf1 differs from the related protein Nrf2 by the presence of an N-terminal domain (NTD, amino acids 1-124) and an acidic/polar region (residues 125-155) . The protein also contains an acidic domain 1 (amino acids 125-295), within which resides a Neh2-like (Neh2L) subdomain (residues 156-242) that shares 43% identity with the N-terminal Neh2 region of Nrf2 .

The Neh2L subdomain in Nrf1 contains DLG and ETGE motifs that, in Nrf2, are responsible for interaction with Keap1 . These structural features are critical for understanding how Nrf1 functions and is regulated within the cell. The modular organization of Nrf1 suggests it may perform multiple functions depending on cellular context and regulatory status.

What are the standard methods for recombinant expression of Nrf1 in S. pombe?

For recombinant expression of Nrf1 in S. pombe, researchers typically employ plasmid-based expression systems. Based on experimental approaches described in the literature, the following methodology can be applied:

• cDNA Cloning: The cDNA fragment coding for full-length Nrf1 can be obtained using RT-PCR systems. For example, in related studies, researchers used the ProSTAR ultra HF RT-PCR system with appropriate primers to amplify the target gene .

• Vector Construction: The amplified cDNA product is purified and subcloned into expression vectors compatible with S. pombe. Vectors like pcDNA3.1/V5His B can be adapted for S. pombe expression by incorporating suitable promoters and selection markers .

• Transformation: The expression constructs are introduced into S. pombe cells using standard transformation protocols. The one-step gene replacement method has been successfully used for related proteins in S. pombe .

• Selection and Verification: Transformants are selected on appropriate media containing selection markers. Expression of the recombinant protein can be verified by Western blotting using antibodies against epitope tags (e.g., V5) incorporated in the expression construct .

This methodology provides a framework for recombinant expression of Nrf1 in S. pombe, although specific optimization may be required depending on experimental objectives.

How does the N-terminal domain of Nrf1 affect its localization and function in S. pombe?

The N-terminal domain (NTD) of Nrf1 plays a critical role in determining its subcellular localization and regulating its function. Experimental evidence has demonstrated that the NTD negatively regulates Nrf1's transactivation activity through its ability to direct the protein to the endoplasmic reticulum instead of the nucleus .

Deletion mutagenesis studies have revealed that removal of residues 2-120 (the NTD) from Nrf1 causes a substantial increase in its transactivation activity in both RL-34 and COS-1 cell lines . Further deletion of N-terminal amino acids up to residue 170 caused no additional increase in transactivation activity, indicating that the NTD is the major negative regulating domain in Nrf1 .

This regulatory mechanism was further confirmed using Gal4D fusion constructs. Transfection experiments with constructs expressing the Gal4D protein fused to various N-terminal truncations of Nrf1 showed that:

• Removal of residues 1-44 had little effect on reporter gene expression

• Removal of residues 1-65 or 1-119 caused a gradual step-like increase in transactivation activity

• Maximal activation occurred when almost the entire NTD was deleted

These findings suggest that in S. pombe, the NTD of Nrf1 would likely function similarly to direct the protein to the endoplasmic reticulum, potentially affecting its involvement in vacuolar transport pathways.

What experimental approaches can distinguish between Nrf1's roles in transcriptional regulation versus vacuolar transport?

To distinguish between Nrf1's potential dual roles in transcriptional regulation and vacuolar transport, several experimental approaches can be employed:

• Domain-specific mutational analysis: Creating mutants with specific alterations in functional domains can help dissect different functions. For example:

  • NTD deletion mutants to assess changes in both transcriptional activity and vacuolar protein sorting

  • Mutations in the Neh2-like domain to disrupt potential protein-protein interactions while preserving localization signals

• Fusion protein approach: Creating fusion proteins between Nrf1 domains and reporter proteins can help track localization and function:

  • NTD-reporter protein fusions to track subcellular localization

  • Similar to experiments where the NTD from Nrf1 was attached to the N-terminus of Nrf2, redirecting it from the nucleus to the endoplasmic reticulum

  • CPY1-SUC2 style fusion constructs (as used with S. pombe carboxypeptidase Y) to identify vacuolar sorting signals

• Colocalization studies: Immunocytochemistry to determine whether Nrf1 colocalizes with:

  • Endoplasmic reticulum markers

  • Vacuolar membrane proteins

  • Vacuolar transport machinery components

• Protein-protein interaction analyses: Techniques such as co-immunoprecipitation, yeast two-hybrid, or proximity labeling to identify Nrf1 interaction partners involved in either transcriptional regulation or vacuolar transport.

These approaches can provide valuable insights into the multifunctional nature of Nrf1 in S. pombe cellular processes.

What is the impact of ppr10 deletion on mitochondrial function, and how might this relate to Nrf1 activity?

The deletion of ppr10 in S. pombe has significant effects on mitochondrial function. Studies have shown that the ppr10 deletion mutant exhibits growth defects in respiratory media and is dramatically impaired for viability during the late-stationary phase . At the molecular level, deletion of ppr10 affects the accumulation of specific mitochondrial mRNAs and severely impairs mitochondrial protein synthesis .

These findings suggest that Ppr10 plays a general role in mitochondrial protein synthesis. Ppr10 interacts with another protein called Mpa1 both in vivo and in vitro, and these proteins colocalize in the mitochondrial matrix . Interestingly, ppr10 and mpa1 deletion mutants exhibit very similar phenotypes, suggesting functional relationships .

The potential relationship between ppr10 deletion effects and Nrf1 activity could be explored through several mechanisms:

• Stress response pathways: Mitochondrial dysfunction caused by ppr10 deletion may trigger cellular stress responses that involve Nrf1-mediated transcriptional regulation.

• Redox signaling: Impaired mitochondrial function often leads to altered redox status, which could affect Nrf1 activity and its regulation of antioxidant response elements.

• Protein quality control: Both mitochondrial protein synthesis (affected by ppr10) and ER protein processing (influenced by Nrf1 localization) are critical for cellular homeostasis and may have regulatory crosstalk.

Future research could investigate whether Nrf1 expression or activity is altered in ppr10 deletion mutants, potentially revealing functional connections between these pathways.

What are the optimal conditions for purifying recombinant Nrf1 from S. pombe?

Purification of recombinant Nrf1 from S. pombe requires careful consideration of the protein's properties and subcellular localization. Based on the available information about Nrf1 and similar proteins in S. pombe, the following purification strategy is recommended:

• Expression construct design:

  • Incorporate an affinity tag (such as V5, His, or FLAG) to facilitate purification

  • Consider using an inducible promoter system to control expression levels

  • The pcDNA3.1/V5His B vector system has been successfully used for similar proteins

• Cell lysis optimization:

  • Since Nrf1 is largely localized to the endoplasmic reticulum due to its NTD , membrane fractionation approaches are essential

  • Use a combination of mechanical disruption (e.g., glass beads) and detergent solubilization

  • Try mild detergents first (e.g., 1% Triton X-100 or 0.5% NP-40) to preserve protein structure and function

• Purification protocol:

  • Affinity chromatography using the incorporated tag (e.g., Ni-NTA for His-tagged proteins)

  • Consider including protease inhibitors throughout purification to prevent degradation

  • Ion exchange chromatography as a secondary purification step

  • Size exclusion chromatography for final polishing and buffer exchange

• Protein stabilization:

  • Include reducing agents (e.g., DTT or β-mercaptoethanol) to maintain cysteine residues

  • Optimize buffer conditions (pH, salt concentration) based on Nrf1's isoelectric point

  • Consider including glycerol (10-15%) in storage buffers to enhance stability

This approach provides a starting point that should be optimized based on experimental results and specific research requirements.

How can researchers effectively generate and validate Nrf1 mutants in S. pombe?

Generating and validating Nrf1 mutants in S. pombe requires systematic approaches to ensure both successful creation of the desired mutations and proper functional characterization. Based on methodologies described in the literature, the following comprehensive strategy is recommended:

• Mutant design and generation:

  • Two primary strategies have proven effective: site-directed mutagenesis for small internal deletions and PCR-based approaches for larger modifications

  • For internal deletions (e.g., Nrf1 Δ125-170, Nrf1 ΔETGE), the Quick-Change Site Directed Mutagenesis kit can be employed with appropriate primer pairs

  • For N- and C-terminal truncations (e.g., Nrf1 Δ2-120, Nrf1 Δ2-150), PCR with specific primers followed by subcloning into expression vectors is effective

  • All constructs should be sequence-verified to confirm the introduced mutations

• Expression validation:

  • Western blot analysis using antibodies against Nrf1 or incorporated epitope tags

  • Quantitative comparison of mutant vs. wild-type expression levels

  • Assessment of protein stability and half-life through cycloheximide chase experiments

• Localization validation:

  • Immunocytochemistry to determine subcellular localization of mutant proteins

  • Comparison with appropriate organelle markers (e.g., endoplasmic reticulum markers)

  • Subcellular fractionation followed by Western blotting as a complementary approach

• Functional validation:

  • Transactivation assays using reporter constructs containing Nrf1-responsive elements

  • Complementation studies in Nrf1-deficient strains to assess functional rescue

  • Protein-protein interaction analyses to evaluate effects on key binding partners

This systematic approach ensures comprehensive characterization of Nrf1 mutants, enabling reliable interpretation of experimental results and mechanistic insights.

What techniques are most effective for studying Nrf1's potential interactions with vacuolar transport machinery?

To investigate potential interactions between Nrf1 and the vacuolar transport machinery in S. pombe, researchers should employ a multi-faceted approach combining genetic, biochemical, and imaging techniques:

• Genetic interaction screening:

  • Systematic analysis of genetic interactions between Nrf1 and known vacuolar transport components

  • Creation of double mutants combining Nrf1 mutations with mutations in vacuolar protein sorting genes

  • Synthetic genetic array (SGA) analysis to identify genome-wide genetic interactions

• Protein-protein interaction studies:

  • Co-immunoprecipitation assays to identify direct protein interactions

  • Yeast two-hybrid screening using Nrf1 domains as bait against vacuolar transport proteins

  • Proximity labeling techniques (BioID or APEX) to identify proteins in close proximity to Nrf1 in living cells

  • Cross-linking mass spectrometry to capture transient interactions

• Functional transport assays:

  • Vacuolar protein sorting assays using reporter proteins like CPY1-invertase fusions

  • Pulse-chase experiments to track protein transport through the secretory pathway

  • Quantitative analysis of vacuolar protein missorting in Nrf1 mutant strains

• Advanced imaging approaches:

  • Fluorescence microscopy with fluorescently tagged Nrf1 and vacuolar markers

  • Live-cell imaging to track protein movement in real-time

  • Super-resolution microscopy to visualize detailed subcellular structures

  • Correlative light and electron microscopy for ultrastructural analysis

• Proteomic profiling:

  • Comparative proteomics of vacuolar fractions from wild-type and Nrf1 mutant strains

  • Quantitative analysis of changes in the vacuolar proteome upon Nrf1 manipulation

  • Post-translational modification analysis to identify regulatory events

These complementary approaches provide a comprehensive toolkit for investigating Nrf1's potential roles in vacuolar transport processes in S. pombe.

How should researchers interpret contradictory results between Nrf1 localization and function studies?

When faced with contradictory results between Nrf1 localization and function studies, researchers should consider several explanations and adopt a structured approach to resolve these discrepancies:

• Multiple functional pools: Nrf1 may exist in different cellular compartments with distinct functions. The N-terminal domain directs Nrf1 primarily to the endoplasmic reticulum , but a fraction might localize to other compartments under specific conditions. Consider quantitative assessment of Nrf1 distribution across cellular compartments under different experimental conditions.

• Processing or post-translational modifications: Nrf1 may undergo proteolytic processing or modifications that alter its localization and function. For example:

  • N-terminal processing could remove the ER-targeting domain

  • Phosphorylation or other modifications might regulate nuclear import/export

  • Validate the presence of potential processed forms via Western blotting with antibodies targeting different regions of Nrf1

• Context-dependent regulation: Experimental conditions or cell state can influence results:

  • Cell cycle phase may affect Nrf1 localization and activity

  • Stress conditions might trigger relocalization of Nrf1

  • Overexpression artifacts may disrupt normal localization patterns

  • Use synchronized cultures and physiologically relevant expression levels to minimize variables

• Technical considerations:

  • Fixation methods for microscopy can alter apparent protein localization

  • Epitope tags may interfere with localization signals or protein interactions

  • Compare results using multiple fixation protocols and different tag positions

  • Validate with both tagged and untagged proteins when possible

• Reconciliation approach: Design experiments that directly address the contradictions:

  • Create chimeric proteins where localization can be experimentally manipulated

  • Use inducible systems to dynamically track Nrf1 movement between compartments

  • Employ proximity labeling to identify interacting partners in different locations

By systematically exploring these possibilities, researchers can develop a more nuanced understanding of Nrf1's complex biology.

What are the key challenges in distinguishing the functions of Nrf1 from other related proteins in S. pombe?

Distinguishing the functions of Nrf1 from related proteins in S. pombe presents several significant challenges that researchers must address through careful experimental design:

• Structural and functional overlap: Nrf1 shares significant structural similarities with other proteins, particularly Nrf2. For example:

  • The Neh2-like subdomain in Nrf1 (residues 156-242) shares 43% identity with the N-terminal Neh2 region of Nrf2

  • Both Nrf1 and Nrf2 regulate ARE (antioxidant response element)-driven genes

  • Develop assays that can distinguish between the activities of these related factors

• Compensatory mechanisms: Deletion or mutation of Nrf1 may lead to compensatory upregulation of related proteins:

  • Perform simultaneous profiling of multiple related proteins when manipulating Nrf1

  • Consider creating multiple knockout/knockdown strains to address redundancy

  • Use acute inactivation strategies (e.g., degron approaches) to minimize compensation

• Differential regulation: Nrf1 and related proteins may be regulated differently:

  • Nrf1 is negatively regulated by its N-terminal domain targeting it to the ER

  • Nrf2 is primarily regulated by Keap1-mediated degradation

  • Different regulatory mechanisms may converge on similar downstream pathways

  • Design experiments that specifically target unique regulatory features of each protein

• Experimental artifacts in recombinant systems:

  • Overexpression may disrupt normal regulatory mechanisms

  • Epitope tags may interfere with protein-specific interactions

  • Use genomic integration of tagged constructs at endogenous loci where possible

  • Validate key findings with multiple experimental approaches

• Comparative analysis approach:

  • Systematic comparison of phenotypes between different mutants

  • Transcriptomic/proteomic profiling to identify protein-specific targets

  • Domain swapping experiments to identify functional determinants

  • Evolutionary analysis to identify conserved and divergent features

This multi-faceted approach can help researchers delineate the specific functions of Nrf1 within the complex regulatory network of S. pombe.

How can researchers troubleshoot low expression or insolubility issues with recombinant Nrf1?

When encountering low expression or insolubility issues with recombinant Nrf1 in S. pombe, researchers should implement a systematic troubleshooting strategy:

• Expression optimization:

  • Evaluate different promoter systems (constitutive vs. inducible)

  • Optimize codon usage for efficient translation in S. pombe

  • Test expression at different growth phases and temperatures

  • Consider using fusion partners known to enhance solubility (e.g., SUMO, MBP, or GST)

  • Experiment with different media compositions and induction conditions

• Solubility enhancement strategies:

  • Since Nrf1 is largely associated with the endoplasmic reticulum membrane due to its N-terminal domain , specialized extraction methods are required

  • Test a panel of detergents at various concentrations (e.g., CHAPS, DDM, or Triton X-100)

  • Add stabilizing agents such as glycerol, specific ions, or low concentrations of denaturants

  • Consider expressing truncated versions lacking the membrane-targeting NTD

  • Explore extraction at different pH values and ionic strengths

• Protein engineering approaches:

  • Design constructs lacking regions predicted to cause aggregation

  • Remove the N-terminal domain (amino acids 1-124) which targets Nrf1 to the ER

  • Introduction of solubility-enhancing mutations based on structural predictions

  • Creation of chimeric proteins with well-expressed S. pombe proteins

• Co-expression strategies:

  • Co-express potential binding partners or chaperones

  • Test co-expression with molecular chaperones specific to the ER

  • Expression of protein disulfide isomerases to assist proper folding

• Expression monitoring and analysis:

  • Analyze expression at both mRNA (RT-qPCR) and protein levels

  • Determine if low protein levels are due to poor transcription, translation, or protein degradation

  • Examine potential toxicity effects using growth curve analysis

  • Monitor for potential proteolytic degradation by adding protease inhibitors

This comprehensive approach addresses multiple aspects of recombinant protein expression and provides strategies to overcome common challenges encountered with membrane-associated proteins like Nrf1.

What are promising approaches for investigating potential connections between Nrf1 and mitochondrial function in S. pombe?

The investigation of potential connections between Nrf1 and mitochondrial function in S. pombe represents an exciting frontier for research, particularly given the observed effects of PPR proteins like Ppr10 on mitochondrial processes . Several promising approaches can be employed:

• Comparative phenotypic analysis:

  • Systematic comparison of growth phenotypes between Nrf1 and mitochondrial gene mutants (e.g., ppr10) in respiratory vs. fermentative conditions

  • Analysis of viability during stationary phase, as ppr10 deletion dramatically impairs late-stationary phase viability

  • Mitochondrial morphology studies using fluorescent markers in Nrf1 mutant backgrounds

  • Measurement of mitochondrial membrane potential and respiratory capacity

• Transcriptional regulation studies:

  • Genome-wide transcriptional profiling to identify Nrf1-dependent regulation of nuclear-encoded mitochondrial genes

  • Chromatin immunoprecipitation (ChIP) analysis to identify direct Nrf1 binding to promoters of mitochondrial genes

  • Reporter assays to validate Nrf1-responsive elements in mitochondrial gene promoters

  • Analysis of how mitochondrial stress affects Nrf1 activation and target gene expression

• Protein-level interactions:

  • Proteomics analysis of mitochondrial fractions in wild-type vs. Nrf1 mutant strains

  • Investigation of potential physical interactions between Nrf1 and mitochondrial proteins

  • Examination of whether Nrf1's ER localization facilitates communication with mitochondria at ER-mitochondria contact sites

  • Study of how Nrf1 might influence the import of nuclear-encoded proteins into mitochondria

• Stress response integration:

  • Analysis of how oxidative stress affects both Nrf1 activity and mitochondrial function

  • Investigation of retrograde signaling from mitochondria to the nucleus via Nrf1

  • Examination of whether Nrf1 coordinates cellular responses to mitochondrial dysfunction

  • Study of potential cross-regulation between Nrf1 and mitochondrial quality control systems

These approaches can provide valuable insights into the potential roles of Nrf1 in maintaining mitochondrial homeostasis in S. pombe, potentially revealing conserved mechanisms relevant to human disease.

How might advanced genetic engineering techniques enhance our understanding of Nrf1 function?

Advanced genetic engineering techniques offer powerful approaches to deepen our understanding of Nrf1 function in S. pombe:

• CRISPR-Cas9 genome editing:

  • Generation of precise point mutations or domain deletions at the endogenous Nrf1 locus

  • Creation of fluorescent protein knock-ins for live-cell imaging of endogenous Nrf1

  • Multiplexed editing to simultaneously modify Nrf1 and potential interaction partners

  • Insertion of inducible degron tags for rapid protein depletion studies

  • Engineering conditional alleles to study essential functions

• Single-cell analysis techniques:

  • Single-cell RNA-seq to explore cell-to-cell variability in Nrf1-dependent responses

  • Time-lapse microscopy with fluorescent reporters to track dynamic Nrf1 activities

  • Microfluidic approaches to study Nrf1 function under precisely controlled conditions

  • Mass cytometry to simultaneously measure multiple parameters in Nrf1 mutant populations

• Synthetic biology approaches:

  • Creation of minimal synthetic circuits to reconstitute Nrf1 regulatory modules

  • Design of orthogonal systems to control Nrf1 activity with external stimuli

  • Engineering of synthetic protein interaction domains to rewire Nrf1 signaling

  • Development of biosensors to monitor Nrf1 activity in real-time

• Comparative evolutionary approaches:

  • Systematic replacement of S. pombe Nrf1 with orthologs from other species

  • Identification of conserved functional domains through complementation studies

  • Analysis of how different selective pressures have shaped Nrf1 function across species

  • Ancestral sequence reconstruction to study the evolution of Nrf1 regulation

• Multi-omics integration:

  • Coordinated analysis of transcriptome, proteome, and metabolome in Nrf1 mutants

  • Network modeling to identify key nodes in Nrf1-dependent regulatory networks

  • Machine learning approaches to predict Nrf1 functions from integrated datasets

  • Systems biology framework to understand emergent properties of Nrf1-regulated processes

These cutting-edge approaches can provide unprecedented insights into Nrf1 biology, potentially revealing novel functions and regulatory mechanisms.

What potential therapeutic applications could emerge from understanding Nrf1 function in S. pombe?

Research on Nrf1 function in S. pombe could lead to several promising therapeutic applications, particularly in areas related to stress response, protein homeostasis, and metabolic regulation:

• Neurodegenerative disease interventions:

  • Understanding how Nrf1's N-terminal domain regulates its localization to the endoplasmic reticulum could inform therapies targeting protein misfolding diseases

  • S. pombe models of Nrf1 regulation could help identify small molecules that modulate Nrf1 activity in specific cellular compartments

  • Since LRPPRC mutations (a mammalian functional homolog of yeast Pet309) cause French-Canadian-type Leigh syndrome , insights from S. pombe Nrf1 studies might inform therapeutic approaches for this neurodegenerative disorder

• Cancer therapy approaches:

  • Elucidating the mechanisms by which Nrf1 regulates antioxidant response elements could reveal novel targets for cancer therapy

  • Understanding differences between Nrf1 and Nrf2 regulation could enable selective modulation of these pathways in cancer cells

  • S. pombe models could serve as platforms for screening compounds that specifically target Nrf1-dependent processes

• Mitochondrial disease treatments:

  • Insights into how Nrf1 might influence mitochondrial protein synthesis (similar to Ppr10's role ) could inform therapeutic strategies for mitochondrial disorders

  • Understanding the relationship between ER-localized Nrf1 and mitochondrial function could reveal new intervention points for diseases involving ER-mitochondria communication

  • Identification of small molecules that enhance mitochondrial function through Nrf1-dependent pathways

• Aging-related interventions:

  • The impaired viability of ppr10 deletion mutants during late-stationary phase suggests connections to cellular aging processes

  • Understanding how Nrf1 regulates cellular stress responses could identify interventions to promote cellular resilience during aging

  • S. pombe as a model system could facilitate high-throughput screening for compounds that extend cellular lifespan through Nrf1-dependent mechanisms

• Metabolic disorder therapies:

  • Exploration of how Nrf1 influences cellular energy metabolism could reveal targets for metabolic disease intervention

  • Understanding the interplay between Nrf1 and vacuolar/lysosomal function could inform approaches to lysosomal storage disorders

  • Identification of conserved regulatory mechanisms could translate to therapeutic strategies in humans