Recombinant Ashbya gossypii Calpain-like protease palB/RIM13 (RIM13), partial

What is Ashbya gossypii and why is it relevant for studying RIM13?

Ashbya gossypii is a riboflavin-overproducing filamentous fungus that shares close phylogenetic ties with unicellular yeasts like Saccharomyces cerevisiae. Its completed genome sequence and ease of genetic manipulation make it an excellent model organism for studying filamentous versus yeast growth regulatory networks . The A. gossypii RIM13 (palB homolog) functions as part of the conserved RIM pathway for pH adaptation, similar to mechanisms found in pathogenic fungi like Candida albicans, where morphological switches between yeast and filamentous forms are important for virulence .

What is the biochemical function of the RIM13 protein in A. gossypii?

RIM13 in A. gossypii functions as a calpain-like cysteine protease that likely processes the RIM101 transcription factor, similar to its homologs in other fungi. As part of the alkaline pH response pathway, RIM13 (annotated as ADR274C in the Ashbya Genome Database) is highly conserved compared to its S. cerevisiae counterpart YMR154C . The protein contains characteristic protease domains and is involved in the cellular response to environmental pH changes, which subsequently affects crucial developmental processes including sporulation .

How is the RIM101 pathway structured in A. gossypii?

The RIM101 pathway in A. gossypii involves several conserved components including RIM13/ADR274C (the calpain-like protease) and RIM20/AER342C . In this pathway, RIM13 is presumed to proteolytically process the RIM101 transcription factor (encoded by AFR190C in A. gossypii) in response to alkaline pH. The RIM101 protein contains conserved C2H2 zinc finger domains characteristic of this transcription factor family . This pathway is essential for proper sporulation at alkaline pH, as deletion of RIM101 in A. gossypii leads to severely reduced sporulation efficiency at pH 8.0 and above, while having minimal effects at acidic pH (6.5) .

What growth conditions are optimal for studying RIM13 function in A. gossypii?

For studying RIM13 function, researchers should culture A. gossypii in Ashbya Full Medium (AFM) containing 1% yeast extract, 1% peptone, and 2% dextrose. For pH-dependent studies, the medium should be buffered with Tris-HCl to specific pH values ranging from 6.5 to 8.5 . For sporulation assays, full media plates supplemented with 1 g/L myo-inositol are recommended. Liquid sporulation assays can be conducted in minimal medium containing 1.7 g/L yeast nitrogen base (without ammonium sulfate and amino acids), 0.69 g/L CSM, 20 g/L glucose, 2 g/L asparagine, and 1 g/L myo-inositol, buffered to pH 8.5 with Tris-HCl .

What genetic tools are available for manipulating RIM13 in A. gossypii?

A. gossypii has well-established PCR-based gene targeting approaches for genetic manipulation . Researchers can employ a versatile toolbox for PCR-based gene targeting that includes markers for selection and various tags for protein visualization or purification . For RIM13 studies, transformants can be selected using G418/geneticin (200 μg/mL) . The efficiency of homologous recombination in A. gossypii facilitates targeted gene deletions, promoter exchanges, and protein tagging, allowing comprehensive functional analysis of RIM13 and its interactions within the RIM pathway .

How can one assess the proteolytic activity of RIM13 in vitro?

Assessing RIM13 proteolytic activity requires careful experimental design considering its limited substrate specificity. Based on approaches used for related calpain-like proteases:

• Protein expression and purification: Express recombinant RIM13 with appropriate tags (His or GST) in heterologous systems.

• Substrate selection: Since calpain-7 (a mammalian ortholog of PalB) shows specificity for certain substrates like domain 1 of calpastatin when fused with the Bro1 domain , design fusion proteins containing potential RIM101 cleavage sites with fluorogenic reporters.

• Activity assay conditions: Include appropriate cofactors (likely Ca²⁺), test various pH conditions (pH 7.0-8.5), and monitor proteolytic activity using SDS-PAGE or fluorescence-based assays.

• Controls: Include inactive mutants with substitutions in the catalytic triad and test RIM13 activity in the presence of cysteine protease inhibitors.

• Analysis: Western blotting with specific antibodies can detect cleavage products to identify precise cutting sites within the substrate .

What molecular mechanisms connect RIM13 activity to riboflavin production in A. gossypii?

The connection between RIM13 activity and riboflavin production likely involves several interrelated mechanisms:

• pH-dependent regulation: As RIM13 functions in the alkaline pH response pathway, it may indirectly regulate metabolic processes linked to riboflavin synthesis through RIM101-mediated transcriptional regulation.

• Succinate dehydrogenase connection: Studies show that succinate dehydrogenase (SDH) activity significantly impacts riboflavin production, with SDH inhibition reducing riboflavin production by up to 78% . The RIM pathway may regulate SDH expression or activity.

• Proteostasis influence: Similar to how proteasome inhibitors (like MG-132) reduce riboflavin production by 79% and alter the levels of flavoproteins like SDH , RIM13 may influence protein turnover or maturation affecting riboflavin biosynthetic enzymes.

• Experimental approach: To investigate this connection, researchers should combine RIM13 deletion/mutation with metabolic flux analysis, proteomic profiling of riboflavin pathway enzymes, and measurement of key flavin intermediates under varying pH conditions.

What are the recommended methods for studying RIM13 localization during pH adaptation?

For studying RIM13 localization during pH adaptation in A. gossypii:

• Fluorescent protein tagging: Use C-terminal tagging with fluorescent proteins (GFP, mCherry) using PCR-based gene targeting . Verify that the fusion protein retains functionality by complementation tests in rim13Δ strains.

• Live cell imaging setup: Employ confocal microscopy with temperature-controlled chambers to maintain A. gossypii at optimal growth temperature (30°C).

• pH shift experiments: Prepare cells in medium buffered to pH 6.5, then shift to medium buffered to pH 8.5, monitoring localization changes over time (0-120 minutes).

• Co-localization studies: Simultaneously tag RIM20 (AER342C) and endosomal markers to track co-localization during pH adaptation.

• Quantitative analysis: Measure fluorescence intensity in different cellular compartments over time to track protein redistribution in response to pH changes.

• Mutational analysis: Examine how mutations in the MIT domains or C2 domain-like regions affect localization patterns, as these domains are critical for proteolytic activity in related proteins .

How does the domain structure of RIM13 influence its substrate specificity?

The domain structure of RIM13/palB influences substrate specificity through several mechanisms:

Research approaches to study domain-substrate relationships:

• Generate domain deletion/substitution constructs to identify which regions determine specificity

• Use yeast two-hybrid or pull-down assays to identify domain-specific binding partners

• Perform comparative structural modeling with calpain-7 to predict substrate binding pockets

• Develop fluorescence resonance energy transfer (FRET) systems with potential substrates to monitor real-time interactions

What considerations are important when designing CRISPR-Cas9 approaches for RIM13 functional studies in A. gossypii?

When implementing CRISPR-Cas9 for RIM13 studies in A. gossypii:

• Guide RNA design:

  • Target sequences with minimal off-target potential across the A. gossypii genome

  • Avoid regions containing essential catalytic residues if partial function is desired

  • Design gRNAs targeting both 5' and 3' regions of the gene for complete deletion

• Delivery method:

  • Optimize transformation protocols for multinucleate A. gossypii cells

  • Consider using AMA1-based autonomously replicating plasmids for transient expression

• Homology-directed repair:

  • Design repair templates with homology arms of ≥45 bp for efficient integration

  • Include selection markers appropriate for A. gossypii (G418/geneticin)

  • Consider incorporating fluorescent tags for functional studies

• Verification of modifications:

  • Account for the coenocytic nature of A. gossypii when screening transformants

  • Purify transformants through several rounds of selection to ensure homogeneity

  • Confirm modifications by sequencing and functional complementation tests

• Phenotypic analysis:

  • Assess growth and sporulation at varying pH values (6.5-8.5)

  • Examine RIM101 processing efficiency using tagged versions of RIM101

How does the proteolytic mechanism of RIM13 compare with other fungal calpain-like proteases?

Fungal calpain-like proteases show important mechanistic differences compared to classical mammalian calpains:

• Activation mechanism:

  • Classical calpains: Require Ca²⁺ for activation

  • RIM13/PalB: Likely activated through protein-protein interactions, particularly with ESCRT components

• Substrate recognition:

  • RIM13 likely recognizes specific structural features rather than strict sequence motifs

  • Limited proteolysis pattern suggests high substrate selectivity, similar to calpain-7 which cleaves domain 1 of calpastatin fused to the Bro1 domain at a specific site

• Regulatory interactions:

  • The MIT domains interact with ESCRT machinery, spatially restricting RIM13 activity

  • C2 domain-like regions are critical for proteolytic function, unlike in classical calpains

• Experimental approach for comparison:

  • Express recombinant versions of various fungal calpain-like proteases

  • Test against a panel of synthetic substrates under identical conditions

  • Compare cleavage patterns using mass spectrometry to identify substrate preferences

  • Perform structural analyses to identify conserved substrate-binding regions

What is the recommended protocol for generating RIM13 deletion mutants in A. gossypii?

To generate RIM13 deletion mutants in A. gossypii:

• PCR-based gene targeting:

  • Design primers with 45 bp homology to sequences flanking the RIM13 coding region

  • Amplify a selection marker cassette (e.g., GEN3/G418 resistance) using these primers

  • Transform A. gossypii with the PCR product using standard protocols

• Transformation procedure:

  • Grow A. gossypii in AFM medium until mid-log phase

  • Prepare protoplasts using enzymatic digestion of the cell wall

  • Transform with the deletion cassette using PEG/CaCl₂ method

  • Plate on selective medium containing G418 (200 μg/mL)

• Transformant verification:

  • Screen primary transformants by analytical PCR with primers binding outside the targeted region

  • Verify the absence of the RIM13 gene by RT-PCR or Southern blotting

  • Purify heterokaryotic transformants through repeated isolation of spores and selection

• Phenotypic confirmation:

  • Test growth and sporulation at various pH values (6.5-8.5)

  • A true rim13Δ mutant should show significantly reduced sporulation efficiency at alkaline pH

How can one establish a quantitative assay for measuring RIM101 processing by RIM13?

To quantitatively measure RIM101 processing by RIM13:

• Epitope tagging of RIM101:

  • Generate strains expressing C-terminally tagged RIM101 (e.g., with HA or FLAG tag)

  • Use PCR-based gene targeting for integration at the native locus

• pH shift experimental setup:

  • Grow cells in medium buffered to pH 6.5

  • Shift cells to medium buffered to pH 8.5

  • Collect samples at various time points (0, 5, 15, 30, 60, 120 minutes)

• Processing detection:

  • Prepare protein extracts with protease inhibitors (except cysteine protease inhibitors)

  • Perform Western blotting with anti-tag antibodies

  • Quantify the ratio of processed to unprocessed RIM101 using densitometry

• Comparison framework:

  • Run extracts from wild-type and rim13Δ strains side by side

  • The rim13Δ strain should show no processing of RIM101

  • Include a time course to determine processing kinetics

• Validation approach:

  • Express mutant versions of RIM13 with mutations in catalytic residues

  • These should show reduced or abolished RIM101 processing

What experimental design would best elucidate the relationship between RIM13 activity and riboflavin production?

To explore the relationship between RIM13 activity and riboflavin production:

• Strain construction:

  • Generate rim13Δ strains and RIM13 catalytic mutants

  • Create strains with varying levels of RIM13 expression using regulated promoters

  • Generate double mutants with disruptions in both RIM13 and SDH1 genes

• Riboflavin production analysis:

  • Grow strains in production medium at different pH values

  • Measure riboflavin production using spectrofluorometric methods

  • Extract and analyze using HPLC for precise quantification

• Enzymatic activity assays:

  • Measure activities of key enzymes in the riboflavin pathway

  • Assess SDH activity, which is linked to riboflavin production (SDH inhibition reduces riboflavin production by up to 78%)

  • Measure NADH dehydrogenase activity as a control enzyme

• Proteomics approach:

  • Perform comparative proteomics between wild-type and rim13Δ strains

  • Focus on changes in riboflavin biosynthetic enzymes and SDH components

  • Monitor levels of ubiquitinated proteins to assess protein homeostasis changes

• Metabolic flux analysis:

  • Use isotope-labeled precursors to trace carbon flow through the riboflavin pathway

  • Compare metabolic flux patterns between wild-type and rim13Δ strains

What controls are essential when performing in vitro proteolysis assays with recombinant RIM13?

Essential controls for in vitro RIM13 proteolysis assays:

• Enzymatic controls:

  • Catalytically inactive RIM13 mutant (mutation in active site cysteine)

  • Heat-inactivated RIM13 enzyme

  • Wild-type enzyme with cysteine protease inhibitors (E-64, leupeptin)

• Substrate controls:

  • Unrelated proteins unlikely to be RIM13 substrates

  • Pre-cleaved substrate to mark exact cleavage products

  • Mutated substrate with altered predicted cleavage sites

• Reaction condition controls:

  • pH gradient series (pH 5.0-9.0) to determine optimal pH

  • Calcium dependency assessment (0-10 mM Ca²⁺)

  • Time course assays (0-24 hours) to establish reaction kinetics

• Buffer and environmental controls:

  • Include all cofactors individually to determine requirements

  • Test with and without reducing agents (DTT, β-mercaptoethanol)

  • Include protease inhibitor cocktail minus cysteine protease inhibitors

• Validation approaches:

  • Mass spectrometry to confirm cleavage site identities

  • N-terminal sequencing of cleavage products

  • Circular dichroism to ensure proper protein folding of recombinant RIM13

What are common challenges when expressing recombinant RIM13 and how can they be addressed?

Common challenges and solutions for recombinant RIM13 expression:

Recommended expression systems:

• For full-length RIM13: Insect cells (Sf9, High Five) with baculovirus system

• For domain studies: E. coli with specialized solubility tags and chaperone co-expression

• For functional studies: Pichia pastoris for proper post-translational modifications

How can researchers address multinucleate challenges when studying RIM13 in A. gossypii?

A. gossypii presents unique challenges due to its multinucleate, coenocytic nature:

• Heterokaryotic state management:

  • Perform multiple rounds of spore isolation and selection to achieve homokaryotic state

  • Use spore purification methods with zymolyase treatment to degrade vegetative cells

  • Verify nuclear genotype using fluorescent markers expressed from each nucleus

• Protein localization studies:

  • Use time-lapse microscopy to track protein dynamics in relation to nuclear position

  • Employ nuclear markers (e.g., Histone-RFP) for co-localization studies

  • Implement deconvolution algorithms to resolve signals from multiple nuclei

• Gene expression analysis:

  • Use single-nucleus RNA sequencing approaches where possible

  • Implement microdissection techniques to isolate specific hyphal segments

  • Account for potential nuclear autonomy in transcriptional responses

• Phenotypic analysis:

  • Focus on terminal phenotypes after multiple generations to ensure complete genotype penetration

  • Use microscopically-guided single spore isolation for genetic purity

  • Implement quantitative phenotypic measurements with statistical controls

• Transformation strategies:

  • Use high concentrations of transformation DNA to increase likelihood of reaching all nuclei

  • Consider targeting autonomously replicating sequences for maintenance in all nuclei

  • Implement strong selection pressure during vegetative growth cycles

What are the key considerations when designing site-directed mutagenesis experiments for RIM13?

Key considerations for RIM13 site-directed mutagenesis:

• Critical residues to target:

  • Catalytic triad residues (Cys, His, Asn) in the protease domain

  • Conserved residues in MIT domains that mediate ESCRT interactions

  • Key residues in C2 domain-like regions critical for proteolytic activity

• Mutation strategy:

  • Conservative substitutions to maintain protein folding

  • Structure-guided mutations based on homology models with calpain-7

  • Alanine-scanning for unbiased functional mapping

• Validation approach:

  • Complementation tests in rim13Δ strains

  • In vitro proteolytic activity assays with recombinant proteins

  • Structural analysis to confirm proper folding

• Functional readouts:

  • RIM101 processing efficiency

  • Growth and sporulation at alkaline pH

  • Protein-protein interactions with pathway components

• Controls:

  • Wild-type RIM13 expression at identical levels

  • Empty vector controls

  • Unrelated mutations outside functional domains

How might high-throughput approaches advance our understanding of RIM13 substrate specificity?

High-throughput approaches for RIM13 substrate identification:

• Proteome-wide screening:

  • Synthetic peptide libraries with fluorogenic reporters

  • Protein microarrays with recombinant A. gossypii proteins

  • PICS (Proteomic Identification of Cleavage Sites) approach with fungal proteome digests

• In vivo substrate trapping:

  • Biotinylated activity-based probes specific for calpain-like proteases

  • Expression of catalytically inactive RIM13 mutants to trap substrates

  • Proximity labeling with TurboID or APEX2 fused to RIM13

• Comparative proteomics:

  • SILAC or TMT labeling to compare proteomes of wild-type and rim13Δ strains

  • N-terminomics to identify new N-termini generated by RIM13 cleavage

  • Quantitative analysis of protein stability in the presence/absence of RIM13

• Computational prediction:

  • Machine learning algorithms trained on known calpain substrates

  • Structural modeling of RIM13-substrate interactions

  • Molecular dynamics simulations to predict energetically favorable cleavage sites

What research questions remain unanswered regarding the evolutionary conservation of RIM13 functions?

Unanswered questions about RIM13 evolutionary conservation:

• Substrate diversity across species:

  • Does RIM13 cleave different sets of substrates in filamentous fungi versus yeasts?

  • How has substrate specificity evolved relative to morphological complexity?

• Functional diversification:

  • Beyond pH adaptation, what additional roles has RIM13 acquired in different fungal lineages?

  • How does RIM13 function relate to riboflavin overproduction in A. gossypii specifically?

• Regulatory mechanisms:

  • How has regulation of RIM13 activity evolved across fungal species?

  • Are there lineage-specific cofactors or inhibitors?

• Structural adaptation:

  • How have the MIT and C2-like domains evolved to accommodate different interaction partners?

  • What structural features determine cleavage specificity differences between species?

• Research approach:

  • Perform complementation studies with RIM13 orthologs from diverse fungi

  • Conduct comparative biochemical analysis of recombinant RIM13 proteins

  • Construct chimeric proteins to map species-specific functional domains

How can systems biology approaches integrate RIM13 function into broader cellular networks?

Systems biology approaches for RIM13 network integration:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data from wild-type and rim13Δ strains

  • Map changes onto known cellular pathways including riboflavin biosynthesis

  • Identify regulatory nodes connecting pH adaptation to metabolic responses

• Network modeling:

  • Construct protein-protein interaction networks centered on RIM13

  • Model RIM101 pathway interactions with other cellular processes

  • Predict synthetic genetic interactions for experimental validation

• Temporal dynamics analysis:

  • Monitor system-wide responses to pH shifts in wild-type and rim13Δ strains

  • Identify immediate versus delayed consequences of RIM13 activity

  • Map causality networks for pH adaptation responses

• Cross-species comparative analysis:

  • Compare RIM pathway architecture between filamentous and unicellular fungi

  • Identify conserved versus species-specific network connections

  • Relate network differences to morphological and metabolic adaptations

• Experimental validation:

  • Test predicted network interactions using synthetic genetic arrays

  • Validate key hub proteins through targeted proteomics

  • Perform conditional RIM13 activation to map rapid system responses