Recombinant Ashbya gossypii GTPase-activating protein BEM3 (BEM3), partial

What is BEM3 and what is its primary function in Ashbya gossypii?

BEM3 is a GTPase-activating protein (GAP) in the filamentous fungus Ashbya gossypii that regulates Cdc42, a Rho GTPase crucial for actin and septin organization. Its primary function involves spatial and temporal control of this small GTPase during polarized growth . BEM3 localizes to sites of polarized growth and plays a significant role in regulating cell polarity and morphogenesis. Unlike its homolog in Saccharomyces cerevisiae, the Ashbya gossypii BEM3 has evolved specific functions related to the filamentous growth pattern of this organism .

What protein domains characterize BEM3 and how do they contribute to its function?

BEM3 contains multiple functional domains that contribute to its localization and activity:

BEM3's localization is guided by two distinct targeting regions: the PX-PH-domain-containing TD1 and the coiled-coil-containing TD2. The TD2 domain mediates localization through interaction with the polarisome component Epo1 via heterotypic coiled-coil interaction, revealing a novel role for the polarisome in linking BEM3 to its functional target, Cdc42 . Additionally, the coiled-coil domain interacts homotypically, which is important for the regulation of Cdc42 by BEM3 .

How should recombinant BEM3 protein be stored and reconstituted for maximum stability?

For optimal stability and activity of recombinant Ashbya gossypii BEM3 protein:

• Store lyophilized protein at -20°C/-80°C (shelf life approximately 12 months)

• Store liquid formulations at -20°C/-80°C (shelf life approximately 6 months)

• Avoid repeated freeze-thaw cycles; working aliquots may be stored at 4°C for up to one week

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) for long-term storage aliquots

Centrifuge vials briefly before opening to ensure contents settle to the bottom . The addition of glycerol helps prevent protein degradation during freeze-thaw cycles and improves long-term stability.

What experimental approaches can be used to study BEM3 localization in Ashbya gossypii?

Several complementary approaches can be employed to study BEM3 localization:

• Fluorescent protein fusion: Generate BEM3-GFP fusion constructs under control of the native promoter. This approach has successfully shown that the C-terminus of BEM3 (residues 638-1478) is sufficient for correct localization, while the N-terminal half is not involved in localization .

• Domain analysis: Generate truncated versions of BEM3 fused to fluorescent proteins to identify specific targeting domains. In similar studies with Bud3 (another landmark protein in A. gossypii), researchers demonstrated that the C-terminal fragment (AgBud3 638–1478–GFP) was sufficient for correct localization .

• Co-localization studies: Combine BEM3-fluorescent protein fusions with markers for cellular structures (actin, septins) or interacting proteins (Cdc42, Epo1) tagged with spectrally distinct fluorescent proteins.

• Live-cell microscopy: Capture the dynamic localization of BEM3 during cellular processes using time-lapse imaging to correlate with functional outcomes.

The experimental design should include appropriate controls, such as verification that the fusion protein maintains functionality by complementing bem3Δ phenotypes.

How do the roles of BEM3 in Ashbya gossypii compare to its homologs in other fungi?

While BEM3 in both A. gossypii and S. cerevisiae functions as a GAP for Cdc42, their developmental roles differ due to the distinct morphologies of these fungi. In A. gossypii (a filamentous fungus), BEM3 likely plays specialized roles in maintaining polarized hyphal growth, while in S. cerevisiae (a budding yeast), it participates in the bud site selection pathway .

Interestingly, another landmark protein in A. gossypii, Bud3, has functional differences compared to its S. cerevisiae homolog. While ScBud3 directs bud site selection, AgBud3 acts as a landmark for septation sites and is involved in positioning the contractile ring but does not direct lateral branching . These differences highlight the evolutionary adaptation of conserved proteins to different fungal growth patterns.

What is the relationship between BEM3 and the polarisome in Ashbya gossypii?

Research has revealed a novel interaction between BEM3 and the polarisome in A. gossypii. The coiled-coil-containing TD2 domain of BEM3 localizes through interaction with the polarisome component Epo1 via heterotypic coiled-coil interaction . This interaction provides a mechanistic link between BEM3 and its functional target, Cdc42.

The polarisome is a protein complex that establishes and maintains cell polarity. The discovery that it interacts with BEM3 reveals a coordinated system for spatial regulation of Cdc42 activity:

• The polarisome helps position BEM3 at sites of polarized growth

• BEM3, in turn, regulates Cdc42 activity through its GAP function

• This creates a feedback mechanism that helps maintain proper polarized growth

This finding suggests that the polarisome is not only important for directing growth but also for regulating the activity of small GTPases at growth sites through recruitment of regulatory proteins like BEM3 .

How can CRISPR-Cas9 be optimized for studying BEM3 function in Ashbya gossypii?

Implementing CRISPR-Cas9 for BEM3 manipulation in Ashbya gossypii requires specialized approaches due to this filamentous fungus's unique genomics and physiology:

• Promoter selection for Cas9 expression: Select promoters with appropriate strength and temporal regulation. Recent research has expanded the molecular toolbox with new promoters for A. gossypii . Consider using inducible promoters to control Cas9 expression temporally.

• gRNA design considerations:

  • Target unique sequences in BEM3 to avoid off-target effects

  • Consider the multinucleate nature of A. gossypii hyphae

  • Design multiple gRNAs targeting different regions of BEM3 to increase editing efficiency

• Repair template design for precise modifications:

  • Include homology arms of 40-60 bp for HDR-mediated editing

  • When tagging BEM3, consider C-terminal tags as they are less likely to disrupt function based on domain analysis studies

  • Incorporate selectable markers flanked by loxP sites for subsequent marker removal

• Verification of editing:

  • PCR-based genotyping to confirm edits

  • Western blotting to verify protein modifications

  • Phenotypic analysis to confirm functional consequences

• Transformation protocol optimization:

  • Use protoplast transformation with PEG/CaCl₂

  • Select transformed spores on appropriate antibiotics

  • Repeat selection cycles to achieve homokaryotic transformants

Given that A. gossypii is multinucleate, multiple rounds of selection may be necessary to achieve homokaryotic strains where all nuclei contain the desired modification.

What methodological approaches can resolve the contradictory data on BEM3's role in septin organization?

Contradictory findings regarding BEM3's role in septin organization can be addressed through several complementary approaches:

• High-resolution temporal analysis:

  • Use live-cell imaging with improved temporal resolution to determine the precise sequence of events during septin ring formation

  • Employ photoactivatable or photoconvertible fluorescent proteins to track specific populations of BEM3 and septins

• Protein-protein interaction studies:

  • Implement proximity-dependent biotinylation (BioID or TurboID) to identify proteins in close proximity to BEM3 during septin ring formation

  • Use fluorescence resonance energy transfer (FRET) to detect direct interactions between BEM3 and septin components

  • Perform co-immunoprecipitation under different growth conditions to identify context-dependent interactions

• Genetic interaction mapping:

  • Create a systematic collection of double mutants combining bem3Δ with mutations in septin genes and regulators

  • Perform quantitative phenotypic analysis to identify synthetic interactions

  • Map the genetic interaction network to resolve functional relationships

• Domain-specific perturbations:

  • Express specific domains of BEM3 (such as the TD1 or TD2) to determine their effects on septin organization

  • Create point mutations in key domains rather than full deletions to distinguish between different functions

  • Use rapid protein degradation systems (e.g., auxin-inducible degron) to observe immediate consequences of BEM3 loss

• Cross-species comparative analysis:

  • Compare BEM3 functions in A. gossypii with related proteins in diverse fungi

  • Perform domain swapping experiments between BEM3 homologs to identify regions responsible for species-specific functions

This multifaceted approach would help resolve contradictions by distinguishing direct from indirect effects and identifying context-dependent functions of BEM3 in septin organization.

How might the interaction between BEM3 and Kss1 MAPK integrate polarized growth with filamentous development pathways?

The reported interaction between TD2 (from Bem3) and Kss1 (a MAPK involved in filamentous growth) suggests an intriguing link between polarized growth regulation and MAPK signaling pathways . This connection can be investigated through:

• Mapping the interaction interface:

  • Perform alanine scanning mutagenesis of the TD2 domain to identify residues critical for Kss1 binding

  • Determine whether the interaction is direct or mediated by other proteins

  • Investigate whether the interaction is regulated by phosphorylation events

• Functional consequences analysis:

  • Examine whether BEM3-Kss1 interaction affects:

    • Kss1 kinase activity

    • BEM3 GAP activity toward Cdc42

    • Subcellular localization of either protein

  • Determine whether the interaction is constitutive or stimulus-dependent

• Signaling pathway integration:

  • Investigate how environmental signals that activate filamentous growth affect BEM3-Kss1 interaction

  • Examine whether Cdc42 activation status influences Kss1 pathway activity

  • Determine if BEM3 serves as a scaffold to bring Kss1 into proximity with other signaling components

• Developmental context investigation:

  • Compare BEM3-Kss1 interaction during:

    • Normal vegetative growth

    • Initiation of filamentous growth

    • Response to nutrient limitation

  • Examine whether the interaction changes during different life cycle stages

• Proposed model for integration:

This model suggests that BEM3 could serve as an integration point where external signals (detected by MAPK pathways) are translated into changes in polarized growth patterns (mediated by Cdc42 regulation). The dual location of Kss1 in both nucleus and polarized growth sites supports a role in coordinating transcriptional responses with cytoskeletal reorganization .

What are the methodological considerations for developing a quantitative assay to measure BEM3 GAP activity in vitro?

Developing a robust in vitro assay for BEM3 GAP activity requires careful consideration of protein preparation, reaction conditions, and detection methods:

• Protein preparation considerations:

  • Express and purify recombinant BEM3 protein with >85% purity using E. coli expression systems

  • Include proper tags for purification while ensuring they don't interfere with activity

  • Express active domains separately (particularly the GAP domain) for comparison with full-length protein

  • Prepare active GTP-bound Cdc42 as the substrate

• Reaction setup optimization:

  • Buffer composition: Test different pH values (7.0-8.0) and salt concentrations

  • Cofactor requirements: Include magnesium for GTPase activity

  • Temperature: Typically 25-30°C for fungal proteins

  • Time course: Establish linear range of activity

• Activity measurement approaches:

• Controls and validations:

  • Positive control: Include a well-characterized GAP domain

  • Negative controls:

    • Heat-inactivated BEM3

    • BEM3 with mutations in the catalytic arginine finger

  • Specificity test: Compare activity with different Rho GTPases

• Data analysis considerations:

  • Calculate initial velocity from linear portion of progress curves

  • Determine kinetic parameters (kcat, KM) through Michaelis-Menten analysis

  • Compare catalytic efficiency (kcat/KM) across different conditions and mutants

For accurate determination of BEM3's role in Cdc42 regulation, compare wild-type BEM3 with truncated versions and point mutations in key domains to correlate GAP activity with biological functions observed in vivo .

What are the key considerations for designing BEM3 knockout and complementation experiments in Ashbya gossypii?

Successful BEM3 knockout and complementation studies require careful planning and methodological rigor:

• Knockout strategy design:

  • Use homologous recombination with 45-60 bp flanking sequences for precise gene replacement

  • Consider using a recyclable selection marker (loxP-KanMX-loxP) that can be removed with Cre recombinase for subsequent manipulations

  • Target the entire coding sequence rather than just catalytic domains to ensure complete functional disruption

  • Multinucleate nature of A. gossypii requires multiple rounds of selection to achieve homokaryotic mutants

• Phenotypic analysis workflow:

  • Growth rate measurements using colony diameter on solid media

  • Hyphal morphology analysis including branching patterns and polarized growth

  • Actin and septin distribution using fluorescent markers

  • Cell wall and septation patterns using chitin staining (as performed in bud3 mutants)

• Complementation design:

  • Create expression constructs with the native BEM3 promoter

  • Include partial and full-length BEM3 variants to map functional domains

  • Test domain deletions to determine essential regions (particularly TD1, TD2, and GAP domains)

  • Consider integration at the original locus or at a neutral site

• Common pitfalls and solutions:

• Verification methods:

  • PCR verification of gene replacement

  • RT-qPCR to confirm absence of transcript

  • Western blotting to verify protein absence

  • Complementation with wild-type gene to confirm phenotype causality

When designing complementation constructs, consider that the C-terminal region (residues 638-1478) is sufficient for proper localization, which may inform the design of functional domain analysis experiments .

How can researchers effectively study the BEM3 interactome in Ashbya gossypii?

A comprehensive approach to mapping the BEM3 interactome involves multiple complementary techniques:

• Affinity purification-mass spectrometry (AP-MS):

  • Tag BEM3 with affinity tags (FLAG, HA, or His) at C-terminus to preserve function

  • Compare results with N-terminal tags to identify tag-sensitive interactions

  • Include crosslinking steps to capture transient interactions

  • Perform stringent controls, including:

    • Tag-only expression

    • Unrelated protein with same tag

    • BEM3 with GAP domain mutations

• Proximity-based labeling:

  • Fuse BEM3 to BioID or TurboID biotin ligase

  • Label proximal proteins in living cells

  • Analyze biotinylated proteins by streptavidin pulldown and MS

  • Map spatial interactome at different cellular locations

• Yeast two-hybrid screening:

  • Use individual domains (TD1, TD2, GAP) as baits

  • Screen against A. gossypii cDNA library

  • Validate positive interactions by co-immunoprecipitation

  • Test for conservation in S. cerevisiae using orthologous proteins

• Co-localization studies:

  • Generate fluorescently tagged candidate interactors

  • Perform quantitative co-localization analysis

  • Use super-resolution microscopy for detailed spatial relationships

  • Implement fluorescence correlation spectroscopy for dynamic interactions

• Functional validation:

  • Create single and double mutants of BEM3 and interactors

  • Perform synthetic genetic array (SGA) analysis if applicable

  • Test whether interactions are regulated by:

    • Cell cycle stages

    • Polarized growth phases

    • Nutrient conditions

Based on existing literature, priority should be given to analyzing interactions with:

• Cdc42 and other Rho GTPases (as targets)

• Epo1 and other polarisome components (for localization)

• Kss1 MAPK pathway components (for signaling integration)

• Septin proteins (for understanding role in septation)

How might novel promoter systems enhance the study of BEM3 function in Ashbya gossypii?

Recent advances in promoter engineering for A. gossypii provide new opportunities for studying BEM3 function :

• Carbon source-regulatable promoters:

  • Enable conditional expression of BEM3 to study temporal requirements

  • Allow induction/repression experiments to determine immediate vs. adaptive responses

  • Facilitate dosage-dependent studies by varying inducer concentration

• Integration with the Dual Luciferase Reporter (DLR) Assay:

  • Quantitatively measure BEM3 promoter activity under different conditions

  • Identify regulatory elements controlling BEM3 expression

  • Develop synthetic promoters with tailored expression profiles

• Application of new promoter tools for BEM3 research:

• Integrated expression systems:

  • Develop dual-control systems combining transcriptional and post-translational regulation

  • Create BEM3 variants with inducible degron tags for rapid protein depletion

  • Design synthetic circuits to express BEM3 in specific spatial patterns within hyphae

• Implementation methodology:

  • Use Golden Gate assembly for modular construction of expression cassettes

  • Integrate stable expression cassettes into the genome using recombinogenic flanks

  • Validate expression using dual luciferase reporters as internal controls

These approaches would overcome limitations of constitutive expression systems and enable precise temporal and spatial control of BEM3 activity, facilitating new insights into its dynamic functions in polarized growth and development.

What are the implications of BEM3's potential roles in metabolic engineering applications using Ashbya gossypii?

A. gossypii is an established industrial organism for riboflavin production and is being developed for other bioproducts . BEM3's role in regulating polarized growth and cytoskeletal organization has several potential implications for metabolic engineering:

• Growth morphology optimization:

  • Manipulating BEM3 activity could alter hyphal morphology to optimize:

    • Surface-to-volume ratio for nutrient uptake

    • Cytoplasmic organization for metabolic compartmentalization

    • Biomass density in bioreactors

• Stress response engineering:

  • The connection between BEM3 and the Kss1 MAPK pathway suggests roles in stress adaptation

  • Engineering BEM3-mediated signaling could enhance:

    • Tolerance to industrial fermentation conditions

    • Adaptation to varying carbon sources

    • Resistance to metabolic stress from high product titers

• Production strategy implications:

• Implementation considerations:

  • BEM3 modifications must balance growth optimization with metabolic productivity

  • Engineering should consider effects on:

    • Central carbon metabolism

    • Energy allocation between growth and production

    • Secretion efficiency for extracellular products

• Preliminary experimental approach:

  • Create a library of BEM3 variants with different activity levels

  • Test growth characteristics and product formation

  • Identify variants that optimize the production-to-growth ratio

  • Integrate with other metabolic engineering strategies

This research direction would bridge fundamental understanding of BEM3 function with applied biotechnology, potentially creating new platforms for sustainable bioproduction.