Recombinant Neurospora crassa Protein transport protein sec-13 (sec-13)

What is the function of SEC-13 in Neurospora crassa?

SEC-13 in Neurospora crassa is a multifunctional protein that serves as a component of the COPII cage-assembly system. Unlike many other COPII components that have more limited functions, SEC-13 simultaneously participates in multiple protein complexes that facilitate different aspects of cellular processes including chromatin organization, transcription, translation, trafficking, and protein degradation pathways. These functions are differentially sensitive to SEC-13 levels, suggesting a concentration-dependent regulatory role .

The protein plays a crucial role in coordinating information flow from the genome to the proteome, effectively linking genetic variation to phenotypic outcomes through its involvement in this continuous program of cellular processes. This makes SEC-13 particularly important for understanding how Neurospora crassa responds to genetic variation and environmental changes.

How does SEC-13 in Neurospora crassa compare to homologs in other organisms?

SEC-13 is evolutionarily conserved across eukaryotes, but with organism-specific adaptations. In Neurospora crassa, SEC-13 exhibits particular importance in the coordination between protein trafficking and degradation pathways, especially at the ER-Golgi intermediate compartment (ERGIC) . Unlike other model organisms where SEC-13 functions have been more narrowly characterized, the Neurospora protein appears to have evolved an expanded regulatory role.

The unique aspects of Neurospora SEC-13 include its apparent master regulatory function in coordinating information flow from genome to proteome. This contrasts with the more specialized functions of SEC-13 homologs documented in other systems, suggesting that Neurospora crassa may serve as an excellent model for understanding the broader integrative functions of this protein across biological domains.

What are the recommended methods for expressing recombinant SEC-13 in Neurospora crassa?

For recombinant expression of SEC-13 in Neurospora crassa, researchers should consider the following methodological approach:

• Gene targeting can be efficiently performed using CRISPR/Cas9 technology, which has been demonstrated to work effectively in Neurospora crassa without requiring mus-51 or mus-52 mutant backgrounds for efficient homologous recombination .

• The gene replacement strategy should utilize the Cas9 endonuclease combined with single crRNA:tracrRNA chimeric guide RNA (gRNA) designed specifically for the sec-13 locus .

• For promoter selection, researchers have successfully used the β-tubulin promoter for strong expression in Neurospora . Alternatively, the native promoter can be maintained for physiological expression levels, depending on experimental requirements.

• For recombinant construct design, the csr-1 locus has been successfully used as a neutral integration site in Neurospora crassa and could be utilized for SEC-13 expression under control of a selected promoter .

The advantage of this CRISPR-based approach is that it enables efficient gene editing in any Neurospora strain, including natural isolates, which expands the experimental possibilities beyond laboratory strains.

What techniques are available for monitoring SEC-13 localization in Neurospora crassa?

Several methods can be employed to track SEC-13 localization in Neurospora crassa:

• Fluorescent protein tagging: SEC-13 can be tagged with GFP or other fluorescent proteins for live-cell imaging. When designing such constructs, care must be taken to ensure that the tag does not interfere with SEC-13's multiple functions. C-terminal tagging is often preferable to avoid disrupting potential N-terminal targeting sequences.

• Nuclear transport assays: Given SEC-13's role in nuclear-cytoplasmic transport pathways, researchers can assess its function using reporter constructs with nuclear localization signals (NLS). For example, a GFP reporter with an N-terminal SV40 monopartite NLS can be used to evaluate nuclear transport efficiency in strains with altered SEC-13 expression or function .

• Immunolocalization: Antibody-based detection methods provide high specificity for examining native SEC-13 localization, particularly in fixed cells where different cellular compartments can be co-stained.

• Subcellular fractionation: Biochemical separation of cellular compartments followed by Western blotting can provide quantitative assessment of SEC-13 distribution between the nucleus, cytoplasm, and membrane-associated compartments.

For optimal results, combining these approaches can provide complementary data on SEC-13 localization dynamics under different experimental conditions.

How does SEC-13 coordinate information flow from genome to proteome in Neurospora crassa?

SEC-13 functions as a master regulatory node that integrates multiple cellular processes spanning from chromatin organization to protein degradation. Research indicates that SEC-13 simultaneously associates with different protein complexes involved in distinct cellular functions . This unique positioning allows SEC-13 to coordinate responses to genetic variation and environmental cues across multiple levels of information processing.

The mechanism appears to involve differential sensitivity of these processes to SEC-13 concentration. This creates a hierarchical response system where changes in SEC-13 levels result in coordinated adjustments across multiple pathways. For example, reduction in SEC-13 expression has been shown to decrease ubiquitination and degradation of cargo proteins like CFTR, suggesting that protein fate decisions (export vs. degradation) are critically dependent on COPII cage assembly at the ERGIC compartment where COPI exchange occurs .

To experimentally investigate this coordination role, researchers should employ:

• Proteomics approaches to identify SEC-13 interaction partners under different conditions

• Transcriptomics to monitor global gene expression changes in response to SEC-13 modulation

• Quantitative analyses of protein trafficking and degradation rates for model cargo proteins

• Systems biology approaches to integrate these multi-level datasets

What is the relationship between SEC-13 levels and protein degradation pathways in Neurospora crassa?

Research has revealed an unexpected relationship between SEC-13 expression levels and protein degradation pathways in Neurospora crassa. Unlike other COPII components, reduction of SEC-13 expression decreases the ubiquitination and degradation of cargo proteins, resulting in increased stability . This suggests SEC-13 plays a critical regulatory role at the intersection of secretory and degradative pathways.

This relationship appears to be particularly important at the ERGIC (ER-Golgi Intermediate Compartment) where decisions about protein recycling, forward transport, or degradation are made. The data indicate that COPII cage assembly at this compartment, which is SEC-13 dependent, is crucial for determining whether cargo proteins are exported or degraded .

To investigate this relationship experimentally, researchers should:

• Develop quantitative assays for cargo protein stability using pulse-chase experiments

• Employ proximity labeling techniques to identify SEC-13 interactors specifically at the ERGIC

• Use ubiquitination assays to directly measure how SEC-13 levels affect this post-translational modification

• Create a panel of SEC-13 mutants with altered expression levels to establish dose-response relationships

What methodological approaches are recommended for studying SEC-13 interactome in Neurospora crassa?

For comprehensive analysis of the SEC-13 interactome in Neurospora crassa, researchers should implement a multi-faceted approach:

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

  • Tag SEC-13 with an epitope tag (e.g., FLAG, HA) that minimally impacts function

  • Perform purifications under different buffer conditions to capture both stable and transient interactions

  • Use crosslinking approaches to stabilize more transient interactions

  • Include appropriate controls (e.g., untagged strains, irrelevant tagged proteins) to filter out non-specific interactions

• Proximity-based labeling methods:

  • Express SEC-13 fused to BioID or APEX2 to biotinylate proximal proteins in living cells

  • This approach captures both direct and indirect interaction partners in their native cellular context

  • Particularly valuable for identifying interactions at specific cellular locations

• Yeast two-hybrid screening:

  • While more prone to false positives, this can complement other approaches by identifying direct binary interactions

  • Use SEC-13 as both bait and prey to ensure comprehensive coverage

• Co-immunoprecipitation validation:

  • Confirm key interactions identified through high-throughput methods

  • Test interactions under different cellular conditions to assess context-dependency

• Functional validation through genetic approaches:

  • Use CRISPR/Cas9-mediated gene editing to create mutants of identified interactors

  • Assess phenotypic consequences on SEC-13-dependent processes

The resulting interactome data should be analyzed using network biology approaches to identify functional modules and regulatory hubs.

How does SEC-13 function differ between nuclear pore complexes and COPII vesicles in Neurospora crassa?

SEC-13 in Neurospora crassa, like in other eukaryotes, functions in both nuclear pore complexes (NPCs) and COPII vesicle formation, but with distinct molecular contexts and functional outcomes:

In COPII vesicles:

• SEC-13 forms part of the outer coat complex, partnering with SEC31

• This complex provides structural scaffolding for vesicle formation at ER exit sites

• Research indicates SEC-13 plays a regulatory role in determining the fate of cargo proteins, particularly at the ERGIC where decisions about export versus degradation occur

• SEC-13's effect on cargo protein ubiquitination suggests it influences quality control mechanisms during early secretory trafficking

In Nuclear Pore Complexes:

• SEC-13 is a component of the Nup84 subcomplex

• It contributes to the structural integrity of the nuclear pore

• Unlike specialized nuclear transport factors such as NUP-6 (Importin α), which directly mediates nuclear import of proteins with nuclear localization signals , SEC-13's role is more structural

• Its dual localization likely enables coordination between nucleocytoplasmic transport and secretory pathway functions

To experimentally distinguish these functions:

• Create separation-of-function mutants that specifically affect one role but not the other

• Use super-resolution microscopy to quantify SEC-13 distribution between these compartments

• Employ domain-specific antibodies or tags to distinguish SEC-13 populations

• Develop cargo-specific trafficking assays to separate effects on nuclear versus secretory transport

What are the experimental considerations for investigating SEC-13's role in the degradation of CFTR in Neurospora crassa?

Investigating SEC-13's role in CFTR degradation in Neurospora crassa requires careful experimental design:

• Expression system establishment:

  • Develop a heterologous expression system for wild-type and F508del CFTR in Neurospora

  • Consider using the CRISPR/Cas9 system for precise genomic integration

  • The β-tubulin promoter has been successfully used for robust expression in Neurospora

• Degradation and stability assays:

  • Implement pulse-chase experiments to measure CFTR half-life

  • Use cycloheximide chase assays to monitor protein stability in the absence of new synthesis

  • Develop a luciferase reporter system for quantitative monitoring (similar to approaches used in other Neurospora studies)

• SEC-13 manipulation strategies:

  • Create a series of strains with varying SEC-13 expression levels

  • Generate conditional SEC-13 mutants using inducible systems

  • Design point mutations in SEC-13 to disrupt specific interaction domains

• Ubiquitination analysis:

  • Develop immunoprecipitation protocols to isolate CFTR and analyze its ubiquitination status

  • Use mass spectrometry to identify ubiquitination sites and quantify modification levels

  • Compare ubiquitination patterns between wild-type and SEC-13-depleted conditions

• Trafficking visualization:

  • Implement live-cell imaging using fluorescently tagged CFTR to track its subcellular localization

  • Pay particular attention to ERGIC and Golgi localization, where SEC-13 appears to play a critical role

  • Use compartment-specific markers to determine where CFTR accumulates when SEC-13 levels are altered

• Controls and validation:

  • Include other COPII components as controls to demonstrate the specificity of SEC-13 effects

  • Use alternative cargo proteins to determine whether the effect is CFTR-specific or generalizable

What are the recommended approaches for genetic manipulation of SEC-13 in Neurospora crassa?

For efficient genetic manipulation of SEC-13 in Neurospora crassa, researchers should consider the following methodological approaches:

• CRISPR/Cas9-based gene editing:

  • This technology has been demonstrated to work efficiently in Neurospora crassa without requiring specialized genetic backgrounds

  • Design guide RNAs targeting the SEC-13 locus using established Neurospora-specific parameters

  • Utilize the SNR52 promoter for gRNA expression, which has been shown to be operational in Neurospora

  • Employ the Cas9 endonuclease under control of the trpC promoter for efficient expression

• Homologous recombination-based approaches:

  • Design donor templates with appropriate homology arms (typically 500-1000 bp)

  • For complex modifications, utilize yeast recombination systems to assemble constructs

  • While traditional approaches required mus-51 or mus-52 mutant backgrounds, CRISPR methods work effectively in wild-type strains

• Promoter replacement strategies:

  • For controlled expression, consider replacing the native SEC-13 promoter with regulatable options

  • The β-tubulin promoter provides strong constitutive expression

  • For inducible expression, Neurospora-adapted versions of qa-2 or other inducible systems can be employed

• Reporter gene integration:

  • For functional studies, luciferase reporters have been successfully used in Neurospora

  • The csr-1 locus provides a neutral integration site that minimizes positional effects

• Validation approaches:

  • Confirm genetic modifications by PCR, sequencing, and Southern blot analysis

  • Verify expression changes using RT-qPCR and Western blotting

  • Assess phenotypic consequences using appropriate functional assays

What techniques are most effective for studying SEC-13's role in coordinating nuclear-cytoplasmic transport in Neurospora crassa?

To effectively study SEC-13's role in nuclear-cytoplasmic transport, researchers should employ a combination of approaches:

• Nuclear transport reporter assays:

  • Utilize GFP reporters with nuclear localization signals (NLS) to assess import efficiency

  • For example, GFP with an N-terminal SV40 monopartite NLS can be expressed to visualize nuclear accumulation

  • Compare transport efficiency between wild-type and SEC-13 mutant strains

  • Quantify nuclear/cytoplasmic fluorescence ratios to detect subtle changes in transport

• Live-cell imaging approaches:

  • Implement time-lapse microscopy to capture dynamic transport events

  • Photoactivatable or photoconvertible fluorescent proteins can be used to track specific protein populations

  • FRAP (Fluorescence Recovery After Photobleaching) experiments can measure transport rates

• Biochemical fractionation:

  • Develop protocols for clean separation of nuclear and cytoplasmic fractions

  • Western blot analysis of fractions can provide quantitative measures of protein distribution

  • Mass spectrometry-based proteomics of fractions can identify global transport defects

• Interaction studies with nuclear transport machinery:

  • Investigate SEC-13 interactions with importins/karyopherins, including NUP-6 (Importin α)

  • Compare these interactions with those of SEC-13 in COPII vesicle formation

  • Map interaction domains to identify separation-of-function mutations

• Electron microscopy:

  • Examine nuclear pore complex structure in SEC-13 mutants

  • Look for defects in nuclear envelope architecture or nuclear pore density

  • Immunogold labeling can precisely localize SEC-13 within the nuclear pore complex

These approaches should be integrated to develop a comprehensive understanding of SEC-13's contribution to nuclear-cytoplasmic transport in Neurospora crassa.

What are the emerging questions about SEC-13's role in response to environmental stress in Neurospora crassa?

SEC-13's position as a coordinator between multiple cellular processes makes it a prime candidate for mediating integrated responses to environmental stress. Future research should address:

• Stress-specific changes in SEC-13 interactome:

  • How do different environmental stresses (temperature, oxidative, nutrient deprivation) affect SEC-13's protein interaction network?

  • Are there stress-specific post-translational modifications of SEC-13 that redirect its function?

• SEC-13's role in stress granule formation and dynamics:

  • Does SEC-13 participate in the formation of stress granules or P-bodies during cellular stress?

  • How does this relate to its roles in nuclear transport and COPII vesicle formation?

• Integration with stress-responsive signaling pathways:

  • How does SEC-13 function intersect with known stress response pathways in Neurospora?

  • Is SEC-13 expression or localization regulated by stress-responsive transcription factors?

• Adaptive responses and evolutionary conservation:

  • How conserved is SEC-13's stress response function across fungal species?

  • Are there strain-specific adaptations in SEC-13 function related to ecological niches?

• Experimental approaches:

  • Develop stress-specific reporter systems to monitor SEC-13 function

  • Implement systems biology approaches to map SEC-13-dependent stress response networks

  • Use evolutionary and comparative genomics to identify conserved stress-responsive elements in SEC-13

How can systems biology approaches enhance our understanding of SEC-13 function in Neurospora crassa?

Systems biology approaches offer powerful frameworks for understanding SEC-13's multifunctional nature:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data from SEC-13 mutants

  • Implement network analysis to identify functional modules affected by SEC-13 perturbation

  • Use temporal profiling to map the sequence of events following SEC-13 modulation

• Mathematical modeling:

  • Develop kinetic models of SEC-13-dependent processes

  • Create predictive models of how SEC-13 concentration affects the balance between secretion and degradation

  • Use these models to identify potential regulatory nodes and feedback mechanisms

• Comparative systems analysis:

  • Compare SEC-13 networks across multiple Neurospora strains

  • Extend comparison to other filamentous fungi to identify conserved and divergent functions

  • Correlate network differences with phenotypic variations

• High-throughput phenotyping:

  • Develop automated phenotyping platforms for Neurospora growth, development, and stress responses

  • Screen SEC-13 mutants against diverse conditions to build comprehensive phenotypic profiles

  • Correlate phenotypic data with molecular profiles to establish causative relationships

• Integration with structural biology:

  • Incorporate structural information about SEC-13 and its complexes into systems models

  • Use structure-based prediction to identify critical interaction interfaces

  • Design structure-guided mutations to test model predictions

These systems approaches will help unravel the complex regulatory networks in which SEC-13 participates and provide a more holistic understanding of its function in Neurospora crassa.