Recombinant Grosmannia clavigera Signal peptidase complex catalytic subunit SEC11 (SEC11)

What is Grosmannia clavigera and why is it significant in pathogen research?

Grosmannia clavigera (Gc) is a bark beetle-vectored fungal pathogen of pine trees that has destroyed wide areas of lodgepole pine forest in western North America, including more than 16 million hectares in British Columbia. It forms a critical component of the mountain pine beetle outbreak as both a symbiont of the beetle and a pathogen of pine trees . G. clavigera causes wood discoloration ("blue stain") in infected trees and can damage the host tree's water transport system, contributing to tree mortality . Due to its economic and ecological importance, G. clavigera has become a model organism for studying fungal-insect-plant interactions and pathogen adaptation mechanisms .

What is the SEC11 protein and what role does it play in Grosmannia clavigera?

SEC11 is a catalytic subunit of the signal peptidase complex (SPC), an essential membrane complex responsible for removing signal peptides from proteins entering the secretory pathway. Based on homology to other eukaryotic systems, the G. clavigera SEC11 protein is likely involved in processing secreted proteins that may play roles in host colonization, nutrient acquisition, and defense against host immune responses .

The SEC11 gene in G. clavigera encodes a protein that belongs to the peptidase S26B family, with significant homology to the yeast SEC11 protein (47% identity in canine SEC11) . The catalytic activity of SEC11 depends on a Ser-His-Asp triad, with mutation studies in human SEC11 confirming that Asp122/134 is essential for catalytic activity while only moderately affecting protein stability .

How does the genomic organization of G. clavigera contribute to its pathogenicity?

The genome of G. clavigera is approximately 30 Mb and has been assembled into 18 supercontigs with 8,314 protein-coding genes . Genome and transcriptome analyses have revealed that G. clavigera possesses a comprehensive set of genes involved in essential metabolic pathways, including:

• Complete ergosterol pathway

• Nearly complete citrate and pentose phosphate cycles (missing only one gene)

• 59 of 95 genes necessary for primary amino acid biosynthesis

The genomic organization of G. clavigera includes specialized gene clusters that enable it to tolerate and even utilize conifer defense compounds, particularly terpenoids. These adaptations include specialized efflux transporters like GcABC-G1 and detoxification enzymes that allow the fungus to overcome host tree defenses .

What are the structural features of the SEC11 protein in G. clavigera?

While the specific structure of G. clavigera SEC11 has not been fully characterized, structural insights can be inferred from related SEC11 proteins in other organisms, particularly human SEC11A/C, which have been studied using cryo-electron microscopy . Based on homology, the G. clavigera SEC11 likely consists of:

• An N-terminal transmembrane helix that anchors the protein in the endoplasmic reticulum membrane

• A catalytic domain containing the Ser-His-Asp catalytic triad essential for peptidase activity

• A C-terminal region that may contribute to protein-protein interactions within the signal peptidase complex

The catalytic domain of SEC11 contains several conserved sequence motifs (boxes A-E) that are characteristic of ER-type signal peptidases. The active site residues are arranged to form a catalytic pocket capable of recognizing and cleaving signal peptides from nascent proteins .

How does G. clavigera SEC11 compare to SEC11 proteins in other organisms?

The SEC11 protein in G. clavigera shares significant homology with SEC11 proteins from other fungi and higher eukaryotes. Comparative analysis reveals:

• G. clavigera SEC11 is functionally similar to the yeast (S. cerevisiae) SEC11, which is essential for signal peptide cleavage, normal rate of secretion, and cell survival

• Mammalian systems have two SEC11 paralogs (SEC11A and SEC11C) that form distinct heterotetrameric complexes (SPC-A and SPC-C) with accessory subunits

• The catalytic mechanism involving a Ser-His-Asp triad is conserved across species, distinguishing eukaryotic SEC11 from bacterial signal peptidases that use a Ser-Lys dyad

These evolutionary relationships suggest that the fundamental function of SEC11 in signal peptide processing has been conserved across diverse taxonomic groups, while species-specific adaptations may reflect different physiological requirements or niche adaptations.

What protein domains and motifs are critical for SEC11 function in G. clavigera?

Based on homology to well-characterized SEC11 proteins in other organisms, the G. clavigera SEC11 likely contains several conserved domains and motifs essential for its function:

• Transmembrane domain: Required for anchoring SEC11 in the endoplasmic reticulum membrane

• Catalytic domain with conserved boxes A-E: Contains the critical residues for catalytic activity

• Active site residues:

  • Serine in box B (corresponding to Ser56/68 in human SEC11A/C)

  • Histidine in box D (corresponding to His96/108 in human SEC11A/C)

  • Aspartic acid in box E (corresponding to Asp122/134 in human SEC11A/C)

Mutation studies in human SEC11 have shown that altering the catalytic Asp122/134 abolishes enzymatic activity while only moderately affecting protein stability, confirming its critical role in the catalytic mechanism .

What expression systems are most effective for producing recombinant G. clavigera SEC11?

While the search results don't specifically mention expression systems for G. clavigera SEC11, commercial suppliers like CUSABIO TECHNOLOGY LLC produce recombinant G. clavigera SEC11 . Based on established protocols for similar proteins, effective expression systems for recombinant G. clavigera SEC11 production likely include:

• Bacterial expression systems (E. coli): Suitable for producing the catalytic domain without the transmembrane region, though may require optimization for proper folding of eukaryotic proteins

• Yeast expression systems (S. cerevisiae or P. pastoris): Advantage of eukaryotic post-translational machinery, potentially yielding more native-like protein

• Insect cell expression systems: May provide better folding environment for complex eukaryotic proteins

For functional studies, heterologous expression in yeast has proven successful for related G. clavigera proteins like GcABC-G1, suggesting this may be a viable approach for SEC11 as well .

What purification strategies yield the highest quality recombinant G. clavigera SEC11 protein?

While specific purification protocols for G. clavigera SEC11 are not detailed in the search results, effective purification strategies for membrane-associated proteases like SEC11 typically include:

• Affinity chromatography: Using tags such as His6, GST, or FLAG for initial capture

• Size exclusion chromatography: For separating monomeric SEC11 from aggregates or contaminants

• Ion exchange chromatography: For further purification based on charge properties

For membrane proteins like SEC11, additional considerations include:

• Use of appropriate detergents to solubilize the protein while maintaining native structure

• Careful buffer optimization to preserve activity

• Potential reconstitution into liposomes or nanodiscs for functional studies

Quality assessment should include verification of purity by SDS-PAGE, confirmation of identity by mass spectrometry, and validation of enzymatic activity using synthetic peptide substrates containing signal peptide sequences .

How can researchers optimize expression conditions to maintain the catalytic activity of recombinant G. clavigera SEC11?

To optimize expression conditions for maintaining catalytic activity of recombinant G. clavigera SEC11, researchers should consider:

• Expression temperature: Lower temperatures (16-25°C) often improve folding of eukaryotic proteins

• Induction parameters: Optimizing inducer concentration and induction time to balance yield and proper folding

• Codon optimization: Adapting the G. clavigera SEC11 coding sequence to the codon usage of the expression host

• Chaperone co-expression: Including molecular chaperones to assist in proper protein folding

• Buffer composition: Including stabilizing agents like glycerol or specific ions required for structural integrity

For functional assays, researchers studying SEC11 in other organisms have used in vitro assays with model substrates like pre-β-lactamase, which could be adapted for G. clavigera SEC11 activity assessment .

What methods are used to assess the enzymatic activity of recombinant G. clavigera SEC11?

While specific assays for G. clavigera SEC11 activity are not detailed in the search results, standard methods for assessing signal peptidase activity that could be adapted include:

• In vitro cleavage assays: Using synthetic peptide substrates containing known signal peptide sequences followed by detection of cleavage products by:

  • HPLC

  • Mass spectrometry

  • Fluorescence-based assays with quenched fluorescent peptides

• Complementation assays: Testing the ability of G. clavigera SEC11 to rescue growth defects in yeast SEC11 mutants

• Cell-free translation systems: Assessing signal peptide cleavage in reconstituted translation systems containing G. clavigera SEC11

For example, studies with human SEC11 paralogs demonstrated that both SPC-A and SPC-C complexes could process pre-β-lactamase in vitro with similar efficiencies, providing a potential model assay system .

How can researchers identify the substrate specificity of G. clavigera SEC11?

To determine the substrate specificity of G. clavigera SEC11, researchers could employ several complementary approaches:

• Bioinformatic analysis: Comparing known signal peptide sequences cleaved by SEC11 from other organisms to predict G. clavigera SEC11 substrates

• Proteomic approaches:

  • Analyzing secreted proteins from G. clavigera wild-type and SEC11 mutant strains

  • Identifying N-terminal peptides to map cleavage sites

  • Quantitative proteomics to identify proteins affected by SEC11 mutation or inhibition

• In vitro cleavage assays: Testing cleavage efficiency of diverse synthetic signal peptides to establish specificity determinants

• Structural analysis: Modeling the substrate binding pocket based on homology to characterized SEC11 proteins to predict substrate preferences

These approaches could help identify both conserved substrates shared with other fungi and unique substrates that may contribute to G. clavigera's specialized lifestyle as a pine pathogen.

What genetic approaches can be used to study SEC11 function in G. clavigera?

Several genetic approaches can be employed to study SEC11 function in G. clavigera:

• Gene knockout or knockdown:

  • CRISPR-Cas9 gene editing

  • RNA interference (RNAi)

  • Homologous recombination-based gene replacement

• Site-directed mutagenesis: Creating specific mutations in catalytic residues (based on the Ser-His-Asp triad identified in other SEC11 proteins) to assess their importance for function

• Promoter replacement: Placing SEC11 under control of inducible promoters to study the effects of altered expression levels

• Fluorescent protein tagging: Creating SEC11-fluorescent protein fusions to study localization and dynamics

• RNA-seq analysis: Comparing transcriptomes of wild-type and SEC11 mutant strains to identify affected pathways

Similar approaches have been successfully used to study other G. clavigera genes, such as the ABC transporter GcABC-G1, where gene knockout increased sensitivity to monoterpenes and delayed symptom development in inoculated pine trees .

How does SEC11 contribute to G. clavigera's pathogenicity in pine trees?

While the specific role of SEC11 in G. clavigera pathogenicity has not been directly studied according to the search results, its function as a signal peptidase suggests it likely plays a critical role in processing secreted proteins essential for virulence. Based on our understanding of secretion systems in fungal pathogens:

• SEC11 likely processes signal peptides of secreted proteins involved in:

  • Extracellular enzymes for nutrient acquisition (proteases, lipases, cellulases)

  • Effector proteins that modulate host defenses

  • Adhesion proteins required for host colonization

  • Detoxification enzymes that neutralize host defense compounds

• Disruption of SEC11 function would potentially impair the fungus's ability to:

  • Secrete virulence factors

  • Acquire nutrients from host tissues

  • Tolerate host defense compounds

  • Establish successful infection

By ensuring proper processing of the secretome, SEC11 likely serves as a fundamental enabler of the pathogenic lifestyle of G. clavigera, even if it doesn't directly interact with host components itself.

What is known about the role of SEC11 in G. clavigera's response to pine defense compounds?

The signal peptidase complex containing SEC11 likely contributes to this adaptation by ensuring proper processing and secretion of:

• Detoxification enzymes that modify terpenoids

• Efflux transporters that export toxic compounds

• Stress response proteins that protect cellular components

RNA-seq studies have shown that terpenoids induce substantial antimicrobial stress in G. clavigera , and the fungus has developed several mechanisms to cope with the host's monoterpene defense:

• A monoterpene efflux system mediated by an ABC transporter (GcABC-G1)

• Enzymes that utilize or modify monoterpenes

The proper function of these detoxification systems likely depends on correct processing by the secretory pathway, including the signal peptidase complex containing SEC11.

How do mutations in SEC11 affect G. clavigera's ability to colonize pine hosts?

While the search results don't provide information about SEC11 mutations in G. clavigera specifically, insights can be drawn from studies of SEC11 in other organisms and related pathogenicity factors in G. clavigera:

In yeast (S. cerevisiae), SEC11 has been shown to be required for signal peptide cleavage, normal rate of secretion, and cell survival . By extension, mutations in G. clavigera SEC11 would likely:

• Disrupt the processing of secreted proteins essential for host colonization

• Impair growth and development, potentially reducing virulence

• Compromise stress tolerance, including resistance to host defense compounds

For comparison, deletion of the GcABC-G1 transporter gene in G. clavigera:

• Increased sensitivity to monoterpenes

• Delayed development of symptoms in inoculated young lodgepole pine trees

• Prevented the fungus from using (+)-limonene as a carbon source

Similar phenotypic effects might be expected for SEC11 mutations, though likely more severe given the fundamental role of signal peptide processing in secretory pathway function.

How can G. clavigera SEC11 be targeted for developing novel antifungal strategies?

SEC11 represents a potential target for antifungal development based on several factors:

• Essential function: As a component of the signal peptidase complex, SEC11 likely plays an essential role in fungal viability, similar to its role in yeast

• Druggability: The catalytic site containing the Ser-His-Asp triad presents a defined pocket that could be targeted by small molecule inhibitors

• Selectivity potential: Differences between fungal and human SEC11 proteins could be exploited to develop selective inhibitors with minimal host toxicity

Potential approaches for targeting SEC11 include:

• Structure-based drug design using homology models based on related SEC11 proteins

• High-throughput screening of chemical libraries against recombinant G. clavigera SEC11

• Peptide-based inhibitors mimicking signal peptide substrates but resistant to cleavage

• RNA interference strategies to downregulate SEC11 expression

The development of SEC11 inhibitors could provide new tools for controlling G. clavigera and potentially other pathogenic fungi that rely on secreted virulence factors.

What advanced analytical techniques are used to study protein-protein interactions involving G. clavigera SEC11?

While the search results don't specifically mention techniques used to study G. clavigera SEC11 interactions, several advanced methods could be applied:

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

  • Tagging SEC11 with affinity tags (His, FLAG, etc.)

  • Purifying SEC11 along with interacting partners

  • Identifying binding partners by mass spectrometry

• Yeast two-hybrid screening:

  • Using SEC11 as bait to screen G. clavigera cDNA libraries

  • Identifying direct protein-protein interactions

• Proximity-based labeling:

  • Fusing SEC11 to enzymes like BioID or APEX

  • Identifying proteins in close proximity through biotinylation

  • Analyzing labeled proteins by mass spectrometry

• Co-immunoprecipitation combined with western blotting:

  • Using antibodies against SEC11 to pull down protein complexes

  • Probing for specific interacting proteins

• Structural techniques:

  • Cryo-electron microscopy to visualize the entire signal peptidase complex

  • X-ray crystallography of SEC11 with binding partners

  • NMR spectroscopy for studying dynamic interactions

These approaches could reveal how SEC11 interacts with other components of the signal peptidase complex and potentially with regulatory proteins specific to G. clavigera.

How can transcriptomic and proteomic approaches enhance our understanding of SEC11 function in G. clavigera?

Integrative -omics approaches offer powerful ways to study SEC11 function in G. clavigera:

• Transcriptomics (RNA-seq):

  • Comparing gene expression between wild-type and SEC11 mutant strains

  • Analyzing SEC11 expression under different growth conditions and during host infection

  • Identifying co-expressed genes that may function in related pathways

• Proteomics:

  • Analyzing the secretome of wild-type vs. SEC11 mutant strains to identify processing defects

  • Quantitative proteomics to measure protein abundance changes

  • N-terminal proteomics to map signal peptide cleavage sites

  • Post-translational modification analysis to identify regulatory mechanisms

• Metabolomics:

  • Measuring changes in metabolite profiles in SEC11 mutants

  • Identifying metabolic pathways affected by SEC11 dysfunction

• Integrative analysis:

  • Correlating changes across multiple -omics datasets

  • Network analysis to identify functional modules connected to SEC11

  • Pathway enrichment analysis to identify biological processes affected by SEC11

Existing RNA-seq datasets for G. clavigera grown under various conditions, including exposure to pine phloem extract, could be mined to understand how SEC11 expression responds to host-derived compounds and stress conditions .

What are common challenges in expression and purification of recombinant G. clavigera SEC11?

Researchers working with recombinant G. clavigera SEC11 likely encounter several challenges:

• Expression challenges:

  • Low expression levels due to membrane protein nature

  • Protein misfolding or aggregation

  • Toxicity to expression host

  • Improper processing by host signal peptidase

• Purification challenges:

  • Maintaining stability during solubilization from membranes

  • Selecting appropriate detergents that preserve structure and activity

  • Separating SEC11 from host cell membrane proteins

  • Preventing aggregation during concentration

• Activity preservation challenges:

  • Maintaining the native conformation of the catalytic domain

  • Preserving the catalytic Ser-His-Asp triad integrity

  • Finding buffer conditions that support enzymatic activity

  • Stabilizing the protein for long-term storage

To address these challenges, researchers might:

• Test multiple expression systems (bacterial, yeast, insect cells)

• Optimize expression conditions (temperature, induction timing)

• Screen various detergents and buffer conditions

• Consider expressing only the catalytic domain without the transmembrane region

• Use fusion partners to enhance solubility and folding

How can researchers troubleshoot issues with enzymatic activity assays for G. clavigera SEC11?

When troubleshooting enzymatic activity assays for G. clavigera SEC11, researchers should consider:

• Substrate-related issues:

  • Testing multiple substrate sequences from known G. clavigera secreted proteins

  • Optimizing substrate concentration to prevent inhibition at high concentrations

  • Ensuring proper substrate solubility and presentation

• Assay condition optimization:

  • Testing different pH values (typically 7-8 for signal peptidases)

  • Varying buffer composition (ionic strength, salt type)

  • Testing different detergents at concentrations below CMC

  • Screening for cofactor requirements (divalent cations)

• Detection method troubleshooting:

  • For fluorogenic substrates: checking for inner filter effects or quenching

  • For HPLC/MS methods: optimizing separation conditions and detection parameters

  • For gel-based assays: ensuring appropriate resolution for cleavage products

• Enzyme quality assessment:

  • Verifying protein purity by SDS-PAGE

  • Confirming proper folding using circular dichroism

  • Checking for inhibitory contaminants from the purification process

Comparison with well-characterized signal peptidases from related organisms could provide baseline expectations for activity levels and optimal conditions.

What strategies can help overcome difficulties in generating viable SEC11 mutants in G. clavigera?

Creating viable SEC11 mutants in G. clavigera may be challenging due to its potentially essential role. Strategies to overcome these difficulties include:

Researchers studying the GcABC-G1 transporter in G. clavigera successfully created gene knockouts using a gene replacement strategy , suggesting that similar approaches could be adapted for SEC11, though with potential modifications if the gene proves essential for viability.

What are promising areas for future investigation of G. clavigera SEC11 function?

Several promising areas for future investigation of G. clavigera SEC11 function include:

• Structural biology:

  • Determining the three-dimensional structure of G. clavigera SEC11

  • Comparing its structure with SEC11 from non-pathogenic fungi

  • Identifying unique structural features that could be exploited for targeted inhibition

• Substrate profiling:

  • Comprehensive identification of proteins processed by SEC11 in G. clavigera

  • Characterizing signal peptide preferences specific to G. clavigera SEC11

  • Identifying virulence factors dependent on SEC11 processing

• Regulation mechanisms:

  • Understanding how SEC11 expression and activity are regulated during infection

  • Investigating post-translational modifications that may modulate SEC11 function

  • Exploring the relationship between SEC11 and stress response pathways

• Host-pathogen interface:

  • Determining how SEC11-processed proteins interact with host defenses

  • Investigating the role of SEC11 in adaptation to different pine species

  • Examining how SEC11 function contributes to the symbiotic relationship with bark beetles

• Comparative studies:

  • Comparing SEC11 function across different ophiostomatoid fungi with varying levels of pathogenicity

  • Investigating evolutionary adaptations in SEC11 that correlate with host range or virulence

These research directions could provide valuable insights into fundamental aspects of G. clavigera biology and potential intervention strategies.

How might emerging technologies enhance our understanding of G. clavigera SEC11?

Emerging technologies that could enhance our understanding of G. clavigera SEC11 include:

• Advanced structural biology techniques:

  • Cryo-electron microscopy for membrane protein complexes

  • Integrative structural biology combining multiple data sources

  • AlphaFold2 and other AI-based structure prediction methods

• Single-cell technologies:

  • Single-cell RNA-seq to study SEC11 expression heterogeneity

  • Single-cell proteomics to analyze protein processing at the individual cell level

  • Spatial transcriptomics to map SEC11 expression during host colonization

• Advanced genome editing:

  • Prime editing for precise genetic modifications

  • Base editing for specific nucleotide changes

  • Inducible CRISPR interference for temporal control of gene expression

• Advanced imaging:

  • Super-resolution microscopy to visualize SEC11 localization

  • Live-cell imaging with genetically encoded sensors to track SEC11 activity

  • Correlative light and electron microscopy to connect function with ultrastructure

• Systems biology approaches:

  • Multi-omics integration with machine learning

  • Genome-scale metabolic modeling to predict effects of SEC11 perturbation

  • Network analysis to position SEC11 in the context of cellular pathways

These technologies could provide unprecedented insights into SEC11 function in G. clavigera and its role in pathogenicity.

What implications does research on G. clavigera SEC11 have for understanding other pathogenic fungi?

Research on G. clavigera SEC11 has several important implications for understanding other pathogenic fungi:

• Comparative fungal pathogenicity mechanisms:

  • Insights into how secreted proteins contribute to virulence in tree pathogens

  • Understanding common adaptations in fungi that overcome plant defenses

  • Identifying conserved vs. specialized roles of the secretory pathway in pathogenesis

• Evolutionary insights:

  • Understanding how signal peptidase systems adapt to different hosts

  • Tracking evolutionary changes in SEC11 across fungal lineages with different lifestyles

  • Identifying selection pressures on secretory pathways in pathogens

• Broad-spectrum antifungal development:

  • Assessing SEC11 as a potential target across multiple pathogenic fungi

  • Identifying conserved features of fungal SEC11 distinct from mammalian counterparts

  • Developing inhibitors that could work against multiple plant pathogens

• Biotechnological applications:

  • Improving heterologous protein secretion in fungi

  • Engineering signal peptidase systems for biotechnological applications

  • Developing fungal expression systems with modified secretory capabilities

• Ecological understanding:

  • Insights into how secretory pathways contribute to fungal adaptation to diverse niches

  • Understanding the role of secreted proteins in complex symbioses involving insects and plants

  • Predicting how fungi might adapt to new hosts or environmental conditions