Recombinant Neurospora crassa pH-response transcription factor pacC/RIM101 (pacc-1), partial

What is the PacC/RIM101 transcription factor in Neurospora crassa and what is its function?

The PacC/RIM101 transcription factor (also referred to as PAC-3 in some studies) is a zinc finger DNA binding protein that functions as the central regulator of the pH signaling pathway in Neurospora crassa. It is encoded by the gene annotated as pacC (ORF NCU00090) .

Functionally, PacC/RIM101 responds to ambient pH changes by activating alkaline-regulated genes and repressing acid-regulated genes. The protein is activated through proteolytic processing in response to alkaline pH conditions, which allows it to translocate to the nucleus and bind to specific DNA sequences in target gene promoters .

Research has shown that PacC regulates numerous biological processes in N. crassa, including:

• Female development and sexual reproduction

• Melanin production via regulation of the tyrosinase gene

• Glycogen metabolism through control of glycogen synthase expression

• Reserve carbohydrate metabolism

• Cell wall architecture

What are the main components of the Neurospora crassa pH signaling pathway?

The N. crassa pH signaling pathway shares core components with those identified in Aspergillus nidulans and Saccharomyces cerevisiae. The pathway includes six primary components known as Pal proteins (in A. nidulans terminology) or Rim proteins (in S. cerevisiae) .

The key components identified in N. crassa include:

These proteins function in a cascade where palH and palF are involved in ambient pH sensing at the plasma membrane, while the remaining components participate in signal transduction that ultimately leads to PacC activation . N. crassa mutants lacking functional pal genes (with the exception of Δpal-9, which is the A. nidulans palI homolog) exhibit low conidiation, are unable to grow at alkaline pH, and accumulate the pigment melanin .

How is the experimental production of recombinant PacC/PACC-1 typically accomplished?

Based on published protocols, recombinant PacC production typically involves the following methodology:

• cDNA cloning: A fragment of the ORF NCU00090 (typically the N-terminal portion containing the DNA-binding domain) is amplified by PCR from a cDNA library using specific primers .

• Vector construction: The amplified fragment is cloned into an expression vector (commonly pET28a) with appropriate restriction sites (e.g., NdeI and BamHI) .

• Expression system: The construct is transformed into a bacterial expression system such as E. coli.

• Protein purification: The recombinant protein, typically fused with a His-tag for purification, is isolated using affinity chromatography.

For instance, one reported method produced a truncated recombinant PACC transcription factor by amplifying a 639-bp fragment of ORF NCU00090 and cloning it into the pET28a vector to express an N-terminal 213 amino acid protein with a His-tag fusion .

How does the proteolytic processing of PacC in Neurospora crassa differ from other fungal models?

The proteolytic processing of PacC exhibits notable differences across fungal species:

• Aspergillus nidulans: PacC undergoes two sequential proteolytic cleavage steps. The first cleavage is pH-dependent and activated by the products of the pal genes, while the second is proteasome-mediated and pH-independent. This processing converts the full-length PacC72 to the active PacC27 form .

• Saccharomyces cerevisiae: Rim101p requires only a single cleavage step to be activated and is processed under both acidic and alkaline conditions .

• Neurospora crassa: PAC-3 is proteolytically processed in a single cleavage step predominantly at alkaline pH, although low levels of the processed protein can be observed at normal growth pH . This suggests that N. crassa employs a mechanism distinct from both A. nidulans and S. cerevisiae.

These differences highlight the evolutionary divergence in pH-responsive signaling mechanisms among fungi and suggest that researchers must be cautious when extrapolating findings across species.

What experimental approaches can be used to study PacC binding to target gene promoters?

Several complementary methodologies have proven effective for investigating PacC-DNA interactions:

• In vitro DNA-binding assays: Using purified recombinant PacC protein (typically the DNA-binding domain) with DNA fragments containing putative PacC binding sites. Electrophoretic mobility shift assays (EMSAs) can be used to detect complex formation .

• Chromatin Immunoprecipitation (ChIP): ChIP-PCR or ChIP-seq can identify genomic binding sites for PacC in vivo. In N. crassa, PAC-3 has been shown to bind to all pal gene promoters, regulating their expression at normal growth pH and/or alkaline pH .

• Reporter gene assays: Constructs containing promoter regions with PacC binding sites driving expression of reporter genes can be used to assess functional relevance of binding.

• Binding site mutation analysis: Systematic mutation of predicted binding sites followed by binding and functional assays can define critical nucleotides for PacC recognition.

For instance, research has demonstrated that PAC-3 binds to the tyrosinase promoter and negatively regulates its gene expression, and also binds to its own promoter (pac-3) only at alkaline pH .

What is the role of the PacC signaling pathway in regulating female development in Neurospora crassa?

The PacC pathway plays a critical role in female development in N. crassa through multiple mechanisms:

The mechanism involves:

• Nutrient mobilization: Perithecia grafting experiments demonstrate that autophagy genes and cell-to-cell fusion genes (the MAK-1 and MAK-2 pathway genes) are needed for the mobilization and movement of nutrients from an established vegetative hyphal network into the developing protoperithecium .

• Signaling complex formation: Protein localization experiments with GFP-tagged PALA construct showed that PALA is localized in a peripheral punctate pattern, consistent with a signaling center associated with the ESCRT complex .

This research provides evidence that the N. crassa PACC signal transduction pathway, similar to the PacC/Rim101 pathway in A. nidulans and S. cerevisiae, plays a key role in regulating female development.

How does PacC regulate glycogen metabolism in Neurospora crassa?

PacC regulates glycogen metabolism primarily through transcriptional control of the glycogen synthase gene (gsn):

• Mechanism of regulation: A DNA motif for the PacC transcription factor was identified in the promoter of the gsn gene in N. crassa, suggesting direct regulation .

• Experimental evidence:

  • At pH. 7.8, pacC was overexpressed while gsn was downregulated in wild-type N. crassa, coinciding with low glycogen accumulation.

  • In the pacC knockout strain, glycogen levels and gsn expression at alkaline pH were similar to and higher than the wild-type strain at normal pH (5.8), respectively .

• Binding confirmation: DNA-protein binding assays confirmed that the truncated recombinant protein containing the DNA-binding domain specifically bound to a gsn DNA fragment containing the PacC motif. DNA-protein complexes were observed with extracts from cells grown at normal and alkaline pH, and binding was confirmed by ChIP-PCR analysis .

These results characterize gsn as an "acidic gene" (repressed at alkaline pH) and demonstrate that the pH signaling pathway controls glycogen accumulation by regulating gsn expression .

What methodologies are most effective for studying the nuclear translocation of PacC in response to alkaline pH?

Several complementary approaches have proven effective for studying PacC nuclear translocation:

• Fluorescent protein fusion constructs: The creation of mCherry-PAC-3 fusion proteins expressed under constitutive promoters (such as ccg-1) allows for visualization of protein localization. For example, a DNA fragment of 2,041 bp can be amplified by PCR with specific primers and cloned into appropriate expression vectors (e.g., pTSL48-B) .

• Live-cell imaging: Fluorescence microscopy to track the translocation of fluorescently-tagged PAC-3 in response to pH changes in real-time.

• Subcellular fractionation: Biochemical separation of nuclear and cytoplasmic fractions followed by Western blot analysis to quantify PAC-3 distribution.

• Nuclear localization signal (NLS) analysis: Studies have shown that PAC-3 preferentially localizes in the nucleus during alkaline pH stress, and that this translocation may require N. crassa importin-α, as the PAC-3 nuclear localization signal has strong in vitro affinity with importin-α .

• Mutation analysis: Targeted mutation of putative nuclear localization signals or regulatory phosphorylation sites to identify regions critical for pH-responsive nuclear import.

These approaches can be combined to provide a comprehensive understanding of the dynamics and regulation of PacC nuclear translocation in response to alkaline pH.

How can researchers investigate the feedback regulation mechanisms involving PAC-3 in Neurospora crassa?

Investigating the feedback regulation mechanisms involving PAC-3 requires a multi-faceted experimental approach:

• Chromatin Immunoprecipitation (ChIP) analysis: PAC-3 has been shown to bind to all pal gene promoters, regulating their expression at normal growth pH and/or alkaline pH. Additionally, PAC-3 binds to its own promoter (pac-3) only at alkaline pH . ChIP-seq can comprehensively identify all genomic binding sites.

• Gene expression analysis: Quantitative RT-PCR or RNA-seq comparing wild-type and ΔpacC strains at different pH conditions can identify genes regulated by PAC-3, including components of the pH signaling pathway itself.

• Promoter reporter constructs: Fusion of pal gene promoters or the pac-3 promoter to reporter genes (such as GFP) can allow visualization of feedback regulation.

• Time-course experiments: Analysis of the temporal dynamics of PAC-3 activation and subsequent regulation of target genes, including components of the pH signaling pathway.

• Protein-protein interaction studies: Investigating interactions between PAC-3 and other components of the signaling pathway using techniques such as co-immunoprecipitation or yeast two-hybrid assays.

These approaches can reveal how PAC-3 participates in feedback loops that fine-tune the pH response in N. crassa.

What are the latest methodological advances in studying the broader metabolic impacts of the PAC-3 signaling pathway?

Recent advances in studying the broader metabolic impacts of the PAC-3 signaling pathway include:

• Transcriptomic analyses: RNA-seq has been used to identify genes regulated by PacC/Rim101 across multiple fungal species. In Ustilago maydis, microarray analyses comparing gene expression in wild-type versus rim101/pacC mutant strains revealed a large number of genes regulated by Rim101/PacCp, including proteins involved in various physiological activities .

• Metabolomic profiling: Mass spectrometry-based approaches to identify metabolites affected by PacC/Rim101 regulation.

• Network analysis: Integration of transcriptomic, proteomic, and metabolomic data to construct regulatory networks centered on PacC/Rim101.

• Chromatin structure analysis: Techniques such as ATAC-seq to investigate how PacC/Rim101 affects chromatin accessibility and structure.

• Single-cell approaches: Analysis of cell-to-cell variability in PacC/Rim101 activation and downstream responses.

Recent findings indicate that the pH signaling pathway in N. crassa affects multiple cellular processes, including:

• Reserve carbohydrate metabolism

• Melanin production

• Holocellulolytic enzyme activities

• Sexual development

Additionally, in some fungal species, the pathway has been implicated in:

• Cell wall remodeling

• Adaptation to various stressors (including oxidative and cell wall stressors)

• Lipid asymmetry and ER stress response

These methodological advances provide a more comprehensive understanding of how the PacC/Rim101 pathway integrates environmental pH signals with diverse cellular processes.