PRO40 is a scaffold protein of the cell wall integrity pathway, linking the MAP kinase module to the upstream activator protein kinase C

Abstract

Mitogen-activated protein kinase (MAPK) pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI) MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK) of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK) MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1). We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.

Figure 1. Complementation of pro30 with full-length mik1.

Strains were grown on BMM plates for 7 days. Mutant pro30 was complemented with gfp-mik1 controlled by the mik1 promoter (T1080C, T1094A). Mature perithecia (arrows) can be observed in the wildtype and complemented strains. Scale bar, 1 mm.

Figure 2. Phenotypic characterization of kinase deletion strains.

(A) Sexual development was assayed after 7 days of growth on BMM slides. Mature perithecia are only generated in wildtype (wt) and complemented deletion strains (Δmik1::mik1, Δmek1::mek1, and Δmak1::mak1). Δmik1, Δmek1, and Δmak1 generate only protoperithecia. White scale bar, 100 µm; black scale bar, 20 µm. (B) DIC microscopy of hyphal fusion in subperipheral regions. Strains were grown on solid MMS with a cellophane layer for 2 days. Hyphal fusion bridges are indicated by black arrowheads, whereas hyphae that grow in close contact without fusion are indicated by white arrowheads. Scale bar, 10 µm.

Figure 3. Growth tests and phosphorylation studies.

(A) Vegetative growth and cell wall stress response of CWI kinase mutants. Sensitivity to Calcofluor White (CFW, 250 µg/ml) was monitored in race tubes for 7 consecutive days. Average growth rates on SWG (black bars) and SWG + CFW (gray bars) and standard deviations from three independent experiments are shown. (B) MAK1 phosphorylation was studied in wildtype (wt), deletion (Δ) and complemented (c) strains by Western blot analysis with anti-phospho-p42/44 antibody. Complemented strains are Δmik1::mik1, IT1042; Δmek1::mek1, E292; Δmak1::mak1, IT1118. A representative example of three independent experiments is shown. MAK1 is not phosphorylated in any of the deletion strains. Phosphorylation is reconstituted after reintroduction of full-length gene copies of mik1, mek1, and mak1 into the respective deletion strains. The signal for tubulin was used as internal standard.

Figure 4. Localization of MIK1, MEK1, and MAK1.

(A) GFP-labeled MIK1 and MEK1 are present in the cytoplasm and absent from nuclei. GFP-labeled MAK1 localizes to the cytoplasm, but is also targeted to the nucleus. (B) Co-localization experiments with MEK1-GFP and H2B-tdTomato show that spherical organelles devoid of MEK1 labeling are nuclei (arrowheads). Scale bar, 10 µm.

Figure 5. Shared interaction network of MEK1 and PRO40.

Venn diagram comparing the three datasets generated from affinity purification and mass spectrometry with strains Δpro40::PRO40-FLAG (PRO40), Δmek1::NTAP-MEK1 (MEK1), and Δpro40::NTAP-MEK1 (MEK1(Δpro40)). The 12 proteins found in all three datasets and the 17 proteins found only in the PRO40 and MEK1 datasets are represented by boxes with S. macrospora locus tag numbers or protein designations. The magenta and blue box color indicates that the transcript of the encoding gene belongs to the top500 transcripts (with respect to read counts) in protoperithecia and vegetative hyphae, respectively (data taken from a previous transcriptomics analysis [41]).

Figure 6. Interactions of PRO40 with components of the CWI pathway.

(A) Structures of proteins used for yeast two-hybrid analysis. Derivatives generated in addition to full-length constructs are shown below the protein structures. (B) Yeast two-hybrid analysis of CWI pathway components and PRO40. Yeast cells were drop-plated on SD medium lacking leucine, tryptophan, histidine, and adenine. Empty squares indicate that interactions were not tested. (C) Schematic overview of signal transduction and protein-protein interactions within the PRO40-CWI complex. Signaling through the pathway is depicted by gray arrows; interactions are depicted by black arrows. (D) Interaction sites between PRO40, PKC1, MIK1, MEK1, and MAK1. Black bars represent interaction sites tested in yeast two-hybrid analyses (A, B). For reasons of clarity, only PRO40 is depicted as homodimer.

Figure 7. The Δmek1/pro40 double mutants shares phenotypic characteristics with Δmek1 and Δpro40.

(A) Sexual development was assayed after 7 days of growth on BMM slides. Δpro40 and the Δmek1/pro40 double mutant generate only protoperithecia. White scale bar, 100 µm; black scale bar, 20 µm. (B) Δpro40 and the Δmek1/pro40 double mutant are unable to undergo hyphal fusion, although hyphae often grow in close contact (white arrowheads). Scale bar, 10 µm. (C) Localization of GFP-tagged MIK1, MEK1, and MAK1 in vegetative hyphae of the pro40 mutant and Δpro40. Scale bar, 10 µm. (D) Localization of GFP-tagged MIK1, MEK1, and MAK1 in three days old protoperithecia of the wildtype, the pro40 mutant, and the pro40 deletion strain Δpro40. Scale bar, 20 µm.

Figure 8. PRO40 is required for correct signaling via the CWI pathway.

(A) Time course of MAK1 phosphorylation in pro40 deletion (Δ; S69656) and overexpression strains (OE; T184.2NS11) in comparison to wildtype. Strains were grown for three to six days, and phosphorylated MAK1 was detected in a Western blot using an anti-phospho-p44/42 antibody. The signal for tubulin was used as internal standard. Representative immunoblots of two to four independent experiments with three technical replicates are shown. (B) Stress-induced MAK1 phosphorylation in pro40 deletion (Δ; S69656) and overexpression strains (OE; T184.2NS11) in comparison to wildtype. Strains were grown for three days and subjected to 0.01% H₂O₂ for 0, 15, 30, and 45 minutes prior to harvesting. Phosphorylated MAK1 was detected using an anti-phospho-p44/42 antibody, and the signal for tubulin was used as internal standard. Representative immunoblots of three independent experiments with three technical replicates are shown. (C) Model of the scaffolding function of PRO40 for the CWI pathway. Details are discussed in the text.