The highly conserved striatin‐interacting phosphatases and kinases (STRIPAK) complex regulates phosphorylation/dephosphorylation of developmental proteins in eukaryotic microorganisms, animals and humans. To first identify potential targets of STRIPAK, we performed extensive isobaric tags for relative and absolute quantification‐based proteomic and phosphoproteomic analyses in the filamentous fungus Sordaria macrospora. In total, we identified 4,193 proteins and 2,489 phosphoproteins, which are represented by 10,635 phosphopeptides. By comparing phosphorylation data from wild type and mutants, we identified 228 phosphoproteins to be regulated in all three STRIPAK mutants, thus representing potential targets of STRIPAK. To provide an exemplarily functional analysis of a STRIPAK‐dependent phosphorylated protein, we selected CLA4, a member of the conserved p21‐activated kinase family. Functional characterization of the ∆cla4 deletion strain showed that CLA4 controls sexual development and polarized growth. To determine the functional relevance of CLA4 phosphorylation and the impact of specific phosphorylation sites on development, we next generated phosphomimetic and ‐deficient variants of CLA4. This analysis identified (de)phosphorylation of a highly conserved serine (S685) residue in the catalytic domain of CLA4 as being important for fungal cellular development. Collectively, these analyses significantly contribute to the understanding of the mechanistic function of STRIPAK as a phosphatase and kinase signaling complex.

Sexual and asexual development in filamentous ascomycetes is controlled by components of conserved signaling pathways. Here, we investigated the development of mutant strains lacking genes for kinases MAK2, MEK2, and MIK2, as well as the scaffold protein HAM5 of the pheromone response (PR) pathway. All had a defect in fruiting body development and hyphal fusion. Another phenotype was a defect in melanin-dependent ascospore germination. However, this deficiency was observed only in kinase deletion mutants, but not in strains lacking HAM5. Notably, the same developmental phenotypes were previously described for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) mutants, but the germination defect was only seen in NOX2 mutants. These data suggest a molecular link between the pheromone signaling pathway and both NOX complexes. Using data from yeast two-hybrid (Y2H) analysis, we found that the scaffolding protein HAM5 interacts with NOR1, the regulator of NOX1 and NOX2 complexes. This interaction was further confirmed using differently fluorescent-labeled proteins to demonstrate that NOR1 and HAM5 co-localize at cytoplasmic spots and tips of mature hyphae. This observation was supported by phenotypic characterization of single and double mutants. The oxidative stress response and the initiation of fruiting bodies were similar in Δham5Δnor1 and Δham5, but distinctly reduced in Δnor1, indicating that the double deletion leads to a partial suppression of the Δnor1 phenotype. We conclude that the PR and NOX1 complexes are connected by direct interaction between HAM5 and NOR1. In contrast, PR kinases are linked to the NOX2 complex without participation of HAM5.

1 from the fungus Sordaria macrospora identifies a conserved serine phosphorylation 2 site in PAK kinase CLA4 to be important for sexual development and polarized 3 growth 4 5 6 Summary 22The highly conserved striatin-interacting phosphatases and kinases (STRIPAK) 23 complex regulates phosphorylation of developmental proteins in eukaryotic 24 microorganisms, animals, and humans. To first identify potential targets of 25 STRIPAK, we performed extensive isobaric tags for relative and absolute 26 quantification (iTRAQ)-based proteomic and phosphoproteomic analyses in the 27 filamentous fungus Sordaria macrospora. In total, we identified 4,193 proteins and 28 2,489 phosphoproteins, which are represented by 10,635 phosphopeptides. By 29 comparing phosphorylation data from wild-type and mutants, we identified 228 30 phosphoproteins to be regulated in all three STRIPAK mutants, thus representing 31 potential targets of STRIPAK. To provide an exemplarily functional analysis of a 32 STRIPAK-dependent phosphorylated protein, we selected CLA4, a member of the 33 conserved p21-activated kinase (PAK) family. Functional characterization of the 34 ∆cla4 deletion strain showed that CLA4 controls sexual development and polarized 35 growth. To determine the functional relevance of CLA4 phosphorylation and the 36 impact of specific phosphorylation sites on development, we next generated 37 phospho-mimetic and -deficient variants of CLA4. This analysis identified 38 (de)phosphorylation of a highly conserved serine (S685) residue in the catalytic 39 domain of CLA4 as being important for fungal cellular development. Collectively, 40 these analyses significantly contribute to the understanding of the mechanistic 41 function of STRIPAK as a phosphatase and kinase signaling complex. 42 43 44 45 46 Results 102 In this work, detailed iTRAQ-based proteomic and phosphoproteomic analyses of 103 the wild-type and three different STRIPAK deletion strains led to the identification of 104 putative dephosphorylation targets of the STRIPAK complex in S. macrospora. 105 Importantly, functional characterization of the PAK kinase CLA4, a target protein of 106 STRIPAK deciphers their role in fungal cellular development. 107 108 iTRAQ-based proteomic and phosphoproteomic analyses revealed putative 109 dephosphorylation targets of the STRIPAK complex 110 To identify dephosphorylation targets of STRIPAK, we performed iTRAQ-based LC-111 MS/MS proteomic and phosphoproteomic analyses to directly compare relative 112 changes in protein expression (Fig. 1). The iTRAQ strategy is based on the N-113 terminal labelling of peptides with up to eight amino group-reactive reagents coupled 114 to different reporter and balancer groups (Ross et al. 2004; Wu et al. 2006). Thus, 115 we were able to conduct a simultaneous analysis of up to eight different cultural 116 samples. While these isobaric tags have the same mass, fragmentation during 117 tandem MS (MS/MS) leads to the generation of mass-specific reporter ions whose 118 abundances are used for protein quantific...