The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex is highly conserved within eukaryotes. In fungi, STRIPAK controls multicellular development, morphogenesis, pathogenicity, and cell-cell recognition, while in humans, certain diseases are related to this signaling complex. To date, phosphorylation and dephosphorylation targets of STRIPAK are still widely unknown in microbial as well as animal systems. Here, we provide an extended global proteome and phosphoproteome study using the wild type as well as STRIPAK single and double deletion mutants (Δpro11, Δpro11Δpro22,

The striatin-interacting phosphatases and kinases (STRIPAK) multi subunit complex is a highly conserved signaling complex that controls diverse developmental processes in higher and lower eukaryotes. In this perspective article, we summarize how STRIPAK controls diverse developmental processes in euascomycetes, such as fruiting body formation, cell fusion, sexual and vegetative development, pathogenicity, symbiosis, as well as secondary metabolism. Recent structural investigations revealed information about the assembly and stoichiometry of the complex enabling it to act as a signaling hub. Multiple organellar targeting of STRIPAK subunits suggests how this complex connects several signaling transduction pathways involved in diverse cellular developmental processes. Furthermore, recent phosphoproteomic analysis shows that STRIPAK controls the dephosphorylation of subunits from several signaling complexes. We also refer to recent findings in yeast, where the STRIPAK homologue connects conserved signaling pathways, and based on this we suggest how so far non-characterized proteins may functions as receptors connecting mitophagy with the STRIPAK signaling complex. Such lines of investigation should contribute to the overall mechanistic understanding of how STRIPAK controls development in euascomycetes and beyond.

We showed recently that the germinal center kinase III (GCKIII) SmKIN3 from the fungus Sordaria macrospora is involved in sexual development and hyphal septation. Our recent extensive global proteome and phosphoproteome analysis revealed that SmKIN3 is a target of the striatin-interacting phosphatase and kinase (STRIPAK) multisubunit complex. Here, using protein samples from the wild type and three STRIPAK mutants, we applied absolute quantification by parallel-reaction monitoring (PRM) to analyze phosphorylation site occupancy in SmKIN3 and other septation initiation network (SIN) components, such as CDC7 and DBF2, as well as BUD4, acting downstream of SIN. For SmKIN3, we show that phosphorylation of S668 and S686 is decreased in mutants lacking distinct subunits of STRIPAK, while a third phosphorylation site, S589, was not affected. We constructed SmKIN3 mutants carrying phospho-mimetic and phospho-deficient codons for phosphorylation sites S589, S668, and S686. Investigation of hyphae in a ΔSmkin3 strain complemented by the S668 and S686 mutants showed a hyper-septation phenotype, which was absent in the wild type, the ΔSmkin3 strain complemented with the wild-type gene, and the S589 mutant. Furthermore, localization studies with SmKIN3 phosphorylation variants and STRIPAK mutants showed that SmKIN3 preferentially localizes at the terminal septa, which is distinctly different from the localization of the wild-type strains. We conclude that STRIPAK-dependent phosphorylation of SmKIN3 has an impact on controlled septum formation and on the time-dependent localization of SmKIN3 on septa at the hyphal tip. Thus, STRIPAK seems to regulate SmKIN3, as well as DBF2 and BUD4 phosphorylation, affecting septum formation. IMPORTANCE Phosphorylation and dephosphorylation of proteins are fundamental posttranslational modifications that determine the fine-tuning of their biological activity. Involved in this modification process is the recently identified striatin-interacting phosphatase and kinase (STRIPAK) multisubunit complex, which is evolutionarily conserved from fungi to humans. STRIPAK functions as a macromolecular assembly communicating through physical interactions with other conserved signaling protein complexes to constitute larger dynamic protein networks. Its function is implied in many cellular processes, such as signal transduction pathways, growth, and cellular differentiation. We applied absolute quantification of protein phosphorylation by parallel-reaction monitoring (PRM) to analyze phosphorylation site occupancy in signaling components that are linked to the STRIPAK complex. Using the filamentous fungus Sordaria macrospora, we provide evidence for the phosphorylation-dependent role of the Hippo-like germinal center kinase SmKIN3, which controls septum formation, and localize it in a time-dependent manner on septa at the hyphal tip.

19 The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex is 20 highly conserved within eukaryotes. In fungi, STRIPAK controls multicellular development, 21 morphogenesis, pathogenicity, and cell-cell recognition, while in humans, certain diseases are 22 related to this signaling complex. To date, phosphorylation and dephosphorylation targets of 23 STRIPAK are still widely unknown in microbial as well as animal systems. Here, we provide 24 an extended global proteome and phosphoproteome study using the wild type as well as 25 STRIPAK single and double deletion mutants from the filamentous fungus 26 Sordaria macrospora. Notably, in the deletion mutants, we identified the differential 27 phosphorylation of 129 proteins, of which 70 phosphorylation sites were previously unknown.28 Included in the list of STRIPAK targets are eight proteins with RNA recognition motifs (RRMs) 29 including GUL1. Knockout mutants and complemented transformants clearly show that GUL1 30 affects hyphal growth and sexual development. To assess the role of GUL1 phosphorylation on 31 fungal development, we constructed phospho-mimetic and -deficient mutants of GUL1 residues 32 S180, S216, and S1343. While the S1343 mutants were indistinguishable from wildtype, 33 phospho-deficiency of S180 and S216 resulted in a drastic reduction in hyphal growth and 34 phospho-deficiency of S216 also affects sexual fertility. These results thus suggest that 35 differential phosphorylation of GUL1 regulates developmental processes such as fruiting body 36 maturation and hyphal morphogenesis. Moreover, genetic interaction studies provide strong 37 evidence that GUL1 is not an integral subunit of STRIPAK. Finally, fluorescence microcopy 38 revealed that GUL1 co-localizes with endosomal marker proteins and shuttles on endosomes.39 Here, we provide a new mechanistic model that explains how STRIPAK-dependent and -40 independent phosphorylation of GUL1 regulates sexual development and asexual growth. 41 3 42 Author Summary43 In eukaryotes, the striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit 44 signaling complex controls a variety of developmental processes, and the lack of single 45 STRIPAK subunits is associated with severe developmental defects and diseases. However, in 46 humans, animals, as well as fungal microbes, the phosphorylation and dephosphorylation 47 targets of STRIPAK are still largely unknown. The filamentous fungus Sordaria macrospora 48 is a well-established model system used to study the function of STRIPAK, since a collection 49 of STRIPAK mutants is experimentally accessible. We previously established an isobaric tag 50 for relative and absolute quantification (iTRAQ)-based proteomic and phosphoproteomic 51 analysis to identify targets of STRIPAK. Here, we investigate mutants that lack one or two 52 STRIPAK subunits. Our analysis resulted in the identification of 129 putative phosphorylation 53 targets of STRIPAK including GUL1, a homolog of the RNA-binding protein SSD1 from yeast. 54 Using f...

We showed recently that the germinal centre kinase III (GCKIII) SmKIN3 from the fungus Sordaria macrospora is involved in sexual development and hyphal septation. Our recent extensive global proteome and phosphoproteome analysis revealed that SmKIN3 is a target of the striatin interacting phosphatase and kinase (STRIPAK) multi-subunit complex. Here, using protein samples from wild type and three STRIPAK mutants, we applied absolute quantification by parallel reaction monitoring (PRM) to analyze phosphorylation site occupancy in SmKIN3 and other septation initiation network (SIN) components, such as CDC7 and DBF2, as well as BUD4, acting downstream of SIN. For SmKIN3, we show that phosphorylation of S668 and S686 is decreased in mutants lacking distinct subunits of STRIPAK, while a third phosphorylation site, S589, was not affected. We constructed SmKIN3 mutants carrying phospho-mimetic and phospho-deficient codons for phosphorylation sites S589, S668 and S686. Investigation of hyphae in a ΔSmKin3 strain complemented by the S668 and S686 mutants showed a hyper-septation phenotype, which was absent in the wild type, the ΔSmKin3 strain complemented with wild type gene, or the mutant S589. Furthermore, localization studies with SmKIN3 phosphorylation variants and STRIPAK mutants showed that SmKIN3 preferentially localizes at the terminal septa, which is distinctly different from the wild type strains. We conclude that STRIPAK-dependent phosphorylation of SmKIN3 has an impact on controlled septum formation and on the time-dependent localization of SmKIN3 on septa at the hyphal tip. Thus, STRIPAK seems to regulate SmKIN3, as well as DBF2 and BUD4 phosphorylation, affecting septum formation.