The STRIPAK signaling complex regulates phosphorylation of GUL1, an RNA-binding protein that shuttles on endosomes

Abstract

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...