The STRIPAK signaling complex regulates phosphorylation of GUL1, an RNA-binding protein that shuttles on endosomes
The STRIPAK signaling complex regulates phosphorylation of GUL1, an RNA-binding protein that shuttles on endosomes
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DOI：
10.1101/2020.05.01.072009
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发表时间：
2020-05
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通讯作者：
V. Stein;B. Blank-Landeshammer;K. Müntjes;R. Märker;I. Teichert;M. Feldbrügge;A. Sickmann;U. Kück
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作者：
V. Stein;B. Blank-Landeshammer;K. Müntjes;R. Märker;I. Teichert;M. Feldbrügge;A. Sickmann;U. Kück
纹状体相互作用磷酸酶和激酶（STRIPAK）多亚基信号复合物在真核生物中高度保守。在真菌中，STRIPAK控制多细胞发育，形态发生，致病性和细胞-细胞识别，而在人类中，某些疾病与这种信号复合物有关。迄今为止，在微生物和动物系统中，STRIPAK的磷酸化和去磷酸化靶点仍然是未知的。在这里，我们提供了一个扩展的全球蛋白质组和磷酸化蛋白质组研究，使用野生型以及STRIPAK单，双删除突变体的丝状真菌大孢囊霉。值得注意的是，在缺失突变体中，我们确定了129个蛋白质的差异磷酸化，其中70个磷酸化位点是以前未知的。在STRIPAK靶标列表中包括八种具有RNA识别基序（RRM）的蛋白质，包括GUL 1。敲除突变体和互补转化体清楚地表明GUL 1影响菌丝生长和性发育。为了评估GUL 1磷酸化对真菌发育的作用，我们构建了GUL 1残基S180，S216和S1343的磷酸模拟和缺陷突变体。虽然S1343突变体与野生型没有区别，但S180和S216的磷酸缺乏导致菌丝生长急剧减少，S216的磷酸缺乏也影响性育性。因此，这些结果表明，GUL 1的差异磷酸化调节发育过程，如子实体成熟和菌丝形态发生。此外，遗传相互作用的研究提供了强有力的证据，GUL 1不是一个完整的亚基的STRIPAK。最后，荧光显微镜显示GUL 1与内体标记蛋白共定位，并在内体上穿梭。在这里，我们提供了一个新的机制模型，解释了如何STRIPAK依赖性和非依赖性磷酸化GUL 1调节性发育和无性生长。在真核生物中，纹状体相互作用磷酸酶和激酶（STRIPAK）多亚基信号复合物控制着各种发育过程，缺乏单一的STRIPAK亚基与严重的发育缺陷和疾病有关。然而，在人类、动物以及真菌微生物中，STRIPAK的磷酸化和去磷酸化靶点在很大程度上仍然未知。丝状真菌Sordaria macrospora是用于研究STRIPAK功能的成熟模型系统，因为实验上可获得一系列STRIPAK突变体。我们之前建立了一个同量异位素标签，用于基于相对和绝对定量（iTRAQ）的蛋白质组学和磷酸化蛋白质组学分析，以识别STRIPAK的靶点。在这里，我们研究突变体，缺乏一个或两个STRIPAK亚基。我们的分析导致了129个STRIPAK的推定磷酸化靶点的鉴定，包括GUL 1，一种来自酵母的RNA结合蛋白SSD 1的同源物。使用荧光显微镜，我们证明GUL 1穿梭于内体。我们还研究了缺失，磷酸化模拟，和缺失突变体，并揭示GUL 1调节性和无性发育的磷酸化依赖的方式。总的来说，我们全面的遗传和细胞分析为GUL 1作为STRIPAK靶标如何控制多种细胞功能的机制提供了新的基本见解。
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 from the filamentous fungus Sordaria macrospora. Notably, in the deletion mutants, we identified the differential phosphorylation of 129 proteins, of which 70 phosphorylation sites were previously unknown. Included in the list of STRIPAK targets are eight proteins with RNA recognition motifs (RRMs) including GUL1. Knockout mutants and complemented transformants clearly show that GUL1 affects hyphal growth and sexual development. To assess the role of GUL1 phosphorylation on fungal development, we constructed phospho-mimetic and -deficient mutants of GUL1 residues S180, S216, and S1343. While the S1343 mutants were indistinguishable from wildtype, phospho-deficiency of S180 and S216 resulted in a drastic reduction in hyphal growth and phospho-deficiency of S216 also affects sexual fertility. These results thus suggest that differential phosphorylation of GUL1 regulates developmental processes such as fruiting body maturation and hyphal morphogenesis. Moreover, genetic interaction studies provide strong evidence that GUL1 is not an integral subunit of STRIPAK. Finally, fluorescence microcopy revealed that GUL1 co-localizes with endosomal marker proteins and shuttles on endosomes. Here, we provide a new mechanistic model that explains how STRIPAK-dependent and - independent phosphorylation of GUL1 regulates sexual development and asexual growth. Author Summary In eukaryotes, the striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex controls a variety of developmental processes, and the lack of single STRIPAK subunits is associated with severe developmental defects and diseases. However, in humans, animals, as well as fungal microbes, the phosphorylation and dephosphorylation targets of STRIPAK are still largely unknown. The filamentous fungus Sordaria macrospora is a well-established model system used to study the function of STRIPAK, since a collection of STRIPAK mutants is experimentally accessible. We previously established an isobaric tag for relative and absolute quantification (iTRAQ)-based proteomic and phosphoproteomic analysis to identify targets of STRIPAK. Here, we investigate mutants that lack one or two STRIPAK subunits. Our analysis resulted in the identification of 129 putative phosphorylation targets of STRIPAK including GUL1, a homolog of the RNA-binding protein SSD1 from yeast. Using fluorescence microscopy, we demonstrate that GUL1 shuttles on endosomes. We also investigated deletion, phospho-mimetic, and -deletion mutants and revealed that GUL1 regulates sexual and asexual development in a phosphorylation-dependent manner. Collectively, our comprehensive genetic and cellular analysis provides new fundamental insights into the mechanism of how GUL1, as a STRIPAK target, controls multiple cellular functions.