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    • This study demonstrates that GUL

    2019-07-13

    This study demonstrates that GUL-1 affects multiple pathways/processes. As GUL-1 is likely to function as an RNA-binding protein, it is conceivable that the mRNAs involved are bound to GUL-1 as part of the regulatory function of the protein. In fact, in S. cerevisiae Ssd1 was shown to bind multiple transcripts (Hogan et al., 2008, Jansen et al., 2009). To what extent this is the case in N. crassa (and maybe other filamentous fungi) has yet to be determined. However, if that is the case, it is likely that GUL-1 would be found in multiple cellular locations. In S. cerevisiae, the GUL-1 homologue Ssd1 is localized predominantly to cytoplasmic puncta and is associated with P-bodies and stress granules under conditions of cellular stress (Jansen et al., 2009, Kurischko et al., 2011a). These complexes lead to the translational repression of Ssd1-associated mRNAs (Kurischko et al., 2011a). Under standard growth conditions, GUL-1-GFP was found to be distributed within the entire cell volume along with a clear presence of GUL-1-GFP Pergolide mesylate structure that traffic within the cytoplasm. This is similar to that observed in S. cerevisiae (Anderson and Kedersha, 2009, ; Buchan et al., 2008, Kurischko et al., 2011a, Kurischko et al., 2011b, Parker and Sheth, 2007). To date, in filamentous fungi, Ssd1 homologues were only described to be uniformly spread in the cytoplasm (Lin et al., 2018, Tanaka et al., 2007), while we have now observed the described aggregates. Unexpectedly, we did not observe any presence of GUL-1 aggregates at foci of polarized growth, such as hyphal tips. This is contrary to that observed in the dimorphic fungus Candida albicans, where Ssd1 was found to localize, among other cellular locations, in polarized sites during hyphal growth (Lee et al., 2015). One explanation for this difference may be due to the absence of nuclei, which GUL-1 apparently associates with (see below), in the apical region. We also expected to find GUL-1 to share a cellular location with its presumed regulator COT-1. The latter has been found to be present in various areas within the cell, including hyphal tips, the cytoplasm, along the plasma membrane, and within nuclei (Gorovits et al., 2000), and has also been suggested to associate with the septum (Seiler et al., 2006). These data indicate that GUL-1 and COT-1 probably interact in areas distal to the hyphal tips. Similarly, in Schizosaccharomyces pombe, Sts5 (the GUL-1 homologue) was observed to localize near non-growing cell tips, which are depleted of Orb6 kinase (Nuñez et al., 2016). Interestingly, in C. albicans Cbk1 has been suggested to be required for Ssd1 localization during yeast and hyphal growth (Lee et al., 2015). Whether the same applies for filamentous fungi remains to be elucidated. We also followed GUL-1 localization under two stress conditions. These were either the presence of Nikkomycin Z or nitrogen starvation, which was based on our current findings suggestion the involvement of gul-1 in the expression of various nitrogen metabolism-related genes. In both cases we observed changes in GUL-1 localization patterns. Similar to that described in yeast (Kurischko et al., 2011a, Lee et al., 2015, Nuñez et al., 2016, Richardson et al., 2012, Tarassov et al., 2008, Zhang et al., 2014), when either of the stress conditions were imposed, we observed more aggregates in the cytoplasm, in comparison to standard growth conditions. Moreover, many of the aggregates appeared to associate with the perinuclear space (Fig. 8, Fig. 9). The likelihood of GUL-1 association with nuclei is supported by the identification of a predicted monopartite NLS as well as a putative NES in the GUL-1 protein. Kurischko and Broach (2017) suggested that Ssd1 can associate with P-body elements or with mRNAs only within the nucleus. Furthermore, in the absence of nuclear import, Ssd1 forms aggregates that coalesce into insoluble protein deposits. While both P-bodies and stress granules have been well studied in yeast, in filamentous fungi their characterization is limited. In A. nidulans, some yeast homologues have been identified to function as P-bodies and stress granules, including Dcp1 and FabM, respectively (Buchan et al., 2008, Huang et al., 2013, Kozubowski et al., 2011, Morozov et al., 2010). At the same time, PbpA, which is the A. nidulans yeast P-body-related protein homologue, does not associate with P-bodies nor stress granules, despite its ability to functionally complement its yeast counterpart (Soukup et al., 2017).