Abstract
Yeast pseudohyphal filamentation is a stress-responsive growth transition relevant to processes required for virulence in pathogenic fungi. Pseudohyphal growth is controlled through a regulatory network encompassing conserved MAPK (Ste20p, Ste11p, Ste7p, Kss1p, and Fus3p), protein kinase A (Tpk2p), Elm1p, and Snf1p kinase pathways; however, the scope of these pathways is not fully understood. Here, we implemented quantitative phosphoproteomics to identify each of these signaling networks, generating a kinase-dead mutant in filamentous S. cerevisiae and surveying for differential phosphorylation. By this approach, we identified 439 phosphoproteins dependent upon pseudohyphal growth kinases. We report novel phosphorylation sites in 543 peptides, including phosphorylated residues in Ras2p and Flo8p required for wild-type filamentous growth. Phosphoproteins in these kinase signaling networks were enriched for ribonucleoprotein (RNP) granule components, and we observe co-localization of Kss1p, Fus3p, Ste20p, and Tpk2p with the RNP component Igo1p. These kinases localize in puncta with GFP-visualized mRNA, and KSS1 is required for wild-type levels of mRNA localization in RNPs. Kss1p pathway activity is reduced in lsm1Δ/Δ and pat1Δ/Δ strains, and these genes encoding P-body proteins are epistatic to STE7. The P-body protein Dhh1p is also required for hyphal development in Candida albicans. Collectively, this study presents a wealth of data identifying the yeast phosphoproteome in pseudohyphal growth and regulatory interrelationships between pseudohyphal growth kinases and RNPs.
Highlights
The pseudohyphal growth response is a complex morphogenetic program in which fungal cells transition from a yeast-like growth form to a filamentous state, with cells remaining physically connected after cytokinesis in elongated structures [1,2,3]
The single-celled budding yeast provides a striking example, undergoing a set of changes under conditions of nitrogen or glucose limitation resulting in the formation of extended cellular chains or filaments
We used a combination of genetics and proteomics to identify proteins that undergo phosphorylation dependent upon kinases required for filamentous growth
Summary
The pseudohyphal growth response is a complex morphogenetic program in which fungal cells transition from a yeast-like growth form to a filamentous state, with cells remaining physically connected after cytokinesis in elongated structures [1,2,3]. This growth transition is evident in several strains of S. cerevisiae (e.g., S1278b and SK1) [4, 5] and is triggered by numerous conditions, including nitrogen limitation, glucose limitation, the presence of starch as a sole carbon source, and elevated levels of fusel alcohols [1, 6,7,8,9]. The ability to form hyphae and to transition between these growth forms is required for virulence in C. albicans [14,15,16]
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