Abstract
The budding yeast Saccharomyces cerevisiae undergoes a stress-responsive transition to a pseudohyphal growth form in which cells elongate and remain connected in multicellular filaments. Pseudohyphal growth is regulated through conserved signaling networks that control cell growth and the response to glucose or nitrogen limitation in metazoans. These networks are incompletely understood, and our studies identify the TORC1- and PKA-regulated kinase Ksp1p as a key stress-responsive signaling effector in the yeast pseudohyphal growth response. The kinase-defective ksp1-K47D allele results in decreased pseudohyphal morphology at the cellular and colony level, indicating that Ksp1p kinase signaling is required for pseudohyphal filamentation. To determine the functional consequences of Ksp1p signaling, we implemented transcriptional profiling and quantitative phosphoproteomic analysis of ksp1-K47D on a global scale. Ksp1p kinase signaling maintains wild-type transcript levels of many pathways for amino acid synthesis and metabolism, relevant for the regulation of translation under conditions of nutrient stress. Proteins in stress-responsive ribonucleoprotein granules are regulated post-translationally by Ksp1p, and the Ksp1p-dependent phosphorylation sites S176 in eIF4G/Tif4631p and S436 in Pbp1p are required for wild-type levels of pseudohyphal growth and Protein Kinase A pathway activity. Pbp1p and Tif4631p localize in stress granules, and the ksp1 null mutant shows elevated abundance of Pbp1p puncta relative to wild-type. Collectively, the Ksp1p kinase signaling network integrates polarized pseudohyphal morphogenesis and translational regulation through the stress-responsive transcriptional control of pathways for amino acid metabolism and post-translational modification of translation factors affecting stress granule abundance.
Highlights
THE eukaryotic cellular response to nutrient limitation is complex, encompassing programmed changes resulting in reduced protein translation, increased autophagy, an elevated
Given the range of functionalities associated with Ksp1p, we sought to determine the contributions of Ksp1p kinase activity toward pseudohyphal growth induced by nutrient limitation
The Lys-to-Asp substitution was achieved by allelic replacement at the native KSP1 locus in a strain of the filamentous S1278b genetic background, and the homozygous diploid ksp1-K47D kinase-defective mutant was assayed for surface-spread filament formation under conditions of nitrogen limitation
Summary
THE eukaryotic cellular response to nutrient limitation is complex, encompassing programmed changes resulting in reduced protein translation, increased autophagy, an elevated. Umekawa and Klionsky (2012) reported that KSP1 negatively regulates autophagy, consistent with the observed hypo-phosphorylation of Atg13p in ksp1D, and that this suppressive function of Ksp1p is partially activated by PKA. These studies suggest that KSP1 contributes to eukaryotic cell signaling through stress-responsive pathways that regulate pseudohyphal growth, among other cell processes; the functional significance of Ksp1p kinase signaling is unclear, and the scope of the Ksp1p kinase signaling network remains to be determined. We present data indicating the relevance of Ksp1p kinase activity in the yeast pseudohyphal response and globally identify changes in transcript abundance and protein phosphorylation dependent upon Ksp1p kinase signaling under filamentation-inducing conditions. The work identifies Ksp1p as part of the mechanism linking highly conserved nutrient stress-responsive signaling pathways with the regulation of RNP granules, while identifying the molecular basis of Ksp1p kinase signaling as a TORC1 and PKA downstream effector
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