TORC1 is a conserved multisubunit kinase complex that regulates many aspects of eukaryotic growth including the biosynthesis of ribosomes. The TOR protein kinase resident in TORC1 is responsive to environmental cues and is potently inhibited by the natural product rapamycin. Recent characterization of the rapamycin-sensitive phosphoproteome in yeast has yielded insights into how TORC1 regulates growth. Here, we show that Sch9, an AGC family kinase and direct substrate of TORC1, promotes ribosome biogenesis (Ribi) and ribosomal protein (RP) gene expression via direct inhibitory phosphorylation of the transcriptional repressors Stb3, Dot6 and Tod6. Deletion of STB3, DOT6 and TOD6 partially bypasses the growth and cell size defects of an sch9 strain and reveals interdependent regulation of both Ribi and RP gene expression, and other aspects of Ribi. Dephosphorylation of Stb3, Dot6 and Tod6 enables recruitment of the RPD3L histone deacetylase complex to repress Ribi/RP gene promoters. Taken together with previous studies, these results suggest that Sch9 is a master regulator of ribosome biogenesis through the control of Ribi, RP, ribosomal RNA and tRNA gene transcription.

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Protein phosphorylation cascades play a central role in the regulation of cell growth and protein kinases PKA, Sch9 and Ypk1 take center stage in regulating this process in S. cerevisiae. To understand how these kinases co-ordinately regulate cellular functions we compared the phospho-proteome of exponentially growing cells without and with acute chemical inhibition of PKA, Sch9 and Ypk1. Sites hypo-phosphorylated upon PKA and Sch9 inhibition were preferentially located in RRxS/T-motifs suggesting that many are directly phosphorylated by these enzymes. Interestingly, when inhibiting Ypk1 we not only detected several hypo-phosphorylated sites in the previously reported RxRxxS/T-, but also in an RRxS/T-motif. Validation experiments revealed that neutral trehalase Nth1, a known PKA target, is additionally phosphorylated and activated downstream of Ypk1. Signaling through Ypk1 is therefore more closely related to PKA- and Sch9-signaling than previously appreciated and may perform functions previously only attributed to the latter kinases.

23Protein phosphorylation cascades play a central role in the regulation of cell growth and protein 24 kinases PKA, Sch9 and Ypk1 take centre stage in regulating this process in S. cerevisiae. To 25 understand how these kinases co-ordinately regulate cellular functions we compared the 26 phospho-proteome of exponentially growing cells without and with acute chemical inhibition of 27 PKA, Sch9 and Ypk1. Sites hypo-phosphorylated upon PKA and Sch9 inhibition were preferentially 28 located in RRxS/T-motifs suggesting that many are directly phosphorylated by these enzymes. 29Interestingly, when inhibiting Ypk1 we not only detected several hypo-phosphorylated sites in the 30 previously reported RxRxxS/T-, but also in an RRxS/T-motif. Validation experiments revealed that 31 neutral trehalase Nth1, a known PKA target, is additionally phosphorylated and activated 32 downstream of Ypk1. Signalling through Ypk1 is therefore more closely related to PKA-and Sch9-33 signalling than previously appreciated and may perform functions previously only attributed to 34 the latter kinases. 35 36 2

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