Characterization of the rapamycin-sensitive phosphoproteome reveals that Sch9 is a central coordinator of protein synthesis

Abstract

The target of rapamycin complex 1 (TORC1) is an essential multiprotein complex conserved from yeast to humans. Under favorable growth conditions, and in the absence of the macrolide rapamycin, TORC1 is active, and influences virtually all aspects of cell growth. Although two direct effectors of yeast TORC1 have been reported (Tap42, a regulator of PP2A phosphatases and Sch9, an AGC family kinase), the signaling pathways that couple TORC1 to its distal effectors were not well understood. To elucidate these pathways we developed and employed a quantitative, label-free mass spectrometry approach. Analyses of the rapamycin-sensitive phosphoproteomes in various genetic backgrounds revealed both documented and novel TORC1 effectors and allowed us to partition phosphorylation events between Tap42 and Sch9. Follow-up detailed characterization shows that Sch9 regulates RNA polymerases I and III, the latter via Maf1, in addition to translation initiation and the expression of ribosomal protein and ribosome biogenesis genes. This demonstrates that Sch9 is a master regulator of protein synthesis.[Keywords: Maf1; Sch9; TOR; phosphoproteomics; rapamycin; ribosome biogenesis] Supplemental material is available at http://www.genesdev.org.

Temperature-sensitive ipl1-2/Aurora B mutation is suppressed by mutations in TOR complex 1 via the Glc7/PP1 phosphatase

“…29). In addition to Gip4, which does not appear to be the relevant target here, we note two Glc7 regulators (Shp1 and Reg1) among the TORC1 targets identified by screening the rapamycin-sensitive phosphoproteome by mass spectrometry (36,39). Shp1 is hyperphosphorylated at Ser106 and Ser108 following rapamycin treatment (36).…”

“…In addition to Gip4, which does not appear to be the relevant target here, we note two Glc7 regulators (Shp1 and Reg1) among the TORC1 targets identified by screening the rapamycin-sensitive phosphoproteome by mass spectrometry (36,39). Shp1 is hyperphosphorylated at Ser106 and Ser108 following rapamycin treatment (36). Shp1, a cofactor for the AAA-ATPase Cdc48, was originally identified by a mutation that suppresses the lethality caused by overexpression of Glc7 (40).…”

“…Reg1 phosphorylation has been reported to be reduced at Ser572 and Ser576 (36) or S570 (39) upon rapamycin treatment. Reg1 is thought to negatively regulate Snf1, the yeast ortholog of AMPactivated protein kinase, by targeting Glc7-dependent dephosphorylation of Thr210 (43).…”

Protein kinase A and TORC1 activate genes for ribosomal biogenesis by inactivating repressors encoded by Dot6 and its homolog Tod6

“…Moreover, Deminoff et al (23) have shown that PKA phosphorylates Dot6 in vitro, and we have recently found that Dot6 and Tod6 exhibit increased phosphorylation on several PKA sites after addition of glucose to glycerol-grown cells (Table S2). Loewith and colleagues (24) have determined that phosphorylation of Dot6 and Tod6 is altered by rapamycin in a Sch9-dependent manner. Finally, both Dot6 and Tod6 have been identified as components of the Rpd3L complex, which likely possesses histone deacetylase activity, an association that may account for their repressive activity (25).…”

Maf1, repressor of tRNA transcription, is involved in the control of gluconeogenetic genes in Saccharomyces cerevisiae

“…Maf1 is regulated by its phosphorylation-dependent cellular localization (Boisnard et al, 2009;Graczyk et al, 2011;Huber et al, 2009;Lee et al, 2009;Moir et al, 2006; A C C E P T E D M A N U S C R I P T…”