Sfp1 interaction with TORC1 and Mrs6 reveals feedback regulation on TOR signaling.

Ribosome biogenesis drives cell growth, and the large transcriptional output underlying this process is tightly regulated. The Target of Rapamycin (TOR) kinase is part of a highly conserved signaling pathway linking nutritional and stress signals to regulation of ribosomal protein (RP) and ribosome biogenesis (Ribi) gene transcription. In Saccharomyces cerevisiae, one of the downstream effectors of TOR is Sfp1, a transcriptional activator that regulates both RP and Ribi genes. Here, we report that Sfp1 interacts directly with TOR complex 1 (TORC1) in a rapamycin-regulated manner, and that phosphorylation of Sfp1 by this kinase complex regulates its function. Sfp1, in turn, negatively regulates TORC1 phosphorylation of Sch9, another key TORC1 target that acts in parallel with Sfp1, revealing a feedback mechanism controlling the activity of these proteins. Finally, we show that the Sfp1-interacting protein Mrs6, a Rab escort protein involved in membrane trafficking, regulates both Sfp1 nuclear localization and TORC1 signaling.

The Vam6 GEF controls TORC1 by activating the EGO complex.

The target of rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is activated by a variety of hormones (e.g., insulin) and nutrients (e.g., amino acids) and is deregulated in various cancers. Here, we report that the yeast Rag GTPase homolog Gtr1, a component of the vacuolar-membrane-associated EGO complex (EGOC), interacts with and activates TORC1 in an amino-acid-sensitive manner. Expression of a constitutively active (GTP-bound) Gtr1(GTP), which interacted strongly with TORC1, rendered TORC1 partially resistant to leucine deprivation, whereas expression of a growth inhibitory, GDP-bound Gtr1(GDP), caused constitutively low TORC1 activity. We also show that the nucleotide-binding status of Gtr1 is regulated by the conserved guanine nucleotide exchange factor (GEF) Vam6. Thus, in addition to its regulatory role in homotypic vacuolar fusion and vacuole protein sorting within the HOPS complex, Vam6 also controls TORC1 function by activating the Gtr1 subunit of the EGO complex.

Mitochondrial genomic dysfunction causes dephosphorylation of Sch9 in the yeast Saccharomyces cerevisiae.

TORC1-dependent phosphorylation of Saccharomyces cerevisiae Sch9 was dramatically reduced upon exposure to a protonophore or in respiration-incompetent ρ(0) cells but not in respiration-incompetent pet mutants, providing important insight into the molecular mechanisms governing interorganellar signaling in general and retrograde signaling in particular.

Caffeine extends yeast lifespan by targeting TORC1.

Dietary nutrient limitation (dietary restriction) is known to increase lifespan in a variety of organisms. Although the molecular events that couple dietary restriction to increased lifespan are not clear, studies of the model eukaryote Saccharomyces cerevisiae have implicated several nutrient-sensitive kinases, including the target of rapamycin complex 1 (TORC1), Sch9, protein kinase A (PKA) and Rim15. We have recently demonstrated that TORC1 activates Sch9 by direct phosphorylation. We now show that Sch9 inhibits Rim15 also by direct phosphorylation. Treatment of yeast cells with the specific TORC1 inhibitor rapamycin or caffeine releases Rim15 from TORC1-Sch9-mediated inhibition and consequently increases lifespan. This kinase cascade appears to have been evolutionarily conserved, suggesting that caffeine may extend lifespan in other eukaryotes, including man.

Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast.

The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases; however, it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as a whole. Here, we used label-free, quantitative phosphoproteomics in a systematically perturbed model organism (Saccharomyces cerevisiae) to determine the relationships between 97 kinases, 27 phosphatases, and more than 1000 phosphoproteins. We identified 8814 regulated phosphorylation events, describing the first system-wide protein phosphorylation network in vivo. Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery-and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis.

Sch9 is a major target of TORC1 in Saccharomyces cerevisiae.

The Target of Rapamycin (TOR) protein is a Ser/Thr kinase that functions in two distinct multiprotein complexes: TORC1 and TORC2. These conserved complexes regulate many different aspects of cell growth in response to intracellular and extracellular cues. Here we report that the AGC kinase Sch9 is a substrate of yeast TORC1. Six amino acids in the C terminus of Sch9 are directly phosphorylated by TORC1. Phosphorylation of these residues is lost upon rapamycin treatment as well as carbon or nitrogen starvation and transiently reduced following application of osmotic, oxidative, or thermal stress. TORC1-dependent phosphorylation is required for Sch9 activity, and replacement of residues phosphorylated by TORC1 with Asp/Glu renders Sch9 activity TORC1 independent. Sch9 is required for TORC1 to properly regulate ribosome biogenesis, translation initiation, and entry into G0 phase, but not expression of Gln3-dependent genes. Our results suggest that Sch9 functions analogously to the mammalian TORC1 substrate S6K1 rather than the mTORC2 substrate PKB/Akt.

The DHHC protein Pfa3 affects vacuole-associated palmitoylation of the fusion factor Vac8.

Vacuole biogenesis depends on specific targeting and retention of peripheral membrane proteins. At least three palmitoylated proteins are found exclusively on yeast vacuoles: the fusion factor Vac8, the kinase Yck3, and a novel adaptor protein implicated in microautophagy, Meh1. Here, we analyze the role that putative acyltransferases of the DHHC family play in their localization and function. We find that Pfa3/Ynl326c is required for efficient localization of Vac8 to vacuoles in vivo, while Yck3 or Meh1 localization is not impaired in any of the seven DHHC deletions. Vacuole-associated Vac8 appears to be palmitoylated in a pfa3 mutant, but this population is refractive to further palmitoylation on isolated vacuoles. Vacuole morphology and inheritance, which both depend on Vac8 palmitoylation, appear normal, although there is a reduction in vacuole fusion. Interestingly, Pfa3 is required for the vacuolar localization of not only an SH4 domain that is targeted by myristate/palmitate (as in Vac8) but also one that is targeted by a myristate/basic stretch (as in Src). Our data indicate that Pfa3 has an important but not exclusive function for Vac8 localization to the vacuole.