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.

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.

The conserved Target Of Rapamycin (TOR) growth control signaling pathway is a major regulator of genes required for protein synthesis. The ubiquitous toxic metalloid arsenic, as well as mercury and nickel, are shown here to efficiently inhibit the rapamycin-sensitive TORC1 (TOR complex 1) protein kinase. This rapid inhibition of the TORC1 kinase is demonstrated in vivo by the dephosphorylation and inactivation of its downstream effector, the yeast S6 kinase homolog Sch9. Arsenic, mercury, and nickel cause reduction of transcription of ribosome biogenesis genes, which are under the control of Sfp1, a TORC1-regulated transcriptional activator. We report that arsenic stress deactivates Sfp1 as it becomes dephosphorylated, dissociates from chromatin, and exits the nucleus. Curiously, whereas loss of SFP1 function leads to increased arsenic resistance, absence of TOR1 or SCH9 has the opposite effect suggesting that TORC1 has a role beyond down-regulation of Sfp1. Indeed, we show that arsenic activates the transcription factors Msn2 and Msn4 both of which are targets of TORC1 and protein kinase A (PKA). In contrast to TORC1, PKA activity is not repressed during acute arsenic stress. A normal level of PKA activity might serve to dampen the stress response since hyperactive Msn2 will decrease arsenic tolerance. Thus arsenic toxicity in yeast might be determined by the balance between chronic activation of general stress factors in combination with lowered TORC1 kinase activity. INTRODUCTIONThe transition metal arsenic has a long history of human exploitation as both a poison and a medicine. In more recent times EhrlichЈs discovery of the antisyphilitic drug arsphenamine (also known as salvarsan) by systematic chemical modification of arsenic derivatives marked the beginning of modern pharmaceutical research. Arsenic trioxide (ATO) is used today in cancer treatment (Evens et al., 2004;Lu et al., 2007;Wang and Chen, 2008).Exposure to arsenic evokes a broad spectrum of cellular reactions in Saccharomyces cerevisiae Haugen et al., 2004;Jin, 2008;Thorsen et al., 2007) and in higher eukaryotes (Salnikow and Zhitkovich, 2008). A number of mechanisms exist for detoxification, probably because arsenic has always been widespread in the environment. These involve reduction of influx through the aquaglyceroporin Fps1p Thorsen et al., 2006); sequestration into the vacuole in the form of glutathione conjugates, metallothionein, and other metal/protein complexes; and active extrusion (Ghosh et al., 1999). In yeast, genome-wide analysis of the transcription patterns in response to arsenic revealed a complex network of transcription factors controlling the expression of several hundred genes (Haugen et al., 2004;Wysocki et al., 2004;Thorsen et al., 2007). Mitogen-activated protein kinases mediate protective responses involving AP-1-and AP-1-like transcription factors in higher eukaryotes and in fungi (Cavigelli et al., 1996;Rodriguez-Gabriel and Russell, 2005;Thorsen et al., 2006).The mechanisms by which arsenic might influence signalin...

TORC1-dependent phosphorylation ofSaccharomyces cerevisiaeSch9 was dramatically reduced upon exposure to a protonophore or in respiration-incompetent ρ0cells but not in respiration-incompetentpetmutants, providing important insight into the molecular mechanisms governing interorganellar signaling in general and retrograde signaling in particular.