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The growth rate of a yeast cell is controlled by the target of rapamycin kinase complex I (TORC1) and cAMP-dependent protein kinase (PKA) pathways. To determine how TORC1 and PKA cooperate to regulate cell growth, we performed temporal analysis of gene expression in yeast switched from a non-fermentable substrate, to glucose, in the presence and absence of TORC1 and PKA inhibitors. Quantitative analysis of these data reveals that PKA drives the expression of key cell growth genes during transitions into, and out of, the rapid growth state in glucose, while TORC1 is important for the steady-state expression of the same genes. This circuit design may enable yeast to set an exact growth rate based on the abundance of internal metabolites such as amino acids, via TORC1, but also adapt rapidly to changes in external nutrients, such as glucose, via PKA. Yeast cells respond to nutrients by altering expression of the protein synthesis genes and thus their growth rate. Here, the authors use microarrays to show that TORC1 controls gene expression during steady state growth while PKA speeds up expression changes when nutrient levels change.

The Target of Rapamycin kinase Complex I (TORC1) is a central hub in the cell growth and metabolic control network of eukaryotes. How its upstream regulators cooperate to tune signaling across environmental conditions remains unclear. Here, we combine phosphoproteomics, TORC1 activity assays, and targeted genetic perturbations to dissect TORC1 regulation in Saccharomyces cerevisiae during transitions from a high-quality nitrogen source (glutamine) to a low-quality nitrogen source (proline), and on to complete nitrogen starvation. In proline medium, Ait1 and Gcn2 attenuate TORC1 activity, establishing a partially inhibited “Low Nitrogen Adaptive” state marked by extensive metabolic reprogramming without growth arrest. In contrast, during nitrogen starvation, SEAC, Ait1, and Gcn2 cooperate to drive TORC1 into a fully inhibited state, triggering widespread dephosphorylation of its downstream targets and entry into quiescence. Our results define a multilayered regulatory circuit that governs graded TORC1 control—a design likely conserved across eukaryotes. Budding yeast use multilayered regulation by Ait1, Gcn2, and the SEAC/GATOR complex to control TORC1 activity. This circuit design ensures that cells can adapt to low-quality nitrogen sources or shut down growth during starvation.

TOR kinase complex I (TORC1) is a key regulator of cell growth and metabolism in all eukaryotes. Previous studies in yeast have shown that three GTPases—Gtr1, Gtr2, and Rho1—bind to TORC1 in nitrogen and amino acid starvation conditions to block phosphorylation of the S6 kinase Sch9 and activate protein phosphatase 2A (PP2A). This leads to downregulation of 450 Sch9-dependent protein and ribosome synthesis genes and upregulation of 100 PP2A-dependent nitrogen assimilation and amino acid synthesis genes. Here, using bandshift assays and microarray measurements, we show that the TORC1 pathway also populates three other stress/starvation states. First, in glucose starvation conditions, the AMP-activated protein kinase (AMPK/Snf1) and at least one other factor push the TORC1 pathway into an off state, in which Sch9-branch signaling and PP2A-branch signaling are both inhibited. Remarkably, the TORC1 pathway remains in the glucose starvation (PP2A inhibited) state even when cells are simultaneously starved for nitrogen and glucose. Second, in osmotic stress, the MAPK Hog1/p38 drives the TORC1 pathway into a different state, in which Sch9 signaling and PP2A-branch signaling are inhibited, but PP2A-branch signaling can still be activated by nitrogen starvation. Third, in oxidative stress and heat stress, TORC1-Sch9 signaling is blocked while weak PP2A-branch signaling occurs. Together, our data show that the TORC1 pathway acts as an information-processing hub, activating different genes in different conditions to ensure that available energy is allocated to drive growth, amino acid synthesis, or a stress response, depending on the needs of the cell.

The target of rapamycin complex I (TORC1) regulates cell growth and metabolism in eukaryotes. Previous studies have shown that nitrogen and amino acid signals activate TORC1 via the highly conserved small GTPases, Gtr1/2 (RagA/C in humans), and the GTPase activating complex SEAC/GATOR. However, it remains unclear if, and how, other proteins/pathways regulate TORC1 in simple eukaryotes like yeast. Here, we report that the previously unstudied GPCR-like protein, Ait1, binds to TORC1-Gtr1/2 in Saccharomyces cerevisiae and holds TORC1 around the vacuole during log-phase growth. Then, during amino acid starvation, Ait1 inhibits TORC1 via Gtr1/2 using a loop that resembles the RagA/C-binding domain in the human protein SLC38A9. Importantly, Ait1 is only found in the Saccharomycetaceae/codaceae , two closely related families of yeast that have lost the ancient TORC1 regulators Rheb and TSC1/2. Thus, the TORC1 circuit found in the Saccharomycetaceae/codaceae , and likely other simple eukaryotes, has undergone significant rewiring during evolution.

The target of rapamycin complex I (TORC1) regulates cell growth and metabolism in eukaryotes. Previous studies have shown that nitrogen and amino acid signals activate TORC1 via the small GTPases, Gtr1/2. However, little is known about the way that other nutrient signals are transmitted to TORC1. Here we report that glucose starvation triggers disassembly of TORC1, and movement of the key TORC1 component Kog1/Raptor to a single body near the edge of the vacuole. These events are driven by Snf1/AMPK-dependent phosphorylation of Kog1 at Ser 491/494 and two nearby prion-like motifs. Kog1-bodies then serve to increase the threshold for TORC1 activation in cells that have been starved for a significant period of time. Together, our data show that Kog1-bodies create hysteresis (memory) in the TORC1 pathway and help ensure that cells remain committed to a quiescent state under suboptimal conditions. We suggest that other protein bodies formed in starvation conditions have a similar function. In humans, yeast and other eukaryotes, a group of proteins called the Target of Rapamycin Complex I (TORC1) promote cell growth and increase metabolic activity when nutrients are plentiful. Previous studies have shown how molecules that contain the nutrient nitrogen – which is needed to make proteins – activate TORC1. However, it is not clear how other nutrients regulate this complex. Bakers yeast is a simple, single celled organism that researchers often use as a model to study how cells work. The yeast TORC1 is made up of three core proteins, including Kog1 and Tor1. Kog1 selectively recruits proteins to the complex, where they are modified by Tor1 to regulate their activity. Here, Hughes Hallett et al. used microscopy to study what effect sugar starvation has on the complex. In the experiments, yeast cells were genetically engineered so that Kog1 and Tor1 appeared fluorescent under the microscope. The experiments reveal that, when sugar is in short supply, Kog1 breaks away from the rest of the TORC1 and moves to another part of the cell where it accumulates to form a cluster called a “body”. This movement is driven by a “kinase” enzyme that adds chemical groups called phosphates to Kog1, and by regions within the Kog1 protein known as prion like domains. When sugar first becomes available again, Kog1 is still in the body so Tor1 cannot immediately trigger cell growth. However, once a steady supply of sugar resumes, Kog1 rejoins the rest of the complex and the cells start to grow. Together, Hughes Hallett et al.’s findings reveal that the formation of Kog1 bodies during sugar starvation creates a “memory” that prevents TORC1 from reactivating cell growth if sugar is only temporarily available. Humans have over 100 proteins that contain prion like domains. Therefore a future challenge is to find out whether any of these proteins form similar bodies that enable our cells to remember past events.

Erin O'Shea and colleagues present a quantitative model of the Hog1 MAPK-dependent osmotic stress response in budding yeast derived from gene expression analyses in single- and multiple-mutant strains. The network reveals interactions involved in signal integration and processing and could serve as model for investigations into other gene regulatory networks. Cells regulate gene expression using a complex network of signaling pathways, transcription factors and promoters. To gain insight into the structure and function of these networks, we analyzed gene expression in single- and multiple-mutant strains to build a quantitative model of the Hog1 MAPK-dependent osmotic stress response in budding yeast. Our model reveals that the Hog1 and general stress (Msn2/4) pathways interact, at both the signaling and promoter level, to integrate information and create a context-dependent response. This study lays out a path to identifying and characterizing the role of signal integration and processing in other gene regulatory networks.

Many proteins populate collapsed intermediate states during folding. In order to elucidate the nature and importance of these species, we have mapped the structure of the on-pathway intermediate of the four-helix protein, Im7, together with the conformational changes it undergoes as it folds to the native state. Kinetic data for 29 Im7 point mutants show that the intermediate contains three of the four helices found in the native structure, packed around a specific hydrophobic core. However, the intermediate contains many non-native interactions; as a result, hydrophobic interactions become disrupted in the rate-limiting transition state before the final helix docks onto the developing structure. The results of this study support a hierarchical mechanism of protein folding and explain why the misfolding of Im7 occurs. The data also demonstrate that non-native interactions can play a significant role in folding, even for small proteins with simple topologies.

The Target of Rapamycin kinase Complex I (TORC1) is a master regulator of cell growth and metabolism in eukaryotes. Studies in yeast and human cells have shown that nitrogen/amino acid starvation signals act through Npr2/Npr3 and the small GTPases Gtr1/Gtr2 (Rags in humans) to inhibit TORC1. However, it is unclear how other stress and starvation stimuli inhibit TORC1, and/or act in parallel with the TORC1 pathway, to control cell growth. To help answer these questions, we developed a novel automated pipeline and used it to measure the expression of a TORC1-dependent ribosome biogenesis gene (NSR1) during osmotic stress in 4700 Saccharomyces cerevisiae strains from the yeast knock-out collection. This led to the identification of 440 strains with significant and reproducible defects in NSR1 repression. The cell growth control and stress response proteins deleted in these strains form a highly connected network, including 56 proteins involved in vesicle trafficking and vacuolar function; 53 proteins that act downstream of TORC1 according to a rapamycin assay—including components of the HDAC Rpd3L, Elongator, and the INO80, CAF-1 and SWI/SNF chromatin remodeling complexes; over 100 proteins involved in signaling and metabolism; and 17 proteins that directly interact with TORC1. These data provide an important resource for labs studying cell growth control and stress signaling, and demonstrate the utility of our new, and easily adaptable, method for mapping gene regulatory networks.

The Target of Rapamycin kinase Complex 1 (TORC1) regulates cell growth and metabolism in eukaryotes. Previous studies have shown that, in Saccharomyces cerevisiae , nitrogen and amino acid signals activate TORC1 via the highly conserved small GTPases, Gtr1/2, and the phosphatidylinositol 3-phosphate binding protein, Pib2. However, it was unclear if/how Gtr1/2 and Pib2 cooperate to control TORC1. Here, we report that this dual regulator system pushes TORC1 into at least three distinct signaling states: (i) a Gtr1/2 on, Pib2 on, rapid growth state in nutrient replete conditions; (ii) a Gtr1/2 inhibited, Pib2 on, adaptive/slow growth state in poor-quality growth medium; and (iii) a Gtr1/2 off, Pib2 off, quiescent state in starvation conditions. We suggest that other signaling pathways work in a similar way to drive a multilevel response via a single kinase, but the behavior has been overlooked since most studies follow signaling to a single reporter protein.

Many proteins populate partially organized structures during folding. Since these intermediates often accumulate within the dead time (2–5 ms) of conventional stopped-flow and quench-flow devices, it has been difficult to determine their role in the formation of the native state. Here we use a microcapillary mixing apparatus, with a time resolution of ∼150 μs, to directly follow the formation of an intermediate in the folding of a four-helix protein, Im7. Quantitative kinetic modeling of folding and unfolding data acquired over a wide range of urea concentrations demonstrate that this intermediate ensemble lies on a direct path from the unfolded to the native state.