Spatially Distinct Pools of TORC1 Balance Protein Homeostasis.

The eukaryotic TORC1 kinase is a homeostatic controller of growth that integrates nutritional cues and mediates signals primarily from the surface of lysosomes or vacuoles. Amino acids activate TORC1 via the Rag GTPases that combine into structurally conserved multi-protein complexes such as the EGO complex (EGOC) in yeast. Here we show that Ego1, which mediates membrane-anchoring of EGOC via lipid modifications that it acquires while traveling through the trans-Golgi network, is separately sorted to vacuoles and perivacuolar endosomes. At both surfaces, it assembles EGOCs, which regulate spatially distinct pools of TORC1 that impinge on functionally divergent effectors: vacuolar TORC1 predominantly targets Sch9 to promote protein synthesis, whereas endosomal TORC1 phosphorylates Atg13 and Vps27 to inhibit macroautophagy and ESCRT-driven microautophagy, respectively. Thus, the coordination of three key regulatory nodes in protein synthesis and degradation critically relies on a division of labor between spatially sequestered populations of TORC1.

Indole-3-acetic acid is a physiological inhibitor of TORC1 in yeast.

Indole-3-acetic acid (IAA) is the most common, naturally occurring phytohormone that regulates cell division, differentiation, and senescence in plants. The capacity to synthesize IAA is also widespread among plant-associated bacterial and fungal species, which may use IAA as an effector molecule to define their relationships with plants or to coordinate their physiological behavior through cell-cell communication. Fungi, including many species that do not entertain a plant-associated life style, are also able to synthesize IAA, but the physiological role of IAA in these fungi has largely remained enigmatic. Interestingly, in this context, growth of the budding yeast Saccharomyces cerevisiae is sensitive to extracellular IAA. Here, we use a combination of various genetic approaches including chemical-genetic profiling, SAturated Transposon Analysis in Yeast (SATAY), and genetic epistasis analyses to identify the mode-of-action by which IAA inhibits growth in yeast. Surprisingly, these analyses pinpointed the target of rapamycin complex 1 (TORC1), a central regulator of eukaryotic cell growth, as the major growth-limiting target of IAA. Our biochemical analyses further demonstrate that IAA inhibits TORC1 both in vivo and in vitro. Intriguingly, we also show that yeast cells are able to synthesize IAA and specifically accumulate IAA upon entry into stationary phase. Our data therefore suggest that IAA contributes to proper entry of yeast cells into a quiescent state by acting as a metabolic inhibitor of TORC1.

Leucyl-tRNA synthetase controls TORC1 via the EGO complex.

The target of rapamycin complex 1 (TORC1) is an essential regulator of eukaryotic cell growth that responds to growth factors, energy levels, and amino acids. The mechanisms through which the preeminent amino acid leucine signals to the TORC1-regulatory Rag GTPases, which activate TORC1 within the yeast EGO complex (EGOC) or the structurally related mammalian Rag-Ragulator complex, remain elusive. We find that the leucyl-tRNA synthetase (LeuRS) Cdc60 interacts with the Rag GTPase Gtr1 of the EGOC in a leucine-dependent manner. This interaction is necessary and sufficient to mediate leucine signaling to TORC1 and is disrupted by the engagement of Cdc60 in editing mischarged tRNA(Leu). Thus, the EGOC-TORC1 signaling module samples, via the LeuRS-intrinsic editing domain, the fidelity of tRNA(Leu) aminoacylation as a proxy for leucine availability.

Initiation of the TORC1-regulated G0 program requires Igo1/2, which license specific mRNAs to evade degradation via the 5'-3' mRNA decay pathway.

Eukaryotic cell proliferation is controlled by growth factors and essential nutrients, in the absence of which cells may enter into a quiescent (G(0)) state. In yeast, nitrogen and/or carbon limitation causes downregulation of the conserved TORC1 and PKA signaling pathways and, consequently, activation of the PAS kinase Rim15, which orchestrates G(0) program initiation and ensures proper life span by controlling distal readouts, including the expression of specific genes. Here, we report that Rim15 coordinates transcription with posttranscriptional mRNA protection by phosphorylating the paralogous Igo1 and Igo2 proteins. This event, which stimulates Igo proteins to associate with the mRNA decapping activator Dhh1, shelters newly expressed mRNAs from degradation via the 5'-3' mRNA decay pathway, thereby enabling their proper translation during initiation of the G(0) program. These results delineate a likely conserved mechanism by which nutrient limitation leads to stabilization of specific mRNAs that are critical for cell differentiation and life span.

Phosphorylation of Bem2p and Bem3p may contribute to local activation of Cdc42p at bud emergence.

Site-specific activation of the Rho-type GTPase Cdc42p is critical for the establishment of cell polarity. Here we investigated the role and regulation of the GTPase-activating enzymes (GAPs) Bem2p and Bem3p for Cdc42p activation and actin polarization at bud emergence in Saccharomyces cerevisiae. Bem2p and Bem3p are localized throughout the cytoplasm and the cell cortex in unbudded G1 cells, but accumulate at sites of polarization after bud emergence. Inactivation of Bem2p results in hyperactivation of Cdc42p and polarization toward multiple sites. Bem2p and Bem3p are hyperphosphorylated at bud emergence most likely by the Cdc28p-Cln2p kinase. This phosphorylation appears to inhibit their GAP activity in vivo, as non-phosphorylatable Bem3p mutants are hyperactive and interfere with Cdc42p activation. Taken together, our results indicate that Bem2p and Bem3p may function as global inhibitors of Cdc42p activation during G1, and their inactivation by the Cdc28p/Cln kinase contributes to site-specific activation of Cdc42p at bud emergence.

Phosphoproteomic responses of TORC1 target kinases reveal discrete and convergent mechanisms that orchestrate the quiescence program in yeast.

The eukaryotic TORC1 kinase assimilates diverse environmental cues, including growth factors and nutrients, to control growth by tuning anabolic and catabolic processes. In yeast, TORC1 stimulates protein synthesis in response to abundant nutrients primarily through its proximal effector kinase Sch9. Conversely, TORC1 inhibition following nutrient limitation unlocks various distally controlled kinases (e.g., Atg1, Gcn2, Npr1, Rim15, Slt2/Mpk1, and Yak1), which cooperate through poorly defined circuits to orchestrate the quiescence program. To better define the signaling landscape of the latter kinases, we use in vivo quantitative phosphoproteomics. Through pinpointing known and uncharted Npr1, Rim15, Slt2/Mpk1, and Yak1 effectors, our study examines the architecture of the distally controlled TORC1 kinase network. Accordingly, this is built on a combination of discrete, convergent, and multilayered feedback regulatory mechanisms, which likely ensure homeostatic control of and/or robust responses by TORC1 and its effector kinases under fluctuating nutritional conditions.

Susi, a negative regulator of Drosophila PI3-kinase.

The Phosphatidylinositol-3 kinase/Protein Kinase B (PI3K/PKB) signaling pathway controls growth, metabolism, and lifespan in animals, and deregulation of its activity is associated with diabetes and cancer in humans. Here, we describe Susi, a coiled-coil domain protein that acts as a negative regulator of insulin signaling in Drosophila. Whereas loss of Susi function increases body size, overexpression of Susi reduces growth. We provide genetic evidence that Susi negatively regulates dPI3K activity. Susi directly binds to dP60, the regulatory subunit of dPI3K. Since Susi has no overt similarity to known inhibitors of PI3K/PKB signaling, it defines a novel mechanism by which this signaling cascade is kept in check. The fact that Susi is expressed in a circadian rhythm, with highest levels during the night, suggests that Susi attenuates insulin signaling during the fasting period.

The cullin Rtt101p promotes replication fork progression through damaged DNA and natural pause sites.

Accurate and complete DNA replication is fundamental to maintain genome integrity. While the mechanisms and underlying machinery required to duplicate bulk genomic DNA are beginning to emerge, little is known about how cells replicate through damaged areas and special chromosomal regions such as telomeres, centromeres, and highly transcribed loci . Here, we have investigated the role of the yeast cullin Rtt101p in this process. We show that rtt101Delta cells accumulate spontaneous DNA damage and exhibit a G(2)/M delay, even though they are fully proficient to detect and repair chromosome breaks. Viability of rtt101Delta mutants depends on Rrm3p, a DNA helicase involved in displacing proteinaceous complexes at programmed pause sites . Moreover, rtt101Delta cells show hyperrecombination at forks arrested at replication fork barriers (RFBs) of ribosomal DNA. Finally, rtt101Delta mutants are sensitive to fork arrest induced by DNA alkylation, but not by nucleotide depletion. We therefore propose that the cullin Rtt101p promotes fork progression through obstacles such as DNA lesions or tightly bound protein-DNA complexes via a new mechanism involving ubiquitin-conjugation.

GUP1 of Saccharomyces cerevisiae encodes an O-acyltransferase involved in remodeling of the GPI anchor.

The anchors of mature glycosylphosphatidylinositol (GPI)-anchored proteins of Saccharomyces cerevisiae contain either ceramide or diacylglycerol with a C26:0 fatty acid in the sn2 position. The primary GPI lipid added to newly synthesized proteins in the ER consists of diacylglycerol with conventional C16 and C18 fatty acids. Here we show that GUP1 is essential for the synthesis of the C26:0-containing diacylglycerol anchors. Gup1p is an ER membrane protein with multiple membrane-spanning domains harboring a motif that is characteristic of membrane-bound O-acyl-transferases (MBOAT). Gup1Delta cells make normal amounts of GPI proteins but most mature GPI anchors contain lyso-phosphatidylinositol, and others possess phosphatidylinositol with conventional C16 and C18 fatty acids. The incorporation of the normal ceramides into the anchors is also disturbed. As a consequence, the ER-to-Golgi transport of the GPI protein Gas1p is slow, and mature Gas1p is lost from the plasma membrane into the medium. Gup1Delta cells have fragile cell walls and a defect in bipolar bud site selection. GUP1 function depends on the active site histidine of the MBOAT motif. GUP1 is highly conserved among fungi and protozoa and the gup1Delta phenotype is partially corrected by GUP1 homologues of Aspergillus fumigatus and Trypanosoma cruzi.

Multilayered Control of Protein Turnover by TORC1 and Atg1.

The target of rapamycin complex 1 (TORC1) is a master regulator of cell homeostasis, which promotes anabolic reactions and synchronously inhibits catabolic processes such as autophagy-mediated protein degradation. Its prime autophagy target is Atg13, a subunit of the Atg1 kinase complex that acts as the gatekeeper of canonical autophagy. To study whether the activities of TORC1 and Atg1 are coupled through additional, more intricate control mechanisms than simply this linear pathway, we analyzed the epistatic relationship between TORC1 and Atg1 by using quantitative phosphoproteomics. Our in vivo data, combined with targeted in vitro TORC1 and Atg1 kinase assays, not only uncover numerous TORC1 and Atg1 effectors, but also suggest distinct bi-directional regulatory feedback loops and characterize Atg29 as a commonly regulated downstream target of both TORC1 and Atg1. Thus, an exquisitely multilayered regulatory network appears to coordinate TORC1 and Atg1 activities to robustly tune autophagy in response to nutritional cues.

TORC1 coordinates the conversion of Sic1 from a target to an inhibitor of cyclin-CDK-Cks1.

Eukaryotic cell cycle progression through G1-S is driven by hormonal and growth-related signals that are transmitted by the target of rapamycin complex 1 (TORC1) pathway. In yeast, inactivation of TORC1 restricts G1-S transition due to the rapid clearance of G1 cyclins (Cln) and the stabilization of the B-type cyclin (Clb) cyclin-dependent kinase (CDK) inhibitor Sic1. The latter mechanism remains mysterious but requires the phosphorylation of Sic1-Thr173 by Mpk1 and inactivation of the Sic1-pThr173-targeting phosphatase (PP2ACdc55) through greatwall kinase-activated endosulfines. Here we show that the Sic1-pThr173 residue serves as a specific docking site for the CDK phospho-acceptor subunit Cks1 that sequesters, together with a C-terminal Clb5-binding motif in Sic1, Clb5-CDK-Cks1 complexes, thereby preventing them from flagging Sic1 for ubiquitin-dependent proteolysis. Interestingly, this functional switch of Sic1 from a target to an inhibitor of cyclin-CDK-Cks1 also operates in proliferating cells and is coordinated by the greatwall kinase, which responds to both Cln-CDK-dependent cell-cycle and TORC1-mediated nutritional cues.

TORC1 controls G1-S cell cycle transition in yeast via Mpk1 and the greatwall kinase pathway.

The target of rapamycin complex 1 (TORC1) pathway couples nutrient, energy and hormonal signals with eukaryotic cell growth and division. In yeast, TORC1 coordinates growth with G1-S cell cycle progression, also coined as START, by favouring the expression of G1 cyclins that activate cyclin-dependent protein kinases (CDKs) and by destabilizing the CDK inhibitor Sic1. Following TORC1 downregulation by rapamycin treatment or nutrient limitation, clearance of G1 cyclins and C-terminal phosphorylation of Sic1 by unknown protein kinases are both required for Sic1 to escape ubiquitin-dependent proteolysis prompted by its flagging via the SCF(Cdc4) (Skp1/Cul1/F-box protein) ubiquitin ligase complex. Here we show that the stabilizing phosphorylation event within the C-terminus of Sic1 requires stimulation of the mitogen-activated protein kinase, Mpk1, and inhibition of the Cdc55 protein phosphatase 2A (PP2A(Cdc55)) by greatwall kinase-activated endosulfines. Thus, Mpk1 and the greatwall kinase pathway serve TORC1 to coordinate the phosphorylation status of Sic1 and consequently START with nutrient availability.

TORC1 regulates Pah1 phosphatidate phosphatase activity via the Nem1/Spo7 protein phosphatase complex.

The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls growth-related processes such as protein, nucleotide, and lipid metabolism in response to growth hormones, energy/ATP levels, and amino acids. Its deregulation is associated with cancer, type 2 diabetes, and obesity. Among other substrates, mammalian TORC1 directly phosphorylates and inhibits the phosphatidate phosphatase lipin-1, a central enzyme in lipid metabolism that provides diacylglycerol for the synthesis of membrane phospholipids and/or triacylglycerol as neutral lipid reserve. Here, we show that yeast TORC1 inhibits the function of the respective lipin, Pah1, to prevent the accumulation of triacylglycerol. Surprisingly, TORC1 regulates Pah1 in part indirectly by controlling the phosphorylation status of Nem1 within the Pah1-activating, heterodimeric Nem1-Spo7 protein phosphatase module. Our results delineate a hitherto unknown TORC1 effector branch that controls lipin function in yeast, which, given the recent discovery of Nem1-Spo7 orthologous proteins in humans, may be conserved.

Yeast endosulfines control entry into quiescence and chronological life span by inhibiting protein phosphatase 2A.

The TORC1 and PKA protein kinases are central elements of signaling networks that regulate eukaryotic cell proliferation in response to growth factors and/or nutrients. In yeast, attenuation of signaling by these kinases following nitrogen and/or carbon limitation activates the protein kinase Rim15, which orchestrates the initiation of a reversible cellular quiescence program to ensure normal chronological life span. The molecular elements linking Rim15 to distal readouts including the expression of Msn2/4- and Gis1-dependent genes involve the endosulfines Igo1/2. Here, we show that Rim15, analogous to the greatwall kinase in Xenopus, phosphorylates endosulfines to directly inhibit the Cdc55-protein phosphatase 2A (PP2A(Cdc55)). Inhibition of PP2A(Cdc55) preserves Gis1 in a phosphorylated state and consequently promotes its recruitment to and activation of transcription from promoters of specific nutrient-regulated genes. These results close a gap in our perception of and delineate a role for PP2A(Cdc55) in TORC1-/PKA-mediated regulation of quiescence and chronological life span.

The Gup1 homologue of Trypanosoma brucei is a GPI glycosylphosphatidylinositol remodelase.

Glycosylphosphatidylinositol (GPI) lipids of Trypanosoma brucei undergo lipid remodelling, whereby longer fatty acids on the glycerol are replaced by myristate (C14:0). A similar process occurs on GPI proteins of Saccharomyces cerevisiae where Per1p first deacylates, Gup1p subsequently reacylates the anchor lipid, thus replacing a shorter fatty acid by C26:0. Heterologous expression of the GUP1 homologue of T. brucei in gup1Delta yeast cells partially normalizes the gup1Delta phenotype and restores the transfer of labelled fatty acids from Coenzyme A to lyso-GPI proteins in a newly developed microsomal assay. In this assay, the Gup1p from T. brucei (tbGup1p) strongly prefers C14:0 and C12:0 over C16:0 and C18:0, whereas yeast Gup1p strongly prefers C16:0 and C18:0. This acyl specificity of tbGup1p closely matches the reported specificity of the reacylation of free lyso-GPI lipids in microsomes of T. brucei. Depletion of tbGup1p in trypanosomes by RNAi drastically reduces the rate of myristate incorporation into the sn-2 position of lyso-GPI lipids. Thus, tbGup1p is involved in the addition of myristate to sn-2 during GPI remodelling in T. brucei and can account for the fatty acid specificity of this process. tbGup1p can act on GPI proteins as well as on GPI lipids.

Cell cycle-dependent nuclear export of Cdh1p may contribute to the inactivation of APC/C(Cdh1).

Cdh1p is a substrate-specific subunit of the anaphase-promoting complex (APC/C), which functions as an E3 ubiquitin ligase to degrade the mitotic cyclin Clb2p and other substrates during the G(1) phase of the cell cycle. Cdh1p is phosphorylated and thereby inactivated at the G(1)/S transition predominantly by Cdc28p-Clb5p. Here we show that Cdh1p is nuclear during the G(1) phase of the cell cycle, but redistributes to the cytoplasm between S phase and the end of mitosis. Nuclear export of Cdh1p is regulated by phosphorylation and requires active Cdc28p kinase. Cdh1p binds to the importin Pse1p and the exportin Msn5p, which is necessary and sufficient to promote efficient export of Cdh1p in vivo. Although msn5delta cells are viable, they are sensitive to Cdh1p overexpression. Likewise, a mutant form of Cdh1p, which is constitutively nuclear, prevents accumulation of Clb2p and leads to cell cycle arrest when overexpressed in wild-type cells. Taken together, these results suggest that phosphorylation-dependent nuclear export of Cdh1p by Msn5p contributes to efficient inactivation of APC/C(Cdh1).

A positive feedback loop stabilizes the guanine-nucleotide exchange factor Cdc24 at sites of polarization.

In Saccharomyces cerevisiae, activation of Cdc42 by its guanine-nucleotide exchange factor Cdc24 triggers polarization of the actin cytoskeleton at bud emergence and in response to mating pheromones. The adaptor protein Bem1 localizes to sites of polarized growth where it interacts with Cdc42, Cdc24 and the PAK-like kinase Cla4. We have isolated Bem1 mutants (Bem1-m), which are specifically defective for binding to Cdc24. The mutations map within the conserved PB1 domain, which is necessary and sufficient to interact with the octicos peptide repeat (OPR) motif of Cdc24. Although Bem1-m mutant proteins localize normally, bem1-m cells are unable to maintain Cdc24 at sites of polarized growth. As a consequence, they are defective for apical bud growth and the formation of mating projections. Localization of Bem1 to the incipient bud site requires activated Cdc42, and conversely, expression of Cdc42-GTP is sufficient to accumulate Bem1 at the plasma membrane. Thus, our results suggest that Bem1 functions in a positive feedback loop: local activation of Cdc24 produces Cdc42-GTP, which recruits Bem1. In turn, Bem1 stabilizes Cdc24 at the site of polarization, leading to apical growth.