Structure-based engineering of Tor complexes reveals that two types of yeast TORC1 produce distinct phenotypes.

Certain proteins assemble into diverse complex states, each having a distinct and unique function in the cell. Target of rapamycin (Tor) complex 1 (TORC1) plays a central role in signalling pathways that allow cells to respond to the environment, including nutritional status signalling. TORC1 is widely recognised for its association with various diseases. The budding yeast Saccharomyces cerevisiae has two types of TORC1, Tor1-containing TORC1 and Tor2-containing TORC1, which comprise different constituent proteins but are considered to have the same function. Here, we computationally modelled the relevant complex structures and then, based on the structures, rationally engineered a Tor2 mutant that could form Tor complex 2 (TORC2) but not TORC1, resulting in a redesign of the complex states. Functional analysis of the Tor2 mutant revealed that the two types of TORC1 induce different phenotypes, with changes observed in rapamycin, caffeine and pH dependencies of cell growth, as well as in replicative and chronological lifespan. These findings uncovered by a general approach with huge potential - model structure-based engineering - are expected to provide further insights into various fields such as molecular evolution and lifespan.

Fission Yeast TORC1 Promotes Cell Proliferation through Sfp1, a Transcription Factor Involved in Ribosome Biogenesis.

Target of rapamycin complex 1 (TORC1) is activated in response to nutrient availability and growth factors, promoting cellular anabolism and proliferation. To explore the mechanism of TORC1-mediated proliferation control, we performed a genetic screen in fission yeast and identified Sfp1, a zinc-finger transcription factor, as a multicopy suppressor of temperature-sensitive TORC1 mutants. Our observations suggest that TORC1 phosphorylates Sfp1 and protects Sfp1 from proteasomal degradation. Transcription analysis revealed that Sfp1 positively regulates genes involved in ribosome production together with two additional transcription factors, Ifh1/Crf1 and Fhl1. Ifh1 physically interacts with Fhl1, and the nuclear localization of Ifh1 is regulated in response to nutrient levels in a manner dependent on TORC1 and Sfp1. Taken together, our data suggest that the transcriptional regulation of the genes involved in ribosome biosynthesis by Sfp1, Ifh1, and Fhl1 is one of the key pathways through which nutrient-activated TORC1 promotes cell proliferation.

Contribution of the yeast bi-chaperone system in the restoration of the RNA helicase Ded1 and translational activity under severe ethanol stress.

Preexposure to mild stress often improves cellular tolerance to subsequent severe stress. Severe ethanol stress (10% v/v) causes persistent and pronounced translation repression in Saccharomyces cerevisiae. However, it remains unclear whether preexposure to mild stress can mitigate translation repression in yeast cells under severe ethanol stress. We found that the translational activity of yeast cells pretreated with 6% (v/v) ethanol was initially significantly repressed under subsequent 10% ethanol but was then gradually restored even under severe ethanol stress. We also found that 10% ethanol caused the aggregation of Ded1, which plays a key role in translation initiation as a DEAD-box RNA helicase. Pretreatment with 6% ethanol led to the gradual disaggregation of Ded1 under subsequent 10% ethanol treatment in wild-type cells but not in fes1Δhsp104Δ cells, which are deficient in Hsp104 with significantly reduced capacity for Hsp70. Hsp104 and Hsp70 are key components of the bi-chaperone system that play a role in yeast protein quality control. fes1Δhsp104Δ cells did not restore translational activity under 10% ethanol, even after pretreatment with 6% ethanol. These results indicate that the regeneration of Ded1 through the bi-chaperone system leads to the gradual restoration of translational activity under continuous severe stress. This study provides new insights into the acquired tolerance of yeast cells to severe ethanol stress and the resilience of their translational activity.

Yeast Tor complex 1 phosphorylates eIF4E-binding protein, Caf20.

Tor complex 1 (TORC1), a master regulator of cell growth, is an evolutionarily conserved protein kinase within eukaryotic organisms. To control cell growth, TORC1 governs translational processes by phosphorylating its substrate proteins in response to cellular nutritional cues. Mammalian TORC1 (mTORC1) assumes the responsibility of phosphorylating the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) to regulate its interaction with eIF4E. The budding yeast Saccharomyces cerevisiae possesses a pair of 4E-BP genes, CAF20 and EAP1. However, the extent to which the TORC1-4E-BP axis regulates translational initiation in yeast remains uncertain. In this study, we demonstrated the influence of TORC1 on the phosphorylation status of Caf20 in vivo, as well as the direct phosphorylation of Caf20 by TORC1 in vitro. Furthermore, we found the TORC1-dependent recruitment of Caf20 to the 80S ribosome. Consequently, our study proposes a plausible involvement of yeast's 4E-BP in the efficacy of translation initiation, an aspect under the control of TORC1.

Involvement of Gtr1p in the oxidative stress response in yeast Saccharomyces cerevisiae.

Yeast Gtr1p is a GTPase that forms a heterodimer with Gtr2p, another GTPase; it is involved in regulating TORC1 activity in nutrient signaling, including amino acid availability and growth control. Gtr1p is a positive regulator of TORC1, a kinase that regulates various cellular functions (e.g., protein synthesis and autophagy) under specific nutrient and environmental conditions, including oxidative stress. In this study, we examined the roles of Gtr1p in oxidative stress responses. We found that yeast cells expressing guanosine diphosphatase (GDP)-bound Gtr1p (Gtr1-S20Lp) were resistant to hydrogen peroxide (H2O2), whereas guanosine triphosphate (GTP)-bound Gtr1p (Gtr1-Q65Lp) was sensitive to H2O2 compared with the wild type. Consistent with these findings, yeast cells lacking Iml1p, a component of the GTPase-activating protein complex for Gtr1p, exhibited the H2O2-sensitive phenotype. In gtr1S20L cells, autophagy was highly induced under oxidative stress. gtr1Q65L cells showed decreased expression of the SNQ2 gene, which encodes a multidrug transporter involved in resistance to oxidative stress, and the overexpression of SNQ2 rescued the oxidative stress sensitivity of gtr1Q65L cells. These results suggest that Gtr1p is involved in oxidative stress responses through mechanisms that include autophagy and SNQ2 expression.

Novel Links between TORC1 and Traditional Non-Coding RNA, tRNA.

Target of rapamycin (TOR) is a serine/threonine kinase that modulates cell growth and metabolism in response to environmental changes. Transfer RNA (tRNA) is an abundant and ubiquitous small non-coding RNA that is essential in the translation of mRNAs. Beyond its canonical role, it has been revealed that tRNAs have more diverse functions. TOR complex 1 (TORC1), which is one of the two TOR complexes, regulates tRNA synthesis by controlling RNA polymerase III. In addition to tRNA synthesis regulation, recent studies have revealed hidden connections between TORC1 and tRNA, which are both essential players in eukaryotic cellular activities. Here, we review the accumulating findings on the regulatory links between TORC1 and tRNA-particularly those links in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe.

A nuclear membrane-derived structure associated with Atg8 is involved in the sequestration of selective cargo, the Cvt complex, during autophagosome formation in yeast.

Macroautophagy (hereafter autophagy) is a conserved intracellular degradation mechanism required for cell survival. A double-membrane structure, the phagophore, is generated to sequester cytosolic cargos destined for degradation in the vacuole. The mechanism involved in the biogenesis of the phagophore is still an open question. We focused on 4 autophagy-related (Atg) proteins (Atg2, Atg9, Atg14, and Atg18), which are involved in the formation of the phagophore in order to gain a more complete understanding of the membrane dynamics that occur during formation of the autophagosome. The corresponding mutants, while defective in autophagy, nonetheless generate the membrane-bound form of Atg8, allowing us to use this protein as a marker for the nascent autophagosome precursor membrane. Using electron microscopy (EM), we discovered in these atg mutants a novel single-membrane structure (~120 to 150 nm in size). Electron tomography revealed that this structure originates from a part of the nuclear membrane, and we have named it the alphasome. Our data suggest that the alphasome is associated with Atg8, and sequesters selective cargo, the Cvt complex, during autophagy. Abbreviations: 3D: three-dimensional; AB: autophagic body; AP: autophagosome; Atg: autophagy-related; Cvt: cytoplasm-to-vacuole targeting; EM: electron microscopy; IEM: immunoelectron microscopy; L: lipid droplet; N: nucleus; NM: nuclear membrane; PAS: phagophore assembly site; PE: phosphatidylethanolamine; prApe1: precursor aminopeptidase I; rER: rough endoplasmic reticulum; TEM: transmission electron microscopy; V: vacuole; VLP: virus-like particle.

Vacuole-mediated selective regulation of TORC1-Sch9 signaling following oxidative stress.

Target of rapamycin complex 1 (TORC1) is a central cellular signaling coordinator that allows eukaryotic cells to adapt to the environment. In the budding yeast, Saccharomyces cerevisiae, TORC1 senses nitrogen and various stressors and modulates proteosynthesis, nitrogen uptake and metabolism, stress responses, and autophagy. There is some indication that TORC1 may regulate these downstream pathways individually. However, the potential mechanisms for such differential regulation are unknown. Here we show that the serine/threonine protein kinase Sch9 branch of TORC1 signaling depends specifically on the integrity of the vacuolar membrane, and this dependency originates in changes in Sch9 localization reflected by phosphatidylinositol 3,5-bisphosphate. Moreover, oxidative stress induces the delocalization of Sch9 from vacuoles, contributing to the persistent inhibition of the Sch9 branch after stress. Thus, our results establish that regulation of the vacuolar localization of Sch9 serves as a selective switch for the Sch9 branch in divergent TORC1 signaling. We propose that the Sch9 branch integrates the intrinsic activity of TORC1 kinase and vacuolar status, which is monitored by the phospholipids of the vacuolar membrane, into the regulation of macromolecular synthesis.

Novel tRNA function in amino acid sensing of yeast Tor complex1.

TOR complex1 (TORC1), a master regulator of cell growth, is regulated by amino acids. Amino acids are fundamental nutrients, and 20 species of amino acids building proteins are not interchangeable with each other. Therefore, TORC1 should sense each amino acid individually. Mammalian mTORC1 is controlled by Rag GTPases and their regulators. However, Rag factors are dispensable for amino acid sensing by TORC1 in the budding yeast, suggesting an alternative mechanism of TORC1 regulation. Here, genetic investigation discovered the involvement of (aminoacyl-)tRNA ((aa-)tRNA) in TORC1 regulation. Biochemical TORC1 assay also showed that tRNA directly inhibits TORC1 kinase activity. Reducing cellular tRNA molecule desensitizes TORC1 inactivation by nitrogen starvation in vivo. Based on these results, I propose a model of the TORC1 regulatory mechanism in which free tRNA released from protein synthesis under amino acid starvation inhibits TORC1 activity. Therefore, TORC1 uses tRNA-mediated mechanism and Rag factors in parallel to sense intracellular amino acids.

Amino acid residues required for Gtr1p-Gtr2p complex formation and its interactions with the Ego1p-Ego3p complex and TORC1 components in yeast.

The yeast Ras-like GTPases Gtr1p and Gtr2p form a heterodimer, are implicated in the regulation of TOR complex 1 (TORC1) and play pivotal roles in cell growth. Gtr1p and Gtr2p bind Ego1p and Ego3p, which are tethered to the endosomal and vacuolar membranes where TORC1 functions are regulated through a relay of amino acid signaling interactions. The mechanisms by which Gtr1p and Gtr2p activate TORC1 remain obscure. We probed the interactions of the Gtr1p-Gtr2p complex with the Ego1p-Ego3p complex and TORC1 subunits. Mutations in the region (179-220 a.a.) following the nucleotide-binding region of Gtr1p and Gtr2p abrogated their mutual interaction and resulted in a loss in function, suggesting that complex formation between Gtr1p and Gtr2p was indispensable for TORC1 function. A modified yeast two-hybrid assay showed that Gtr1p-Gtr2p complex formation is important for its interaction with the Ego1p-Ego3p complex. GTP-bound Gtr1p interacted with the region containing the HEAT repeats of Kog1p and the C-terminal region of Tco89p. The GTP-bound Gtr2p suppressed a Kog1p mutation. Our findings indicate that the interactions of the Gtr1p-Gtr2p complex with the Ego1p-Ego3p complex and TORC1 components Kog1p and Tco89p play a role in TORC1 function.

The role of autophagy in genome stability through suppression of abnormal mitosis under starvation.

The coordination of subcellular processes during adaptation to environmental change is a key feature of biological systems. Starvation of essential nutrients slows cell cycling and ultimately causes G1 arrest, and nitrogen starvation delays G2/M progression. Here, we show that budding yeast cells can be efficiently returned to the G1 phase under starvation conditions in an autophagy-dependent manner. Starvation attenuates TORC1 activity, causing a G2/M delay in a Swe1-dependent checkpoint mechanism, and starvation-induced autophagy assists in the recovery from a G2/M delay by supplying amino acids required for cell growth. Persistent delay of the cell cycle by a deficiency in autophagy causes aberrant nuclear division without sufficient cell growth, leading to an increased frequency in aneuploidy after refeeding the nitrogen source. Our data establish the role of autophagy in genome stability through modulation of cell division under conditions that repress cell growth.

A novel mechanism regulates H(2) S and SO(2) production in Saccharomyces cerevisiae.

Sulfite (SO(2) ) plays an important role in flavour stability in alcoholic beverages, whereas hydrogen sulfide (H(2) S) has an undesirable aroma. To discover the cellular processes that control SO(2) and H(2) S production, we screened a library of Saccharomyces cerevisiae deletion mutants. Deletion of 12 genes led to increased H(2) S productivity. Ten of these genes are known to be involved in sulfur-containing amino acid metabolism, whereas UBI4 functions in the ubiquitin-proteasome system and SKP2 encodes an F-box-containing protein whose function is unknown. We found that the skp2 mutant accumulated H(2) S and SO(2) , because the adenosylphophosulfate kinase Met14p is a substrate of SCF(Skp2) and more stable in the skp2 mutant than in the wild-type strain. Furthermore, the skp2 mutant grew more slowly than the wild-type strain under nutrient-limited conditions. Metabolome analysis showed that the concentration of intracellular cysteine is lower in the skp2 mutant than in the wild-type strain. The slow growth of the skp2 mutant was due to a lower concentration of intracellular cysteine, because the addition of cysteine suppressed the slow growth. In the skp2 mutant, the cysteine biosynthesis proteins Str2p, Str3p and Str4p are more stable than in the wild-type strain. Moreover, supplementation with methionine, S-adenosylmethionine, S-adenosylhomocysteine and homocysteine also suppressed the slow growth. Overexpression of STR1 or STR4 caused a more severe defect in the skp2 mutant. These results suggest that the balance of methionine and cysteine biosynthesis is important for yeast cell growth. Thus, Skp2p is one of the key components regulating this balance and H(2) S/SO(2) production.

Prime-numbered Atg proteins act at the primary step in autophagy: unphosphorylatable Atg13 can induce autophagy without TOR inactivation.

Autophagy is induced by inactivation of Tor complex 1 (TORC1), such as what happens during nutrient limitation and rapamycin treatment. However, the mechanism by which TORC1 regulates autophagy remains unclear. The Atg1 kinase complex that comprises Atg1 and its binding prime-numbered Atg proteins (Atg11, Atg13, Atg17, Atg29 and Atg31) has long been a candidate for TORC1's downstream target. This is especially the case for Atg13, a regulatory component of the Atg1 complex, which is highly phosphorylated in a TORC1-dependent manner. We find that yeast TORC1 directly phosphorylates Atg13 on at least eight Ser residues. Strikingly, expression of an unphosphorylatable Atg13 (Atg13- 8SA) mutant bypasses the TORC1 pathway to induce autophagy in vegetatively growing cells. Induction of autophagy by Atg13-8SA is accompanied by molecular events involving Atg proteins, such as formation of the Atg1 complex, activation of Atg1, and the organization of the pre-autophagosomal structure (PAS). These findings suggest that formation of the Atg1 complex is a primary step at induction of autophagy, and that dephosphorylation of Atg13 acts as a molecular switch to turn on starvation-induced autophagy.

Tor directly controls the Atg1 kinase complex to regulate autophagy.

Autophagy is a bulk proteolytic process that is indispensable for cell survival during starvation. Autophagy is induced by nutrient deprivation via inactivation of the rapamycin-sensitive Tor complex1 (TORC1), a protein kinase complex regulating cell growth in response to nutrient conditions. However, the mechanism by which TORC1 controls autophagy and the direct target of TORC1 activity remain unclear. Atg13 is an essential regulatory component of autophagy upstream of the Atg1 kinase complex, and here we show that yeast TORC1 directly phosphorylates Atg13 at multiple Ser residues. Additionally, expression of an unphosphorylatable Atg13 mutant bypasses the TORC1 pathway to induce autophagy through activation of Atg1 in cells growing under nutrient-rich conditions. Our findings suggest that the direct control of the Atg1 complex by TORC1 induces autophagy.

Dynamics and diversity in autophagy mechanisms: lessons from yeast.

Autophagy is a fundamental function of eukaryotic cells and is well conserved from yeast to humans. The most remarkable feature of autophagy is the synthesis of double membrane-bound compartments that sequester materials to be degraded in lytic compartments, a process that seems to be mechanistically distinct from conventional membrane traffic. The discovery of autophagy in yeast and the genetic tractability of this organism have allowed us to identify genes that are responsible for this process, which has led to the explosive growth of this research field seen today. Analyses of autophagy-related (Atg) proteins have unveiled dynamic and diverse aspects of mechanisms that underlie membrane formation during autophagy.

The yeast Tor signaling pathway is involved in G2/M transition via polo-kinase.

The target of rapamycin (Tor) protein plays central roles in cell growth. Rapamycin inhibits cell growth and promotes cell cycle arrest at G1 (G0). However, little is known about whether Tor is involved in other stages of the cell division cycle. Here we report that the rapamycin-sensitive Tor complex 1 (TORC1) is involved in G2/M transition in S. cerevisiae. Strains carrying a temperature-sensitive allele of KOG1 (kog1-105) encoding an essential component of TORC1, as well as yeast cell treated with rapamycin show mitotic delay with prolonged G2. Overexpression of Cdc5, the yeast polo-like kinase, rescues the growth defect of kog1-105, and in turn, Cdc5 activity is attenuated in kog1-105 cells. The TORC1-Type2A phosphatase pathway mediates nucleocytoplasmic transport of Cdc5, which is prerequisite for its proper localization and function. The C-terminal polo-box domain of Cdc5 has an inhibitory role in nuclear translocation. Taken together, our results indicate a novel function of Tor in the regulation of cell cycle and proliferation.

Organization of the pre-autophagosomal structure responsible for autophagosome formation.

Autophagy induced by nutrient depletion is involved in survival during starvation conditions. In addition to starvation-induced autophagy, the yeast Saccharomyces cerevisiae also has a constitutive autophagy-like system, the Cvt pathway. Among 31 autophagy-related (Atg) proteins, the function of Atg17, Atg29, and Atg31 is required specifically for autophagy. In this study, we investigated the role of autophagy-specific (i.e., non-Cvt) proteins under autophagy-inducing conditions. For this purpose, we used atg11Delta cells in which the Cvt pathway is abrogated. The autophagy-unique proteins are required for the localization of Atg proteins to the pre-autophagosomal structure (PAS), the putative site for autophagosome formation, under starvation condition. It is likely that these Atg proteins function as a ternary complex, because Atg29 and Atg31 bind to Atg17. The Atg1 kinase complex (Atg1-Atg13) is also essential for recruitment of Atg proteins to the PAS. The assembly of Atg proteins to the PAS is observed only under autophagy-inducing conditions, indicating that this structure is specifically involved in autophagosome formation. Our results suggest that Atg1 complex and the autophagy-unique Atg proteins cooperatively organize the PAS in response to starvation signals.

Characterization of a novel autophagy-specific gene, ATG29.

Autophagy is a process whereby cytoplasmic proteins and organelles are sequestered for bulk degradation in the vacuole/lysosome. At present, 16 ATG genes have been found that are essential for autophagosome formation in the yeast Saccharomyces cerevisiae. Most of these genes are also involved in the cytoplasm to vacuole transport pathway, which shares machinery with autophagy. Most Atg proteins are colocalized at the pre-autophagosomal structure (PAS), from which the autophagosome is thought to originate, but the precise mechanism of autophagy remains poorly understood. During a genetic screen aimed to obtain novel gene(s) required for autophagy, we identified a novel ORF, ATG29/YPL166w. atg29Delta cells were sensitive to starvation and induction of autophagy was severely retarded. However, the Cvt pathway operated normally. Therefore, ATG29 is an ATG gene specifically required for autophagy. Additionally, an Atg29-GFP fusion protein was observed to localize to the PAS. From these results, we propose that Atg29 functions in autophagosome formation at the PAS in collaboration with other Atg proteins.

Tor2 directly phosphorylates the AGC kinase Ypk2 to regulate actin polarization.

The target of rapamycin (TOR) protein kinases, Tor1 and Tor2, form two distinct complexes (TOR complex 1 and 2) in the yeast Saccharomyces cerevisiae. TOR complex 2 (TORC2) contains Tor2 but not Tor1 and controls polarity of the actin cytoskeleton via the Rho1/Pkc1/MAPK cell integrity cascade. Substrates of TORC2 and how TORC2 regulates the cell integrity pathway are not well understood. Screening for multicopy suppressors of tor2, we obtained a plasmid expressing an N-terminally truncated Ypk2 protein kinase. This truncation appears to partially disrupt an autoinhibitory domain in Ypk2, and a point mutation in this region (Ypk2(D239A)) conferred upon full-length Ypk2 the ability to rescue growth of cells compromised in TORC2, but not TORC1, function. YPK2(D239A) also suppressed the lethality of tor2Delta cells, suggesting that Ypks play an essential role in TORC2 signaling. Ypk2 is phosphorylated directly by Tor2 in vitro, and Ypk2 activity is largely reduced in tor2Delta cells. In contrast, Ypk2(D239A) has increased and TOR2-independent activity in vivo. Thus, we propose that Ypk protein kinases are direct and essential targets of TORC2, coupling TORC2 to the cell integrity cascade.