TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae

Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously.     

Ubiquitin regulates TORC1 in yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae the TOR complex 1 (TORC1) controls many growth‐related cellular processes and is essential for cell growth and proliferation. Macrolide antibiotic rapamycin, in complex with a cytosol protein named FKBP12, specifically inhibits TORC1, causing growth arrest. The FKBP12‐rapamycin complex interferes with TORC1 function by binding to the FRB domain of the TOR proteins. In an attempt to understand the role of the FRB domain in TOR function, we identified a single point mutation (Tor2^W2041R) in the FRB domain of Tor2 that renders yeast cells rapamycin resistant and temperature sensitive. At the permissive temperature, the Tor2 mutant protein is partially defective for binding with Kog1 and TORC1 is impaired for membrane association. At the restrictive temperature, Kog1 but not the Tor2 mutant protein, is rapidly degraded. Overexpression of ubiquitin stabilizes Kog1 and suppresses the growth defect associated with the tor2 mutant at the nonpremissive temperature. We find that ubiquitin binds non‐covalently to Kog1, prevents Kog1 from degradation and stabilizes TORC1. Our data reveal a unique role for ubiquitin in regulation of TORC1 and suggest that Kog1 requires association with the Tor proteins for stabilization.     

Bidirectional regulation between TORC1 and autophagy in Saccharomyces cerevisiae

It has been reported in various model organisms that autophagy and the target of rapamycin complex 1 (TORC1) signaling are strongly involved in eukaryotic cell aging and decreasing TORC1 activity extends longevity by an autophagy-dependent mechanism. Thus, to expand our knowledge of the regulation of eukaryotic cell aging, it is important to understand the relationship between TORC1 signaling and autophagy. Many researchers have shown that TORC1 represses autophagy under normal growth conditions, and TORC1 inactivation contributes to the upregulation of autophagy. However, it is poorly understood how autophagy is regulated or terminated when starvation is prolonged. Here, we report that bidirectional regulation between autophagy and TORC1 exists in the yeast Saccharomyces cerevisiae. We show that mutant cells with weak TORC1 activity maintain autophagy longer than wild-type cells, and TORC1 is partially reactivated under ongoing nitrogen starvation by an autophagy-dependent mechanism. In addition, we found that Atg13 is gradually rephosphorylated during prolonged nitrogen starvation, and the kinase activity of Atg1 is required for Atg13 rephosphorylation. Our data suggest that TORC1 can be substantially, if not fully, reactivated in an autophagy-dependent manner under ongoing starvation, and that partially reactivated TORC1 eventually plays a role in the attenuation of autophagy.     

Bidirectional regulation between TORC1 and autophagy in Saccharomyces cerevisiae

It has been reported in various model organisms that autophagy and the target of rapamycin complex 1 (TORC1) signaling are strongly involved in eukaryotic cell aging and decreasing TORC1 activity extends longevity by an autophagy-dependent mechanism. Thus, to expand our knowledge of the regulation of eukaryotic cell aging, it is important to understand the relationship between TORC1 signaling and autophagy. Many researchers have shown that TORC1 represses autophagy under normal growth conditions, and TORC1 inactivation contributes to the upregulation of autophagy. However, it is poorly understood how autophagy is regulated or terminated when starvation is prolonged. Here, we report that bidirectional regulation between autophagy and TORC1 exists in the yeast Saccharomyces cerevisiae. We show that mutant cells with weak TORC1 activity maintain autophagy longer than wild-type cells, and TORC1 is partially reactivated under ongoing nitrogen starvation by an autophagy-dependent mechanism. In addition, we found that Atg13 is gradually rephosphorylated during prolonged nitrogen starvation, and the kinase activity of Atg1 is required for Atg13 rephosphorylation. Our data suggest that TORC1 can be substantially, if not fully, reactivated in an autophagy-dependent manner under ongoing starvation, and that partially reactivated TORC1 eventually plays a role in the attenuation of autophagy.     

Mapping the interaction of Snf1 with TORC1 in Saccharomyces cerevisiae

Nutrient sensing and coordination of metabolic pathways are crucial functions for all living cells, but details of the coordination under different environmental conditions remain elusive. We therefore undertook a systems biology approach to investigate the interactions between the Snf1 and the target of rapamycin complex 1 (TORC1) in Saccharomyces cerevisiae. We show that Snf1 regulates a much broader range of biological processes compared with TORC1 under both glucose‐ and ammonium‐limited conditions. We also find that Snf1 has a role in upregulating the NADP⁺‐dependent glutamate dehydrogenase (encoded by GDH3) under derepressing condition, and therefore may also have a role in ammonium assimilation and amino‐acid biosynthesis, which can be considered as a convergence of Snf1 and TORC1 pathways. In addition to the accepted role of Snf1 in regulating fatty acid (FA) metabolism, we show that TORC1 also regulates FA metabolism, likely through modulating the peroxisome and β‐oxidation. Finally, we conclude that direct interactions between Snf1 and TORC1 pathways are unlikely under nutrient‐limited conditions and propose that TORC1 is repressed in a manner that is independent of Snf1.     

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.     

Catalases protect cellular proteins from oxidative modification in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae cells had higher antioxidant enzyme activities under growth in ethanol than that in glucose as a carbon and energy source. The correlations between catalase activity and protein carbonyl level (r(2)=0.857), between catalase and glucose-6-phosphate dehydrogenase activities (r(2)=0.924) and between protein carbonyl levels and glucose-6-phosphate dehydrogenase activity (r(2)=0.988) under growth in ethanol were found. Growing in ethanol the strain deficient in cytosolic and peroxisomal catalases had 7.1-fold higher level of carbonyl proteins than that of wild-type strain. Our data suggest that in vivo catalases may protect glucose-6-phosphate dehydrogenase against oxidative inactivation.     

Multiple species of single-stranded nucleic acid-binding proteins in Saccharomyces cerevisiae

DNA affinity chromatography has been used to identify the major single-stranded nucleic acid binding proteins (SSBs) of Saccharomyces cerevisiae. There are five abundant species having molecular masses of 50, 45, 31, 23, and 20 kDa. Four of these proteins are cytoplasmic and one is mitochondrial. To date, three of the proteins have been purified to homogeneity. The purified proteins are designated SSB-m, SSB-1, and SSB-2, with molecular masses of 20, 45, and 50 kDa, respectively. SSB-m is found only in mitochondrial subcellular fractions. SSB-1 stimulates purified yeast DNA polymerase I, while SSB-2 inhibits DNA polymerase I. An antibody against SSB-1 has been prepared in rabbits and purified by SSB-1-Sepharose affinity chromatography. The purified antibody specifically inhibits DNA synthesis in an in vitro replication system, suggesting that SSB-1 may be involved in DNA replication in vivo. SSB-2 has the highest affinity for single-stranded DNA of all three proteins. It may represent a new class of eukaryotic SSB, on the basis of molecular weight, inhibition of DNA polymerase and antigenicity. Antibodies have also been prepared against SSB-2. The immunological reagents have been used to show that SSB-1, SSB-2, and SSB-m are antigenically distinct, as well as to study the relationship of these three SSBs to other proteins in yeast.     

State Transitions in the TORC1 Signaling Pathway and Information Processing in Saccharomyces cerevisiae

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.     

Phosphorylation of ribosomal proteins during differentiation of Saccharomyces cerevisiae

Four major phosphoproteins of yeast ribosomes: S2, S6, L44 and L45, were identified in germinating spores. It was found that protein S6 became phosphorylated at an early stage of germination and that only the phosphorylation of this protein responded well to changes in growth conditions of yeast culture. The results obtained give support for the suggested functional role of modification of S6 protein.     

MAPKs regulate mitophagy in Saccharomyces cerevisiae

The autophagy-dependent selective degradation of mitochondria (mitophagy) plays an important role in removing excessive, damaged and dysfunctional mitochondria to maintain a proper cellular homeostasis. Relative to its significance in cell physiology, very little is known about the molecular machinery and regulatory mechanism of mitophagy in mammalian cells or yeast. We found that two mitogen-activated protein kinases (MAPKs), Slt2 and Hog1, are required for mitophagy in Saccharomyces cerevisiae. Slt2 is involved in both mitophagy and pexophagy (the selective degradation of peroxisomes through autophagy), whereas Hog1 functions specifically in mitophagy.     

MAPKs regulate mitophagy in Saccharomyces cerevisiae

The autophagy-dependent selective degradation of mitochondria (mitophagy) plays an important role in removing excessive, damaged and dysfunctional mitochondria to maintain a proper cellular homeostasis. Relative to its significance in cell physiology, very little is known about the molecular machinery and regulatory mechanism of mitophagy in mammalian cells or yeast. We found that two mitogen-activated protein kinases (MAPKs), Slt2 and Hog1, are required for mitophagy in Saccharomyces cerevisiae. Slt2 is involved in both mitophagy and pexophagy (the selective degradation of peroxisomes through autophagy), whereas Hog1 functions specifically in mitophagy.     

TORC1 is essential for NF1-associated malignancies

Inactivating mutations in NF1 underlie the prevalent familial cancer syndrome neurofibromatosis type 1 [1]. The NF1-encoded protein is a Ras GTPase-activating protein (RasGAP) [2]. Accordingly, Ras is aberrantly activated in NF1-deficient tumors; however, it is unknown which effector pathways critically function in tumor development. Here we provide in vivo evidence that TORC1/mTOR activity is essential for tumorigenesis. Specifically, we show that the mTOR inhibitor rapamycin potently suppresses the growth of aggressive NF1-associated malignancies in a genetically engineered murine model. However, in these tumors rapamycin does not function via mechanisms generally assumed to mediate tumor suppression, including inhibition of HIF-1alpha and indirect suppression of AKT, but does suppress the mTOR target Cyclin D1 [3]. These results demonstrate that mTOR inhibitors may be an effective targeted therapy for this commonly untreatable malignancy. Moreover, they indicate that mTOR inhibitors do not suppress all tumor types via the same mechanism, suggesting that current biomarkers that rely on HIF-1alpha suppression may not be informative for all cancers. Finally, our results reveal important differences between the effects of mTOR inhibition on the microvasculature in genetically engineered versus xenograft models and indicate that the former may be required for effective preclinical screening with this class of inhibitors.     

Multiple pathways regulate endocytic coat disassembly in Saccharomyces cerevisiae for optimal downstream trafficking

Recently, a pathway involving the highly choreographed recruitment of endocytic proteins to sites of clathrin/actin-mediated endocytosis has been revealed in budding yeast. Here, we investigated possible roles for candidate disassembly factors in regulation of the dynamics of the endocytic coat proteins Sla2p, Ent1p, Ent2p, Sla1p, Pan1p and End3p, each of which has mammalian homologues. Live cell imaging analysis revealed that in addition to the synaptojanin, Sjl2p, the Ark1p and Prk1p protein kinases, the putative Arf GTPase-activating protein, Gts1p and the Arf GTPase-interacting protein, Lsb5p, also arrive at endocytic sites late in the internalization pathway, consistent with roles in coat disassembly. Analysis of coat dynamics in various mutant backgrounds revealed that multiple pathways, including the ones mediated by an Arf guanosine triphosphatase and a synaptojanin, facilitate efficient disassembly of different endocytic coat proteins. In total, at least four separate processes are important for disassembly of endocytic complexes and efficient downstream trafficking of endocytic cargo.     

SURVEY AND SUMMARY: Saccharomyces cerevisiae basic helix-loop-helix proteins regulate diverse biological processes

Basic helix–loop–helix (bHLH) proteins are among the most well studied and functionally important regulatory proteins in all eukaryotes. The HLH domain dictates dimerization to create homo- and heterodimers. Dimerization juxtaposes the basic regions of the two monomers to create a DNA interaction surface that recognizes the consensus sequence called the E-box, 5′-CANNTG-3′. Several bHLH proteins have been identified in the yeast Saccharomyces cerevisiae using traditional genetic methodologies. These proteins regulate diverse biological pathways. The completed sequence of the yeast genome, combined with novel methodologies allowing whole-genome expression studies, now offers a unique opportunity to study the function of these bHLH proteins. It is the purpose of this review to summarize the current knowledge of bHLH protein function in yeast.     

Multiple mechanisms regulate expression of low temperature responsive (LOT) genes in Saccharomyces cerevisiae

Using cDNA subtraction screening, we identified five Saccharomyces cerevisiae genes whose expressions is up-regulated when culture temperature was down-shifted from 30 to 10 degrees C. Among these LOT (low temperature-responsive) genes, three (LOT1, LOT2, and LOT3) were identical to FBA1, RPL2B, and NOP1, encoding a fructose biphosphate aldolase, a ribosomal protein L2B, and a nucleolar protein for rRNA processing, respectively. No functions were assigned for LOT5 and LOT6, which are identical to YKL183w and YLR011w, respectively. Northern hybridization analysis revealed that these genes are not uniformly regulated in response to the change of growth temperature. In addition, all the LOT genes, except for LOT1/FBA1, were induced by a low concentration of cycloheximide. The data indicate that multiple mechanisms, including translational functionality may be involved in the regulation of LOT gene expression in yeast.     

Calmodulin-binding proteins of Saccharomyces cerevisiae

The subcellular distribution of calmodulin-binding proteins in the soluble, plasma membrane, and nuclear fractions of Saccharomyces cerevisiae was analyzed with a gel binding assay using 125I-labeled calmodulin. Over 20 binding proteins were detected. The calmodulin-binding protein profiles were markedly different among the fractions. Calmodulin-binding proteins were most abundant in the nuclear fraction, followed by the membrane fraction and the soluble fraction in decreasing order. The amounts of certain calmodulin-binding proteins increased after treatment with alpha-mating factor.