The TOR protein kinases exhibit a conserved role in regulating cellular growth and proliferation. In the fission yeast two TOR homologs are present. tor1 1 is required for starvation and stress responses, while tor2 1 is essential. We report here that Tor2 depleted cells show a phenotype very similar to that of wild-type cells starved for nitrogen, including arrest at the G 1 phase of the cell cycle, induction of nitrogen-starvationspecific genes, and entrance into the sexual development pathway. The phenotype of tor2 mutants is in a striking contrast to the failure of tor1 mutants to initiate sexual development or arrest in G 1 under nitrogen starvation conditions. Tsc1 and Tsc2, the genes mutated in the human tuberous sclerosis complex syndrome, negatively regulate the mammalian TOR via inactivation of the GTPase Rheb. We analyzed the genetic relationship between the two TOR genes and the Schizosaccharomyces pombe orthologs of TSC1, TSC2, and Rheb. Our data suggest that like in higher eukaryotes, the Tsc1-2 complex negatively regulates Tor2. In contrast, the Tsc1-2 complex and Tor1 appear to work in parallel, both positively regulating amino acid uptake through the control of expression of amino acid permeases. Additionally, either Tsc1/2 or Tor1 are required for growth on a poor nitrogen source such as proline. Mutants lacking Tsc1 or Tsc2 are highly sensitive to rapamycin under poor nitrogen conditions, suggesting that the function of Tor1 under such conditions is sensitive to rapamycin. We discuss the complex genetic interactions between tor1 1 , tor2 1 , and tsc1/2 1 and the implications for rapamycin sensitivity in tsc1 or tsc2 mutants.

The Target Of Rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol-3-kinase-related kinases (PIKKs). TOR proteins are found at the core of two distinct evolutionarily conserved complexes, TORC1 and TORC2. Disruption of TORC1 or TORC2 results in characteristically dissimilar phenotypes. TORC1 is a major cell growth regulator, while the cellular roles of TORC2 are not well understood. In the fission yeast Schizosaccharomyces pombe, Tor1 is a component of the TORC2 complex, which is particularly required during starvation and various stress conditions. Our genome-wide gene expression analysis of ⌬tor1 mutants indicates an extensive similarity with chromatin structure mutants. Consistently, TORC2 regulates several chromatin-mediated functions, including gene silencing, telomere length maintenance, and tolerance to DNA damage. These novel cellular roles of TORC2 are rapamycin insensitive. Cells lacking Tor1 are highly sensitive to the DNA-damaging drugs hydroxyurea (HU) and methyl methanesulfonate, similar to mutants of the checkpoint kinase Rad3 (ATR). Unlike Rad3, Tor1 is not required for the cell cycle arrest in the presence of damaged DNA. Instead, Tor1 becomes essential for dephosphorylation and reactivation of the cyclin-dependent kinase Cdc2, thus allowing reentry into mitosis following recovery from DNA replication arrest. Taken together, our data highlight critical roles for TORC2 in chromatin metabolism and in promoting mitotic entry, most notably after recovery from DNA-damaging conditions. These data place TOR proteins in line with other PIKK members, such as ATM and ATR, as guardians of genome stability.

Here we provide evidence that YbeY, a conserved heat shock protein with unknown function, is involved in the translation process. ybeY deletion mutants are temperature sensitive and have a significantly reduced thermotolerance. Nonetheless, there appears to be no damage of the protein quality control of mature polypeptides, as the levels of chaperones and proteases are normal and there is no accumulation of aggregates. Rather, the mutation results in a significant reduction in the level of polysomes, and upon a shift to a restrictive temperature (42°C), there is an immediate and severe slowdown of translation. Taken together, the data indicate that YbeY is an important factor for bacterial translation even at 37°C but becomes essential at high temperatures.Heat shock results in a dramatic increase in the rate of synthesis of a set of proteins called heat shock proteins (30), many of which are chaperones (3) and proteases (14, 34), protecting cells against damage induced by protein unfolding. Heat shock proteins are also induced by conditions that lead to protein unfolding even at low temperatures, such as exposure to heavy metals, denaturing alcohols (30), or amino acid analogs (12).Recently, microarray experiments defining the heat shock regulon in Escherichia coli (7,20,23,26,32,39) revealed the existence of novel heat shock genes. These experiments led to the characterization of heat shock proteins that function in different stages of the translation process (2,5,6,18,19,21,35). As with chaperones and proteases, the function of these genes is important under all growth conditions but is more critical at higher temperatures.One of the genes identified as a heat shock gene by global transcriptional analysis is ybeY, whose function has not yet been determined. YbeY is a 17-kDa protein, highly conserved among bacteria, that belongs to the UPF0054 family. The sequence similarity of YbeY to metal-dependent hydrolases suggests a potential hydrolytic function. The structures of YbeY in E. coli (38), Aquifex aeolicus (24), and Haemophilus influenzae (36) was determined and suggest that YbeY is a metalloprotein with an active site located at the C terminus of the protein. In A. aeolicus (24), YbeY structure homology analysis showed similarity to eukaryotic extracelleular proteinases such as colagenase and gelatinase. However, in vitro YbeY did not have colagenease or gelatinase activity, and no other hydrolase activity could be detected.Recently, a mutant with a deletion of SMc01113, the Sinorhizobium meliloti homolog of ybeY, was characterized in vivo (9). The SMc01113 protein is required for symbiosis of S. meliloti with Medicago sativa (alfalfa) and the SMc01113 deletion mutant is sensitive to UV, oxidative stress, and cell wall inhibitors.Here we show that ybeY deletion mutants have a severe growth defect at higher temperatures and essentially no thermotolerance at lethal temperatures. However, the mutants do not appear to be defective in protein quality control of mature polypeptides. Instead, the mutants are impa...

Background: TOR complex 2 (TORC2) is a conserved protein complex that regulates multiple aspects of cell survival and proliferation. Results: DNA metabolism and DNA damage response are impaired upon disruption of TORC2. Conclusion: TORC2 affects replication-associated damages, independent of checkpoint activation. Significance: TORC2 is required to maintain genome stability in fission yeast, a function that may be evolutionarily conserved.