Saccharomyces cerevisiae cells treated with the immunosuppressant rapamycin or depleted for the targets of rapamycin TOR1 and TOR2 arrest growth in the early Gi phase of the cell cycle. Loss of TOR function also causes an early inhibition of translation initiation and induces several other physiological changes characteristic of starved cells entering stationary phase (GO). A Gi cyclin mRNA whose translational control is altered by substitution of the UBI4 5' leader region (UBI4 is normally translated under starvation conditions) suppresses the rapamycin-induced Gl arrest and confers starvation sensitivity. These results suggest that the block in translation initiation is a direct consequence of loss of TOR function and the cause of the Gl arrest. We propose that the TORs, two related phosphatidylinositol kinase homologues, are part of a novel signaling pathway that activates eIF-4E-dependent protein synthesis and, thereby, Gl progression in response to nutrient availability. Such a pathway may constitute a checkpoint that prevents early Gl progression and growth in the absence of nutrients. INTRODUCTIONThe immunosuppressant rapamycin and the related compound FK506 exert their immunosuppressive effects by inhibiting intermediate steps in signal transduction that lead to T cell activation and proliferation (Heitman et al

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contributed equally to this work 4Corresponding author Following DNA damage or a block to DNA synthesis, checkpoint pathways act to arrest mitosis and prevent the attempted segregation of damaged or unreplicated DNA. The radl7 locus of Schizosaccharomyces pombe is one of seven known radiation-sensitive (rad) loci which are absolutely required to prevent mitosis following DNA damage in fission yeast. Six of these (radi, rad3, rad9, radl 7, rad26 and husi) are also required for the checkpoint which prevents mitosis from occurring before DNA replication is complete. We report here that the predicted radl7 gene product is a basic hydrophilic protein of 606 amino acids which contains five domains with sequence homology to replication factor C (RF-C)/activator 1 subunits. Western analysis and fusion with Green Fluorescent Protein indicate that the abundance and electrophoretic mobility of Radl7 is not significantly modified following a block to DNA synthesis or following DNA damage, and that Radl7 is localized in the nucleus. Radl7 function is not essential for growth, but is required for the function of the DNA structure-dependent checkpoints. Sitedirected mutagenesis has been used to demonstrate the biological significance of the RF-C/activator 1-related domains. These studies have also defined an element of the radiation sensitivity caused by loss of Radl7 function which is not associated with the radiationinduced G2 arrest defect seen in the radl7.d null mutant cells.

The Saccharomyces cerevisiae genes TOR1 and TOR2 encode phosphatidylinositol kinase homologs. TOR2 has two essential functions. One function overlaps with TOR1 and mediates protein synthesis and cell cycle progression. The second essential function of TOR2 is unique to TOR2 and mediates the cell-cycle-dependent organization of the actin cytoskeleton. We have isolated temperature-sensitive mutants that are defective for either one or both of the two TOR2 functions. The three classes of mutants were as follows. Class A mutants, lacking only the TOR2-unique function, are defective in actin cytoskeleton organization and arrest within two to three generations as small-budded cells in the G2/M phase of the cell cycle. Class B mutants, lacking only the TOR-shared function, and class C mutants, lacking both functions, exhibit a rapid loss of protein synthesis and a G1 arrest within one generation. To define further the two functions of TOR2, we isolated multicopy suppressors that rescue the class A or B mutants. Overexpression of MSS4, PKC1, PLC1, RHO2, ROM2, or SUR1 suppressed the growth defect of a class A mutant. Surprisingly, overexpression of PLC1 and MSS4 also suppressed the growth defect of a class B mutant. These genes encode proteins that are involved in phosphoinositide metabolism and signaling. Thus, the two functions (readouts) of TOR2 appear to involve two related signaling pathways controlling cell growth.

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Summary. Ten DNA repair (rad) genes from the fission yeast, Schizosaccharomyces pombe were mapped to the 17 NotI fragments of the three chromosomes. Nine of the genes map to chromosome I, but there is no evidence for significant clustering.