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

No abstract

Gap1p, the general amino acid permease of Saccharomyces cerevisiae, is regulated by intracellular sorting decisions that occur in either Golgi or endosomal compartments. Depending on nitrogen source, Gap1p is transported to the plasma membrane, where it functions for amino acid uptake, or to the vacuole, where it is degraded. We found that overexpression of Bul1p or Bul2p, two nonessential components of the Rsp5p E3–ubiquitin ligase complex, causes Gap1p to be sorted to the vacuole regardless of nitrogen source. The double mutant bul1Δ bul2Δ has the inverse phenotype, causing Gap1p to be delivered to the plasma membrane more efficiently than in wild-type cells. In addition, bul1Δ bul2Δ can reverse the effect of lst4Δ, a mutation that normally prevents Gap1p from reaching the plasma membrane. Evaluation of Gap1p ubiquitination revealed a prominent polyubiquitinated species that was greatly diminished in a bul1Δ bul2Δ mutant. Both a rsp5-1 mutant and a COOH-terminal truncation of Gap1p behave as bul1Δ bul2Δ, causing constitutive delivery of Gap1p to the plasma membrane and decreasing Gap1p polyubiquitination. These results indicate that Bul1p and Bul2p, together with Rsp5p, generate a polyubiquitin signal on Gap1p that specifies its intracellular targeting to the vacuole.

The Saccharomyces cerevisiae genes TORI and TOR2 were originally identified by mutations that confer resistance to the immunosuppressant rapamycin. TOR2 was previously shown to encode an essential 282-kDa phosphatidylinositol kinase (PI kinase) homologue. The TORI gene product is also a large (281 kDa) PI kinase homologue, with 67% identity to TOR2. TORI is not essential, but a TORI TOR2 double disruption uniquely confers a cell cycle (Gi) arrest as does exposure to rapamycin; disruption of TOR2 alone is lethal but does not cause a cell cycle arrest. TOR1-TOR2 and TOR2-TOR1 hybrids indicate that carboxy-terminal domains of TOR1 and TOR2 containing a lipid kinase sequence motif are interchangeable and therefore functionally equivalent; the other portions of TOR1 and TOR2 are not interchangeable. The TORI-I and TOR2-1 mutations, which confer rapamycin resistance, alter the same potential protein kinase C site in the respective protein's lipid kinase domain. Thus, TOR1 and TOR2 are likely similar but not identical, rapamycinsensitive PI kinases possibly regulated by phosphorylation. TOR1 and TOR2 may be components of a novel signal transduction pathway controlling progression through G1.

Ten class E Vps proteins in yeast are known components of the ESCRT complexes I, II and III, which are required for the sorting of proteins to the lumenal membranes of multivesicular bodies. We used the yeast 2 hybrid system to analyze the protein-protein interactions of all 17 soluble class E Vps proteins, as well as proteins thought to be required for the ubiquitination and deubiquitination of cargo proteins at multivesicular bodies. We identified novel interactions between yeast ESCRT complex components suggesting that ESCRTI binds to both ESCRTII and ESCRTIII. These interactions were confirmed by GST pull-down experiments. Our data indicate that the link between ESCRTI and ESCRTIII is via Vps28p and Vps37p/Srn2p binding directly to Vps20p, as well as through indirect interactions via ESCRTII. This is in contrast to the situation in mammalian cells where ESCRTI and ESCRTIII interact indirectly via ALIX, the mammalian homologue of yeast proteins Vps31p/Bro1p and Rim20p. Our data also enable us to link all soluble class E Vps proteins to the ESCRT complexes. We propose the formation of a large multimeric complex on the endosome membrane consisting of ESCRTI, ESCRTII, ESCRTIII and other associated proteins.