TOR1 exhibits synthetically lethal and reduced fitness interactions with genes involved in protein sorting and vacuolar functions. (A) Schematic of the distinctive functional categories, according to the published literature, identified by the tor1 dSLAM screen. (B) Heterozygous diploid strains tor1 pep3 (SZY26), tor1 pep5 (SZY27), tor1 vps16 (SZY29), and tor1 vps33 (SZY49) (see Table 1 for complete genotypes) were sporulated and dissected on YPD solid medium. After 3 days of incubation at 30°, colonies were replica plated onto YPD containing G418 (200 μg/ml) and SC-ura media. Plates were incubated for 2 days and photographed. (C) Heterozygous diploid strains tor1 vps15 (SZY28), tor1 vps34 (SZY30), tor1 vac7 (SZY31), tor1 vac17 (SZY33), tor1 vac8 (SZY32), tor1 vps39 (RPY50), and tor1 vps41 (RPY51) were sporulated and dissected as indicated above. Photographs show the colony size on the YPD plate of representative segregants from individual tetrads.

Class C vps mutants are hypersensitive to rapamycin and fail to recover from rapamycin-induced growth arrest. (A) The WT (BY4742), tor1 (#16864), and isogenic strains bearing mutations in different components of the class C Vps and PAS protein complexes as well as in genes involved in vacuolar inheritance, were grown overnight in liquid YPD medium. Equivalent numbers of cells were serially diluted and aliquots were spotted onto YPD and YPD containing 20 nm rapamycin. Plates were photographed following 3 days incubation at 30°. (B) Cultures of isogenic WT (BY4742), tor1 (#16864), pep5 (#10817), vps16 (#12783), and vps33 (SZY36) were grown to exponential phase. Cultures were divided in half and treated with drug vehicle alone or with 100 nm rapamycin for 6 hr. Cells were pelleted by centrifugation, washed twice, and equivalent numbers of cells were spotted on YPD medium. After incubation photographs were taken at 48 hr for both the untreated (YPD) and rapamycin-treated cells and 72 hr for the rapamycin-treated cells.

Tor1 does not regulate protein sorting from the ER to Golgi complex or protein cycling between the Golgi complex and endosomes. (A) Schematic of a fraction of the genes (grouped in functional categories according to the published literature) that when mutated alter rapamycin sensitivity and showed reduced fitness and synthetically lethal interactions in combination with mutation of PEP3. Interactions of the genes indicated in bold were confirmed by tetrad analysis. Genes enclosed by a box were synthetically lethal in combination with the pep3 mutation. (B) The WT (BY4742), tor1 (#16864), and pep3 (#14105) mutants were grown to early exponential phase and treated with drug vehicle (−) or 100 nm rapamycin for 20 min (+). Cell were pulse labeled with Trans ³⁵S-LABEL for 8 min (min) and chased with unlabeled amino acids for 0, 5, 6, 14, 16, and 20 min as indicated in the figure (for further detail, see materials and methods). The mature (m) and processed (p) forms of α-factor (top) and CpY (bottom) were immunoprecipitated with α-factor and CpY-specific antibodies, respectively. Immunoprecipitated proteins were separated by SDS-PAGE and visualized by autoradiography. The migration of precursor forms characteristic for the Golgi complex and the ER compartment are indicated.

Expression of TOR1 but not TOR2 rescues viability of tor1 pep5 segregants. (A) The tor1 ssd1 mutant strain SZY21 was transformed with vector pRS315 and its derivatives encoding TOR1, TOR2, and the following TOR2–TOR1 hybrids (depicted in C): hybrid 1, Tor2 amino acids 1–1688 fused to Tor1 amino acids 1682–2470; hybrid 2, Tor2 amino acids 1–796 fused to Tor1 amino acids 788–2470; hybrid 3, Tor2 amino acids 1–483 fused to Tor1 amino acids 475–2470; hybrid 4, the Tor1 amino acid sequence from 1772–1815 was replaced by the homologous amino acid sequence of Tor2 from 1780–1818. Protein extracts of the different transformants were analyzed by Western blot with antibodies specific to an N-terminal region of Tor1 or Tor2. In these extracts, Cpr1 was also detected and serve as a loading control. (B) Equivalent cell numbers of the SZY21 transformants as indicated above were serially diluted and spotted in SD-leu medium. Plates were photographed after incubation at 30° or 39° and Tor1 function was scored by the ability to complement the conditional synthetic lethal phenotype of the tor1 ssd1 strain at 39°. (C) The tor1 pep5 diploid strain SZY27 transformed with the plasmids depicted at the left was sporulated and dissected on YPD medium. Following incubation at 30° for 3 days the plates were photographed and replica plated to YPD containing 200 μg/ml G418, SC-ura, and SC-leu media to score genotypes. The numbers on the right indicate the percent of tetrads (from a total of 10 scored) with a 4:0, 3:1, and 2:2 viable:inviable ratio.

Tor1 signaling does not control class C Vps complex functions. (A) Tor1 does not control the maturation of vacuolar hydrolases. Exponentially growing cultures of the WT (BY4742), tor1 (#16864), and pep3 (#14105) strains in YPD medium were treated with either drug vehicle (−) or 100 nm rapamycin (+) for 1 hr. Protein extracts were prepared and analyzed by Western blot with antibodies specific for CpY, Ape1, and Alp1. (B) Tor 1 does not regulate endocytosis of the high-affinity ammonium permease Mep2. The WT, tor1, and pep3 strains indicated in A were transformed with plasmid pMep2-GFP. Transformants were grown to early exponential phase in SC-ura, washed twice, and resuspended in SLAD medium supplemented with the required amino acids to satisfy auxotrophic requirements. After 4 hr incubation in this medium, 50 mm ammonium sulfate was added to the cultures and incubation continued for 30 min. Cell samples were collected prior to and after addition of ammonium sulfate () and imaged for direct epifluorescence as indicated under experimental procedures. (C) Effects of TOR1 mutation in autophagy-mediated maturation of Ape1. The WT (BY4742), and the tor1 (#16864), pep3 (#14105), vac8 (#10253), and the tor1 vac8 (SZY43) mutant strains were grown and treated with rapamycin as indicated in A. Protein extracts were prepared and analyzed by Western blot with specific Ape1 antibodies.

Class C vps mutants show severe defects in nitrogen metabolism. (A) The class C Vps complex is required for adaptation to nitrogen limitation. Isogenic WT (BY4742), and pep5 (#10817), vps16 (#12783), vps33 (SZY36), and tor1 (#16864) mutant strains were grown and spotted on YPD medium as indicated in the legend to Figure 2A. Following overnight incubation, the plate was photographed (see overnight image) and then starved for nitrogen by replica-plating to YNB without nitrogen medium. After incubating for 10 days, cells were replica plated back to YPD, incubated for 24 hr, and photographed. (B) Class C Vps complex mutations induce the general amino acid control response. Exponentially growing cultures of the WT (BY4742), and the pep5 (#10817), pep3 (#14105) mutant strains harboring the GCN4-lacZ reporter plasmid p180 were grown to exponential phase in SC-ura medium. Cultures were treated with 100 nm rapamycin for 0, 1, and 2 hr and analyzed for β-galactosidase activity. (C) Growth of the class C vps mutants at 37° is rescued by addition of glutamine, glutamate, and to a lesser extent by arginine. Isogenic WT (BY4742), and tor1 (#16864), pep3 (#14105), tor1 pep3 (strain SZY40-4 transformed with plasmid pBJ9113 expressing the pep3-108ts allele), pep5 (#10817), vps16 (#12783), vps33 (SZY36), vps39 (#13774), and vps41 (#14015) mutant strains were grown and spotted on SC medium (−) or SC medium supplemented with the indicated amino acid. Plates were incubated at 37° for 2 days and photographed. (D) Supplementation with glutamine or glutamate does not rescue the growth defect of the tor1 vac8, tor1 gtr1, and tor1 ego3 double mutants. Isogenic WT (BY4742), tor1 vac8 (SZY43), tor1 gtr1(RPY50), and tor1 ego3 (RPY51) double mutant strains were assayed as indicated in C except that plates were incubated at 30°.