Figure 4. TORC1-SCH9 signaling from the new vacuole is required for cell-cycle progression.

(A) The vac17Δ tor1Δ double mutant exhibits an accumulation of G1 phase cells. Flow cytometry analysis with PI staining of yeast strains; wild-type, vac17Δ, tor1Δ, and vac17Δ tor1Δ. (B) Quantification of percent cells in G1 and G2 phase. Error bars; SD calculated from four independent experiments. (C) A new vacuole is synthesized in the new daughter cells of the vac17Δ tor1Δ double mutant. Wild-type, vac17Δ, tor1Δ, and vac17Δ tor1Δ cells which express Vph1-GFP from its endogenous locus, were pulse labeled with FM4-64. Arrowheads; new vacuole in daughter cells. (D) The kinase activity of target of rapamycin 1 (Tor1) is required for growth of the vacuole inheritance mutant, vac17Δ. Plasmids were transformed into a vac17Δ tor1Δ mutant containing pRS416 [URA3] TOR1. Plasmids tested were pRS315 [LEU2] (mock), pRS315 HA-TOR1, pRS315 HA-tor1-D2275A, or pRS315 HA-tor1-D2294E. Transformed colonies were cultured in liquid media and serial dilutions spotted onto SC+5-FOA or SC-Leu-Ura plates. Plates were incubated at 24°C for 4 days. (E) TORC1 signals from the new vacuole via Sch9. The phospho-mimetic sch9-2D3E mutant partially rescues the growth defect of the vac17Δ tor1Δ mutant. pRS413 (mock), pRS413 VAC17, pRS413 TOR1, pVT102-H (mock), pVT102-H SCH9, pVT102-H sch9-2D3E, or pVT102-H sch9-5A expressed in vac17Δ tor1Δ with pRS416 TOR1. Transformed colonies were cultured in liquid media and serial dilutions were spotted onto SC-His+5-FOA or SC-His-Ura plates, and incubated at 24°C for 4 days. (F) Sch9 signaling requires a functional vacuole. The phospho-mimetic sch9-2D3E mutant does not rescue the growth defect of the vac17Δ pep12Δ mutant. pRS413 (mock), pRS413 VAC17, pRS413 TOR1, pVT102-H (mock), pVT102-H SCH9, pVT102-H sch9-2D3E, or pVT102-H sch9-5A plasmids were expressed in a vac17Δ pep12Δ strain. Transformed colonies were cultured in liquid media and serial dilutions spotted onto an SC-His plate, and incubated at 24°C for 3 to 4 days.

DOI: http://dx.doi.org/10.7554/eLife.08160.009

Figure 4—figure supplement 1. TORC1-SCH9 is required for the viability of vacuole inheritance mutants.

(A) The tor1Δ mutant exhibits a synthetic growth defect with vac17Δ and vac8Δ. Results of tetrad dissection of heterozygous diploids, VAC17/vac17Δ TOR1/tor1Δ and VAC8/vac8Δ TOR1/tor1Δ. vac17Δ = 17Δ; vac8Δ = 8Δ; tor1Δ = 1Δ; vac17Δ tor1Δ or vac8Δ tor1Δ double mutants = ΔΔ are indicated. (B) Quantification of colony size, relative to average of wild-type colonies. A total of 34 tetrads and 39 tetrads were analyzed for vac17Δ tor1Δ and vac8Δ tor1Δ, respectively. (C) The tor1Δ mutant exhibits a synthetic growth defect with the myo2-N1304D mutant. Plasmids were transformed into a myo2Δ and myo2Δ tor1Δ strain containing YCp50 MYO2. Plasmids tested were pRS413 (mock), pRS413 MYO2, or pRS413 myo2-N1304D. Transformed colonies were cultured in liquid media and serial dilutions were spotted onto SC+5-FOA or SC-His-Ura plate, and incubated at 24°C for 4 days. (D) The vac17Δ kog1-105 mutant showed synthetic growth defects. pRS313 (mock) and pRS415 (mock), pRS313 (mock) and pRS415 VAC17, pRS313 KOG1 and pRS415 (mock), pRS313 KOG1 and pRS415 VAC17, pRS313 kog1-105 and pRS415 VAC17, or pRS313 kog1-105 and pRS415 (mock) expressed in vac17Δ kog1Δ with pRS316 KOG1. Transformed colonies were cultured in liquid media and serial dilutions were spotted on SC-His-Leu+5-FOA or SC-His-Leu-Ura plates, and incubated at 24°C for 5 days.

DOI: http://dx.doi.org/10.7554/eLife.08160.010