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. 2007 Apr;27(8):3154-64.
doi: 10.1128/MCB.01039-06. Epub 2007 Jan 29.

Loss of the TOR kinase Tor2 mimics nitrogen starvation and activates the sexual development pathway in fission yeast

Tomohiko Matsuo  1 , Yoko Otsubo, Jun Urano, Fuyuhiko Tamanoi, Masayuki Yamamoto
Affiliations

Loss of the TOR kinase Tor2 mimics nitrogen starvation and activates the sexual development pathway in fission yeast

Tomohiko Matsuo et al. Mol Cell Biol. 2007 Apr.

Abstract

Fission yeast has two TOR (target of rapamycin) kinases, namely Tor1 and Tor2. Tor1 is required for survival under stressed conditions, proper G(1) arrest, and sexual development. In contrast, Tor2 is essential for growth. To analyze the functions of Tor2, we constructed two temperature-sensitive tor2 mutants. Interestingly, at the restrictive temperature, these mutants mimicked nitrogen starvation by arresting the cell cycle in G(1) phase and initiating sexual development. Microarray analysis indicated that expression of nitrogen starvation-responsive genes was induced extensively when Tor2 function was suppressed, suggesting that Tor2 normally mediates a signal from the nitrogen source. As with mammalian and budding yeast TOR, we find that fission yeast TOR also forms multiprotein complexes analogous to TORC1 and TORC2. The raptor homologue, Mip1, likely forms a complex predominantly with Tor2, producing TORC1. The rictor/Avo3 homologue, Ste20, and the Avo1 homologue, Sin1, appear to form TORC2 mainly with Tor1 but may also bind Tor2. The Lst8 homologue, Wat1, binds to both Tor1 and Tor2. Our analysis shows, with respect to promotion of G(1) arrest and sexual development, that the loss of Tor1 (TORC2) and the loss of Tor2 (TORC1) exhibit opposite effects. This highlights an intriguing functional relationship among TOR kinase complexes in the fission yeast Schizosaccharomyces pombe.

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Figures

FIG. 1.
FIG. 1.
Loss of tor2 function causes G1 arrest. (A) Temperature-sensitive growth of the tor2-ts strains. Cells of JY878 (wt), JT177 (tor2-ts6), and JT178 (tor2-ts10) were streaked on nutrient medium YE and incubated either at 25°C or 30°C for 3 days. (B) Cells of JY476 (wt), JV302 (tor2-ts6), and JV305 (tor2-ts10) were cultured in liquid YE medium at 25°C to exponential phase and shifted to 30°C at a concentration of about 1 × 106 cells/ml. Aliquots were taken at indicated time points, and the cellular DNA content of each sample was measured by FACS analysis. (C) Cells of JV981, in which tor2 is driven by the nmt81 promoter, were grown in liquid MM medium to exponential phase (2 × 106 cells/ml), and thiamine was added to a half of the culture to a concentration of 2 μM to shut off the transcription from the nmt81 promoter. Aliquots were taken at indicated time points, and the cellular DNA content of each sample with (+) or without (−) thiamine was measured by FACS analysis.
FIG. 2.
FIG. 2.
Sexual development of tor2-ts cells grown at the restrictive temperature. (A) The three homothallic haploid strains analyzed in the experiment shown in Fig. 1B were examined microscopically after 24 h of incubation at the restrictive temperature. Bar, 10 μm. (B) Calculated mating efficiency of the three strains shown in panel A. (C) Cells of heterothallic haploid strains incubated at the restrictive temperature for 24 h. wt, JY333; tor2-ts6, JV304; and tor2-ts10, JV306. Bar, 10 μm. (D) Cells of a homothallic strain JT300, in which tor2 is driven by the nmt81 promoter, were grown vegetatively on MM plates (− thiamine). They have undergone mating and sporulation even before shutoff of the promoter. Bar, 10 μm. (E) Expression of starvation-responsive genes in tor2-ts cells. Expression of three nitrogen starvation-responsive genes (ste11, isp6, and fnx1), and one glucose starvation-responsive gene (fbp1) was measured in wt (JY333), tor2-ts6 (JV304), and tor2-ts10 (JV306) cells at time zero and 3.5 and 7 h after the shift to the restrictive temperature. Their expression in pka1-defective cells (JX384) was also examined. rRNA stained with ethidium bromide is shown as a loading control.
FIG. 3.
FIG. 3.
Microarray analysis of loss of tor2 activity. Global expression profiles from tor2+ (JY450) or tor2-ts6 (JV303) incubated for the indicated times at 34°C were assessed using the GeneChip Yeast Genome 2.0 Array. The raw data were normalized and modeled using dChip. Genes showing a variation (standard deviation/mean) of >0.5 were clustered. The cluster of genes that are induced by loss of tor2 is indicated. Red indicates induction, and green indicates suppression relative to expression of the gene in tor2+ cells at 0 h.
FIG. 4.
FIG. 4.
Physical interaction of TOR kinases with raptor Mip1, rictor Ste20, and two other TORC members. (A) IP assay to detect interaction of Mip1 with Tor1 or Tor2. Detailed procedures are given in Materials and Methods. (B) IP assay to detect interaction of Ste20 with Tor1 or Tor2. (C) IP assay to detect interaction of Wat1 with Tor1 or Tor2. (D) IP assay to detect interaction of Sin1 with Tor1 or Tor2. (E) A summary of known components of TORC1 and TORC2 in three kinds of organisms. α, anti.
FIG. 5.
FIG. 5.
Interaction of the temperature-sensitive Tor2 proteins with Mip1. (A) Physical interaction of Tor2-ts6 with Mip1, examined by IP as described in the legend of Fig. 4A. (B) Physical interaction of Tor2-ts10 with Mip1, examined by IP as described in the legend of Fig. 4A. Only samples prepared from cells grown at the permissive temperature 25°C are shown. (C) Genetic interaction: suppression of tor2-ts6 by overexpression of mip1. JV304 (tor2-ts6) and JV306 (tor2-ts10) were transformed with vector pREP41, pREP41-tor2, and pREP41-mip1. Transformants of JV304 were incubated on MM at 32°C for 9 days, whereas transformants of JV306 were incubated on MM at 28°C for 8 days.
FIG. 6.
FIG. 6.
Comparison of phenotypes between tor1Δ and ste20Δ. (A) Comparison of cell morphology. Cells of each homothallic haploid strain were grown on YE medium at 30°C for 4 days. (B) Osmo-sensitive growth was examined on YE medium containing 1 M KCl. Incubation was done at 30°C for 4 days. wt, JY450; tor1Δ, JW950; ste20Δ, JT293; and gad8Δ, JW944. (C) Suppression of tor1Δ (JW950) and ste20Δ (JT293) by gad8-S527D/S546D. The wt and mutant gad8 alleles were expressed from plasmid pR3C (25). Growth on MM containing 0.6 M KCl at 30°C was followed for 4 days. (D) Examination of mutual suppression between tor1Δ and tor2-ts. The double mutants and control strains were tested for growth at 32°C. (E) Suppression of the growth defect of ade6-M216 tsc2Δ under low adenine supply by tor2-ts mutations. Indicated strains were grown on Edinburgh minimal medium plates supplemented with either 1 mg/ml or 0.25 mg/ml of adenine.
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