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Comparative Study
. 2007 Mar;175(3):1153-62.
doi: 10.1534/genetics.106.064170. Epub 2006 Dec 18.

Opposite effects of tor1 and tor2 on nitrogen starvation responses in fission yeast

Ronit Weisman  1 , Irina Roitburg, Miriam Schonbrun, Rona Harari, Martin Kupiec
Affiliations
Comparative Study

Opposite effects of tor1 and tor2 on nitrogen starvation responses in fission yeast

Ronit Weisman et al. Genetics. 2007 Mar.

Abstract

The TOR protein kinases exhibit a conserved role in regulating cellular growth and proliferation. In the fission yeast two TOR homologs are present. tor1(+) is required for starvation and stress responses, while tor2(+) is essential. We report here that Tor2 depleted cells show a phenotype very similar to that of wild-type cells starved for nitrogen, including arrest at the G(1) phase of the cell cycle, induction of nitrogen-starvation-specific genes, and entrance into the sexual development pathway. The phenotype of tor2 mutants is in a striking contrast to the failure of tor1 mutants to initiate sexual development or arrest in G(1) under nitrogen starvation conditions. Tsc1 and Tsc2, the genes mutated in the human tuberous sclerosis complex syndrome, negatively regulate the mammalian TOR via inactivation of the GTPase Rheb. We analyzed the genetic relationship between the two TOR genes and the Schizosaccharomyces pombe orthologs of TSC1, TSC2, and Rheb. Our data suggest that like in higher eukaryotes, the Tsc1-2 complex negatively regulates Tor2. In contrast, the Tsc1-2 complex and Tor1 appear to work in parallel, both positively regulating amino acid uptake through the control of expression of amino acid permeases. Additionally, either Tsc1/2 or Tor1 are required for growth on a poor nitrogen source such as proline. Mutants lacking Tsc1 or Tsc2 are highly sensitive to rapamycin under poor nitrogen conditions, suggesting that the function of Tor1 under such conditions is sensitive to rapamycin. We discuss the complex genetic interactions between tor1(+), tor2(+), and tsc1/2(+) and the implications for rapamycin sensitivity in tsc1 or tsc2 mutants.

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Figures

F<sc>igure</sc> 1.—
Figure 1.—
tor2 phenotypes. (A) Left and middle: tor2− cells expressing nmt1-tor2+ (TA313) were grown to midlog phase and transferred to fresh minimal medium in the absence (derepressed) or presence (repressed) of thiamine. After 12 hr, cells were visualized by light microscopy. Right: wild-type cells (TA001) were starved in minimal medium containing no nitrogen for 21 hr. (B) Δtor2 mutant cells (TA313) were grown as described above. At time zero thiamine was added and cells were counted. (C) FACS analysis of the cells sampled in B. (D) Northern blot analysis. Total RNA was prepared from samples taken at the indicated time points (hours). Northern blots were probed with fnx1+ and with adh1+ as a loading control. (E) Wild-type (TA001), Δtsc2 (TA450), and Δtor2 carrying nmt1-tor2+ (TA313) cells were grown in EMM to midlog phase. Four different dilutions were spotted on EMM plates with and without 60 μg/ml canavanine. Plates were incubated at 30° for 3 days. (F) Cells auxotrophic for leucine were transformed with vector only or with vector containing the nmt1-tor2+ construct. Slow-growth phenotype is observed only when the nmt1 promoter is derepressed (no thiamine). Cells were streaked on minimal (M) plates supplemented with 75 μg/ml leucine and incubated at 30° for 4 days.
F<sc>igure</sc> 2.—
Figure 2.—
Analysis of Δtor1 Δtor2 double mutants. (A) Schematic of the opposite phenotypes of Δtor1 and Δtor2 mutations. (B) Terminal phenotype of Δtor1 Δtor2 cells expressing the nmt1-tor2+ construct (TA426) in the presence or absence of thiamine. (C) FACS analysis. Δtor2 or Δtor1 Δtor2 mutant cells expressing nmt1-tor2+ (TA313 and TA426, respectively) were grown to midlog phase and thiamine was added.
F<sc>igure</sc> 3.—
Figure 3.—
Additive effects of Δtor1 and Δtsc2 mutations. (A) Wild-type (TA001), Δtor1 (TA390), Δtsc2 (TA450), and Δtor1 Δtsc2 (TA459) cells were grown to midlog phase in EMM (M) medium, washed, resuspended in proline (P) medium, and further incubated for 15 min at 30°. RNA was extracted and subjected to Northern blot (top two sections) or semiquantitative RT–PCR analyses (bottom two sections). Northern blots were probed with isp5+ and adh1+ (loading control). For RT–PCR analysis, primers were specific for the amino acid permease 7G5.06 or FKBP12 (control). (B) Δtor1 (TA390), Δtsc2 (TA450), and Δtor1 Δtsc2 (TA459) cells were grown to midlog phase in EMM medium. Incorporation of l-3H-leucine was measured after 6 min using the protocol detailed in materials and methods.
F<sc>igure</sc> 4.—
Figure 4.—
Δtsc2 mutants are sensitive to rapamycin on medium containing a poor nitrogen source. (A) Δtsc1 or Δtsc2 mutants (TA419 and TA418, respectively) were streaked onto rich yeast extract (YE) or minimal plates containing ammonia (EMM) or proline (Pro) as the sole nitrogen source. Addition of rapamycin to the proline medium inhibited the growth on proline but not on rich medium. (B) Sensitivity to rapamycin on proline medium requires FKBP12. Prototrophic Δtsc2 mutants (TA450) or Δtsc2 Δfkh1 double mutants (TA468) were streaked on yeast extract (YE), proline (Pro), or glutamic acid (Glu) plates in the presence or the absence of 0.1 μg/ml rapamycin. Plates were incubated for 4 days at 30°. (C) Δtsc2 mutant cells were unable to grow on proline plates when combined with the Δtor1 mutation. The indicated strains, corresponding to TA001, TA390, TA449, TA481, TA450, and TA459, were streaked on proline plates in the presence or the absence of 0.1 μg/ml rapamycin. (D) Deletion of tor1+ reverses the small cell size and G1 accumulation of Δtsc2 mutants. Wild-type (TA001), Δtor1 (TA390), Δtsc2 (TA450), and Δtor1 Δtsc2 (TA459) cells were grown to late log phase in minimal EMM media at 30° before being photographed. Deletion of tor1+ also reverses the small cell size and G1 accumulation of Δtsc1mutants (data not shown). (E) FACS analysis of the strains indicated in D. Cells were grown to midlog phase in EMM (M) media and then shifted to either proline (Pro) or proline with 0.1 mg/ml rapamycin (Pro + R).
F<sc>igure</sc> 5.—
Figure 5.—
Rapamycin confers canavanine and thialysine resistance, while deletion of tor1+ confers hypersensitivity to these toxic amino acid analogs. (A) Serial dilutions of exponentially growing wild-type (TA001), Δtsc2 (TA450), or Δfkh1 (TA597) cells were spotted on EMM medium containing canavanine or thialysine in the absence or presence of rapamycin. (B) Serial dilutions of TA001, TA450, TA390, TA568, TA561, and TA459 were spotted on EMM plates in the presence of either canavanine or thialysine. (C) Working model. Tor2 positively regulates growth, allowing the G1/S transition under conditions of sufficient nutrients. Tor2 lies downstream of Rhb1 and is negatively regulated by Tsc1–2, while Tor1 acts in parallel with the Tsc1/2–Rhb1 module. The functions that are inhibited by rapamycin (R) are indicated in italics.
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