Regulation of Leucine Uptake by tor1+ in Schizosaccharomyces pombe Is Sensitive to Rapamycin

Abstract

TOR protein kinases are key regulators of cell growth in eukaryotes. TOR is also known as the target protein for the immunosuppressive and potentially anticancer drug rapamycin. The fission yeast Schizosaccharomyces pombe has two TOR homologs. tor1 ϩ is required under starvation and a variety of stresses, while tor2 ϩ is an essential gene. Surprisingly, to date no rapamycin-sensitive TOR-dependent function has been identified in S. pombe. Herein, we show that S. pombe auxotrophs, in particular leucine auxotrophs, are sensitive to rapamycin. This sensitivity is suppressed by deletion of the S. pombe FKBP12 or by introducing a rapamycin-binding defective tor1 allele, suggesting that rapamycin inhibits a tor1p-dependent function. Sensitivity of leucine auxotrophs to rapamycin is observed when ammonia is used as the nitrogen source and can be suppressed by its replacement with proline. Consistently, using radioactive labeled leucine, we show that cells treated with rapamycin or disrupted for tor1 ϩ are defective in leucine uptake when the nitrogen source is ammonia but not proline. Recently, it has been reported that tsc1 ϩ and tsc2 ϩ , the S. pombe homologs for the mammalian TSC1 and TSC2, are also defective in leucine uptake. TSC1 and TSC2 may antagonize TOR signaling in mammalian cells and Drosophila. We show that reduction of leucine uptake in tor1 mutants is correlated with decreased expression of three putative amino acid permeases that are also downregulated in tsc1 or tsc2. These findings suggest a possible mechanism for regulation of leucine uptake by tor1p and indicate that tor1p, as well as tsc1p and tsc2p, positively regulates leucine uptake in S. pombe. inhibits TOR kinases when in complex with FKBP12, a PI-3 and PI-4 kinases and are therefore referred to as PI3K-ubiquitous 12-kD prolyl-isomerase. The inhibition of related kinases. Accumulation of data in yeast, Drosophila, TOR by FKBP12-rapamycin complexes accounts for the and mammalian cells suggests that TOR is a central inhibition of many growth-related functions that are regulator of cellular growth. TOR controls growth in TOR dependent. Nevertheless, rapamycin also affects response to changes in the environment, particularly cellular processes through direct inhibition of the cellunutrient availability and cellular energetic status. et al. (1996) strates that rapamycin does not inhibit all TOR kinase- 1996) vation. tor1 ϩ is also required for growth at extreme tem-

The Birt-Hogg-Dube and Tuberous Sclerosis Complex Homologs Have Opposing Roles in Amino Acid Homeostasis in Schizosaccharomyces pombe

“…To determine whether the previously demonstrated rapamycin inhibition of permease expression is Bhd dependent, we treated wild-type and ⌬bhd S. pombe with the same dose of rapamycin used by Weisman et al (100 ng/ml), and measured the expression of the four permeases (869.10 ϩ , 7G5.06 ϩ , isp4 ϩ , and isp5 ϩ ) using real-time RT-PCR. In contrast to the work of Weisman et al (25), we found that in wild-type cells, rapamycin increased the expression of the permeases. These rapamycin-induced increases were confirmed by Northern blot for 7G5.06 ϩ and isp5 ϩ (Fig.…”

“…In S. pombe, the effects of rapamycin are not well understood, but it is believed to inhibit both Tor1-and Tor2-related functions and to inhibit the expression of isp5 ϩ , 7G5.06 ϩ , and 869.10 ϩ permeases (25,29). To determine whether the previously demonstrated rapamycin inhibition of permease expression is Bhd dependent, we treated wild-type and ⌬bhd S. pombe with the same dose of rapamycin used by Weisman et al (100 ng/ml), and measured the expression of the four permeases (869.10 ϩ , 7G5.06 ϩ , isp4 ϩ , and isp5 ϩ ) using real-time RT-PCR.…”

“…Down-regulation of Tor2 activity leads to up-regulation of nitrogen responsive genes including membrane transporters and amino acid permeases (24). In contrast, deletion of Tor1 protein leads to down-regulation of amino acid permeases (25) and the combination with Tsc1/Tsc2 deletion leads to even lower levels of permease expression (23) indicating that these proteins function in parallel pathways.…”

The TSC/Rheb/TOR Signaling Pathway in Fission Yeast and Mammalian Cells: Temperature Sensitive and Constitutive Active Mutants of TOR

“…23,24 Involvement of the Tor1 complex in stress response, mating and leucine uptake has been reported. 22,25,30 In contrast to fission yeast, budding yeast (S. cerevisiae) appears to differ with the mammalian system in some key aspects (Fig. 1).…”

Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrient-independent mammalian TOR signaling in mammalian cells

“…Although Tor2p, like Rhb1p, is essential for growth, tor1⌬ cells are sterile and unable to arrest in G 1 in response to nitrogen starvation (26,27). Tor1p also is implicated in the regulation of stress response and uptake of some amino acids (26)(27)(28). We recently have reported that fission yeast Rhb1p associates with Tor2p (25).…”