| The Tsc/Rheb/Tor pathway is a highly conserved signaling cassette involved in the regulation of various cellular processes including cell growth and proliferation. Aberrant regulation of this signal transduction pathway leads to various diseases including Tuberous Sclerosis Complex (TSC), Peutz-Jegher Syndrome (PJS), and is implicated in various cancers as well. Therefore, understanding the interplay between the members of the pathway and finding new phenotypes associated with the pathway may prove insightful into the etiology of these diseases.;Previous studies have suggested that Rheb is involved in the regulation of arginine uptake. However, the mechanism, as well as the role of the upstream and downstream components of Rheb in this process, has not been elucidated. Taking advantage of the genetically malleable organism Schizosaccharomyces pombe, we have found that loss of Tsc2, the Rheb GTPase activating protein (GAP), leads to a defect in the uptake of basic amino acids, which is highlighted by a resistance to toxic analogues of arginine and lysine, canavanine and thialysine, respectively. To further understand this process, we identified the cationic amino acid transporter (CAT) in fission yeast via genetic complementation and disruption in budding yeast and fission yeast, respectively. SPAC869.11 was found to be required for the uptake of basic amino acids and its overexpression in wild type yeast led to an increase in the uptake of basic amino acids and sensitivity to canavanine. SPAC869.11 was thusly dubbed Cat1. Interestingly, loss of Tsc2 affected neither Cat1 RNA nor protein levels, rather, led to a drastic mislocalization of Cat1 from the cell tips to the Golgi apparatus. This mislocalization could be suppressed by the expression of RhebD60K, as well as the introduction of a temperature sensitive Tor2 allele (tor2ts). In addition, we found that loss of the E3 ubiquitin ligase, Pub1, also restores proper localization. On the other hand, loss of Tsc1 and rapamycin treatment of wild type cells leads to an inhibition of arginine uptake and a similar mislocalization of Cat1 to the Golgi albeit not as severe to that of tsc2 - mutants or by overexpression of Tor2. Pub1 is also required for the rapamycin inhibition of arginine uptake. Therefore, it appears that Tor1 and Rheb/Tor2 have counteracting functions that impinge upon Pub1 in the control of Cat1.;We have also found that overactivation of Rheb, either by mutation or loss of Tsc2, leads to the resistance of brefeldin A (BFA) and cerulenin, inhibitors of the Golgi apparatus and fatty acid synthase, respectively. This can be suppressed by a tor2ts mutant as well as by the loss of Pub1. The ABC transporter, Bfr1, is also required for this resistance, and whose protein levels were slightly elevated in tsc2 - mutants. Therefore, it appears that the Tsc/Rheb/Tor pathway controls both the import of amino acids as well as the export of xenobiotics via two different mechanisms. |
