The Tsc/Rheb signaling pathway controls basic amino acid uptake via the Cat1 permease in fission yeast

Abstract

The Tsc/Rheb signaling pathway plays critical roles in the control of growth and cell cycle. Studies in fission yeast have also implicated its importance in the regulation of amino acid uptake. Disruption of tsc2+, one of the tsc+ genes, has been shown to result in decreased arginine uptake and resistance to canavanine. A similar effect is also seen with other basic amino acids. We have identified a permease responsible for the uptake of basic amino acids by genetic complementation and disruption. SPAC869.11 (termed Cat1 for cationic amino acid transporter) contains 12 predicted transmembrane domains and its overexpression in wild type fission yeast leads to the increased uptake of basic amino acids and sensitivity to canavanine. Disruption of cat1+ in the deltatsc2 background interfered with the suppression of the canavanine-resistant phenotype of Atsc2 mutants by a dominant negative Rheb. In deltatsc2 mutant strains, the amount of Cat1 was not altered, but instead was mislocalized. This mislocalization was suppressed by the expression of dominant negative Rheb. In addition, we found that the loss of the E3 ubiquitin ligase, Pub1, also restores proper localization. These results provide a crucial link between Tsc/Rheb signaling and the regulation of the basic amino acid permease in fission yeast.

Fig. 1

Δtsc2 Cells defect in basic amino acid uptake and resistance to canavanine can be suppressed by the expression of dominant negative RhbD60K. a Δtsc2 cells transformed with pREP1-tsc2⁺ or pREP1 were assayed for the uptake of arginine, lysine and histidine. Assays were done in triplicate. b Δtsc2 cells transformed with pREP1-tsc2⁺ or pREP1 were spotted onto EMM + Ade plates either with canavanine or thialysine. c Δtsc2 cells transformed with pREP41-rhb1D60K⁺ or pREP41 were assayed for the uptake of arginine, lysine and histidine. Assays were done in triplicate

Fig. 2

Amino acid sequence analysis using DNAStar was performed on ScCan1 and 12 hypothetical Schizosaccharomyces pombe amino acid permeases. a Percent identity table shows low amino acid identity between the Schizosaccharomyces pombe amino acid permeases and ScCan1. b Phylogenetic tree based upon the divergence from ScCan1

Fig. 3

A scheme for the two-step assay to identify the Schizosaccharomyces pombe cationic amino acid transporter (CAT)

Fig. 4

ScCan1 complementation assay. Budding yeast Δcan1 mutant cells were transformed with putative fission yeast amino acid permeases that are placed under control of the ADH promoter. a Three positive transformants (spbpb2b2.01⁺, spbc359.03⁺ and spac869.11⁺), a negative transformant (spac869.10⁺), and a vector control were streaked onto SD–URA with and without canavanine. Strains that complement ScCan1 restore sensitivity to canavanine. b Spotting of three positives (spbpb2b2.01⁺, spbc359.03⁺ and spac869.11⁺) on a plate containing canavanine. Two clones each were tested for spbpb2b2.01⁺ and spbc359.03⁺

Fig. 5

Loss of SPAC869.11 results in canavanine resistance and a defect in basic amino acid uptake. a Schizosaccharomyces pombe strains with spac869.11⁺, spac359.03⁺, and spbpb2b2.01⁺ disrupted with the kanMX or URA cassette were serially diluted and spotted onto EMM + Ade plates with and without 60 mg/l canavanine. Δspac869.11 as well as Δspac869.11 double-mutant cells were resistant to canavanine. b and c Arginine and lysine uptake assays were performed on Δspac869.11 and Δspac359.03 cells. Δspac869.11 Δspbc359.03 double-mutant cells were also assayed for their ability to take up arginine. Cells lacking spac869.11⁺ have a defect in arginine and lysine uptake. Wild type and Δtsc2 cells were used as positive and negative controls, respectively. Assays were done in triplicate

Fig. 6

Transmembrane architecture analysis of SPAC869.11 and ScCan1. The amino acid sequences of both permeases were analyzed by ConPredII (Arai et al. 2004), a transmembrane prediction software program. In and out designate intracellular and extracellular matrices, respectively. The conserved glutamate residue is marked by an asterisk

Fig. 7

Overexpression of cat1⁺ in wild type cells increases basic amino acid uptake and canavanine sensitivity. a spac869.11⁺ was overexpressed under the control of the pREP41 promoter in wild type cells. Cells were assayed for their ability to take up [³H] arginine, lysine, histidine, and proline. Cells overexpressing spac869.11⁺ had a significantly higher uptake of basic amino acids. Assays were done in triplicate. b The same cells were spotted onto EMM + Ade plates with and without 10 mg/l canavanine to test for sensitivity

Fig. 8

Cat1 is required for RhebD60K to suppress the resistance of Δtsc2 to canavanine. Δtsc2 and Δtsc2 Δcat1 cells transformed with pREP1-tsc2⁺, pREP1, or pREP41-rhb1D60K⁺ were spotted onto EMM + Ade plates with and without 60 mg/l canavanine. Plates were photographed after 3 days of incubation

Fig. 9

Loss of tsc2⁺ does not affect cat1⁺ expression, but results in the mislocalization of Cat1. a Northern analysis of cat1⁺ in wild type and Δtsc2 mutant cells. tub1⁺ is used as a loading control. b Western analysis of Cat1−3xHA in wild type and Δtsc2 mutant cells. PCNA is used as a loading control. c Fluorescence microscopy of Cat1-GFP in wild type, Δtsc2 mutant cells transformed with or without pREP1-RhebD60K⁺, and Δtsc2Δpub1 cells