Constitutive Tor2 Activity Promotes Retention of the Amino Acid Transporter Agp3 at Trans-Golgi/Endosomes in Fission Yeast

Abstract

Amino acid transporters are located at specific subcellular compartments, and their localizations are regulated by the extracellular availability of amino acids. In yeast, target of rapamycin (TOR) activation induces the internalization of amino acid transporters located at the plasma membrane. However, whether and how TOR signaling regulates other amino acid transporters located at intracellular compartments remains unknown. Here, we demonstrate that in the fission yeast, the TOR inhibitor Torin-1 induces the transfer of several yellow fluorescent protein (YFP)-fused intracellular amino acid transporters, including Agp3, Isp5, Aat1, and Put4, from trans-Golgi/endosomes into the vacuoles. By contrast, the localizations of YFP-fused Can1, Fnx1, and Fnx2 transporter proteins were unaffected upon Torin-1 treatment. There are two TOR isoforms in fission yeast, Tor1 and Tor2. Whereas tor1 deletion did not affect the Torin-1-induced transfer of Agp3-YFP, Tor2 inhibition using a temperature-sensitive mutant induced the transfer of Agp3-YFP to the vacuolar lumen, similar to the effects of Torin-1 treatment. Tor2 inhibition also induced the transfer of the YFP-fused Isp5, Aat1, and Put4 transporter proteins to the vacuoles, although only partial transfer of the latter two transporters was observed. Under nitrogen depletion accompanied by reduced Tor2 activity, Agp3-YFP was transferred from the trans-Golgi/endosomes to the plasma membrane and then to the vacuoles, where it was degraded by the vacuolar proteases Isp6 and Psp3. Mutants with constitutively active Tor2 showed delayed transfer of Agp3-YFP to the plasma membrane upon nitrogen depletion. Cells lacking Tsc2, a negative regulator of Tor2, also showed a delay in this process in a Tor2-dependent manner. Taken together, these findings suggest that constitutive Tor2 activity is critical for the retention of amino acid transporters at trans-Golgi/endosomes. Moreover, nitrogen depletion suppresses Tor2 activity through Tsc2, thereby promoting the surface expression of these transporters.

Fig 1. Systematic analysis of the localizations of several representative amino acid transporters with or without TOR inhibition in fission yeast.

Representative fluorescent images of the wild-type (WT) cells expressing YFP-fused Agp3 (KP6154), Isp5 (KP6153), Aat1 (KP6156), Put4 (KP6155), Fnx1 (KP6157), Can1 (KP6158), or Fnx2 (KP6159) protein with vehicle (Veh) or Torin–1 (Torin) treatment for 2 h. The cells were grown to the log phase in EMM, and were subjected to vehicle or Torin–1 treatment. To visualize the vacuolar membranes, FM4-64 was loaded for 1 h before the Torin–1 treatment. YFP and FM4-64 signals are shown in green and red, respectively, and the merged signals are shown in yellow. Scale bar, 10 μm.

Fig 2. TOR inhibition induces the transfer of Agp3, Isp5, Aat1, and Put4 from trans-Golgi/endosomes into the vacuoles.

(A) The co-localization of Agp3, Isp5, Aat1, and Put4 with Krp1, a marker for trans-Golgi/endosomes, in normal culture condition. Representative fluorescent images of the wild-type cells co-expressing Krp1-RFP and YFP-fused Agp3 (KP6624), Isp5 (KP6662), Aat1 (KP6665), or Put4 (KP6664) protein are shown. YFP and RFP signals are shown in green and red, respectively, and the merged signals are yellow. Arrowheads indicate the co-localization of Agp3-positive dots with the Krp1-positive structures. Scale bar, 10 μm. (B) The co-localization of Agp3 with Fnx1, a vacuole-enriched amino acid transporter, after Torin–1 treatment. Representative fluorescent images of the wild-type cells co-expressing Agp3-RFP and Fnx1-YFP (KP6644) with vehicle (Veh) or Torin–1 (Torin) treatment for 2 h. RFP and YFP signals are shown in red and green, respectively, and the merged signals are yellow. Scale bar, 10 μm.

Fig 3. Tor2 inhibition induces the transfer of Agp3, Isp5, Aat1, and Put4 from trans-Golgi/endosomes into the vacuoles.

(A) The effect of tor1 deletion on the localization of amino acid transporters. Representative fluorescent images of Δtor1 cells expressing Agp3-YFP (KP6239) with vehicle (Veh) or Torin–1 (Torin) treatment for 2 h are shown. To visualize the vacuolar membranes, FM4-64 was loaded for 1 h before the Torin–1 treatment. YFP and FM4-64 signals are shown in green and red, respectively, and the merged signals are yellow. Scale bar, 10 μm. (B) The effect of tor2-287 mutation on the localization of amino acid transporters. Representative fluorescent images of tor2-287 cells expressing Agp3-YFP (KP6244), Isp5 (KP6651), Aat1 (KP6653), or Put4 (KP6652) with vehicle (Veh) or Torin–1 (Torin) treatment for 2 h. Scale bar, 10 μm. (C) Expression of intact Agp3-YFP protein in the strains used in this study. The wild-type cells (KP6154, WT(+)), tor2-287 cells (KP6244), and Δtor1 cells (KP6239) expressing Agp3-YFP, as well as the wild-type cells without Agp3-YFP expression (KP5080, WT(-)), were collected, and the proteins were extracted for western blotting. Agp3-YFP was detected using the GFP antibody. Tubulin expression was used as an internal control.

Fig 4. Constitutive Tor2 activity is sufficient for Agp3 localization at trans-Golgi/endosomes.

Representative images of Δtsc2 cells expressing Agp3-YFP with vehicle (Veh) or Torin–1 (Torin) treatment are shown. The cells were grown to the log phase in EMM, and subjected to drug treatment. In this experiment, Torin–1 treatment for 4 h rather than 2 h was required for it to exert its effect, which was perhaps due to the elevated Tor2 activity induced by tsc2 deletion. YFP and FM4-64 signals are shown in green and red, respectively, and the merged signals are yellow. Scale bar, 10 μm.

Fig 5. Nitrogen depletion induces the transfer of Agp3 from trans-Golgi/endosomes to the plasma membrane, and then into the vacuoles.

(A) The effect of nitrogen depletion on Agp3 localization. Representative images of wild-type cells expressing Agp3-YFP (KP6154) in nitrogen-rich medium (N-rich) or after the shift to nitrogen-depleted medium for 1 h, 4 h, and 9 h are shown. Scale bar, 10 μm. (B) The localization of Agp3 in FM4-64-stained vacuoles under nitrogen depletion for 9 h. Wild-type cells expressing Agp3-YFP were cultured in nitrogen-depleted medium for 8 h, and FM6-64 was loaded for 1 h in recycled nitrogen-depleted medium to avoid nutrient re-supplementation. Scale bar, 10 μm. (C) The expression of Agp3-YFP and its degradation by vacuolar proteases under nitrogen depletion. The wild-type cells (KP6154, WT, (+)) and Δisp6Δpsp3 cells (KP6635, Δisp6Δpsp3, (+) expressing Agp3-YFP were collected without (0 h) or with nitrogen depletion (1 h, 4 h, and 9 h), and the proteins were extracted for western blotting. Agp3-YFP was detected with green fluorescent protein antibody. The wild-type cells without Agp3-YFP expression (KP5080, WT, (-)) were similarly analyzed as negative controls. The same immunoblot is shown with shorter and longer exposures. Tubulin expression was used as an internal control.

Fig 6. Increased Tor2 activity delays the transfer of Agp3 to the plasma membrane under nitrogen depletion.

(A, B) The effect of constitutively active tor2 mutants on the transfer of Agp3 to the plasma membrane under nitrogen depletion. Representative fluorescent images of wild-type cells (KP6154, WT), tor2 ^L1310P cells (KP6637), and tor2 ^E2221K cells (KP6638) expressing Agp3-YFP in nitrogen-rich medium (N-rich) or after a shift to nitrogen-depleted medium (N-depletion) for 10 min, 20 min, 30 min, and 60 min (A), and of the same cells of another batch under nitrogen depletion for 4 h (B). Scale bar, 10 μm. (C, D) The effect of tsc2 deletion on the transfer of Agp3 to the plasma membrane under nitrogen depletion. Representative fluorescent images of the wild-type cells (KP6154, WT) and Δtsc2 cells (KP6434) expressing Agp3-YFP in nitrogen-rich medium (N-rich) or after the shift to nitrogen-depleted medium (N-depletion) for 1 h (C) and 4 h (D). Scale bar, 10 μm. (E) Rescue of the nitrogen depletion-induced transfer of Agp3 to the plasma membrane in Δtsc2 cells by the tor2-287 mutation. Representative images of the wild-type cells (KP6154), Δtsc2 cells (KP6434), tor2-287 cells (KP6244), and tor2-287Δtsc2 cells (KP6448) expressing Agp3-YFP under nitrogen depletion for 1 h are shown. Scale bar, 10 μm.