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. 2010 Nov 23;20(22):1975-82.
doi: 10.1016/j.cub.2010.10.026. Epub 2010 Oct 28.

Rab-family GTPase regulates TOR complex 2 signaling in fission yeast

Hisashi Tatebe  1 , Susumu Morigasaki, Shinichi Murayama, Cui Tracy Zeng, Kazuhiro Shiozaki
Affiliations

Rab-family GTPase regulates TOR complex 2 signaling in fission yeast

Hisashi Tatebe et al. Curr Biol. .

Abstract

Background: From yeast to human, TOR (target of rapamycin) kinase plays pivotal roles in coupling extracellular stimuli to cell growth and metabolism. TOR kinase functions in two distinct protein complexes, TOR complex 1 (TORC1) and 2 (TORC2), which phosphorylate and activate different AGC-family protein kinases. TORC1 is controlled by the small GTPase Rheb, but little is known about TORC2 regulators.

Results: We have identified the Ryh1 GTPase, a human Rab6 ortholog, as an activator of TORC2 signaling in the fission yeast Schizosaccharomyces pombe. Mutational inactivation of Ryh1 or its guanine nucleotide exchange factor compromises the TORC2-dependent phosphorylation of the AGC-family Gad8 kinase. In addition, the effector domain of Ryh1 is important for its physical interaction with TORC2 and for stimulation of TORC2 signaling. Thus, GTP-bound Ryh1 is likely to be the active form stimulatory to TORC2-Gad8 signaling. Consistently, expression of the GTP-locked mutant Ryh1 is sufficient to promote interaction between TORC2 and Gad8 and to induce Gad8 hyperphosphorylation. The loss of functional Ryh1, TORC2, or Gad8 brings about similar vacuolar fragmentation and stress sensitivity, further corroborating their involvement in a common cellular process. Human Rab6 can substitute Ryh1 in S. pombe, and therefore Rab6 may be a potential activator of TORC2 in mammals.

Conclusions: In its GTP-bound form, Ryh1, an evolutionarily conserved Rab GTPase, activates TORC2 signaling to the AGC kinase Gad8. The Ryh1 GTPase and the TORC2-Gad8 pathway are required for vacuolar integrity and cellular stress resistance in S. pombe.

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Figures

Figure 1
Figure 1. Sat1, Sat4 and Ryh1/Sat7 function in the same pathway that stimulates TORC2–Gad8 signaling
(A) TORC2-dependent phosphorylation of Gad8 Ser-546 is reduced in sat1-11, sat4-86 and sat7-110 cells. Wild type, Δtor1, sat1-11, sat4-86 and sat7-110 strains carrying the gad8:6HA allele were grown at 30°C and their crude cell lysate was analyzed by immunoblotting with anti-phospho-Ser546 and anti-HA antibodies. (B) Like TORC2 and gad8 mutants, Δryh1/sat7, Δsat1 and Δsat4 mutants are hypersensitive to high osmolarity and calcium. Wild type, Δtor1, Δsin1, Δgad8, Δryh1, Δsat1 and Δsat4 cells were spotted in serial dilution onto YES agar containing 0.4 M KCl (“Osm”) or 50 mM CaCl2 (“Ca2+”) at 28°C. (C) Gad8 kinase activity is decreased in Δryh1 cells. Wild type, Δryh1, Δtor1, Δsin1 and Δste20 strains carrying the gad8:3HA allele were grown in YES medium. Gad8 kinase was immunoprecipitated and assayed for its activity using the GST-Fkh2 protein as substrate. Quantified phosphorylation levels of GST-Fkh2 (relative to that of WT) are shown below. (D) The reduced Gad8 Ser-546 phosphorylation in the Δryh1 mutant is not further decreased by the Δsat1 or Δsat4 mutations. Wild-type, Δtor1, Δsin1, Δryh1, Δsat1, Δsat4, Δryh1 Δsat1 and Δryh1 Δsat4 strains were grown in YES medium. Gad8 Ser-546 phosphorylation was examined by anti-phospho-Ser546 immunoblotting. (E) The high osmolarity- and Ca2+-sensitive phenotypes of the Δryh1 Δsat1 and Δryh1 Δsat4 double mutants are similar to those of the Δryh1 single mutant.
Figure 2
Figure 2. Sat1 and Sat4 form a complex that may function as GEF for the Ryh1 GTPase
(A, B) Physical association of the Sat1 and Sat4 proteins. TAP-tagged Sat1 (A) and FLAG-tagged Sat4 (B) were affinity-purified from the cell lysate of sat1:TAP sat4:FLAG, sat1:TAP and sat4:FLAG strains, and the Sat1 and Sat4 proteins in the lysate and the purified fractions were detected by immunoblotting. (C) Sat4 is delocalized in the Δsat1 mutant. The chromosomal sat4 gene was tagged with the GFP sequence in wild type, Δsat1 and Δryh1 strains for fluorescence microscopy. Scale bar, 5 μm. (D) The GTP-locked mutant of ryh1 complements the Δsat1 and Δsat4 phenotype. Serial dilutions of the Δryh1 Δsat1 and Δryh1 Δsat4 strains transformed by plasmids carrying the wild-type, Q70L or T25N mutant of ryh1 were spotted onto YES agar with or without 50 mM CaCl2. (E) The nucleotide-free G23A mutant of Ryh1 can stably bind to Sat4. Ryh1Q70L (“QL”) and Ryh1G23A (“GA”) fused to GST were expressed in the sat4:FLAG strain and purified by glutathione-beads, and the proteins were detected by immunoblotting.
Figure 3
Figure 3. The GTP-bound form of Ryh1 stimulates TORC2–Gad8 signaling
(A) TORC2-dependent phosphorylation of Gad8 Ser-546 is stimulated by the GTP-bound form of Ryh1 through its effector domain. A wild type strain was transformed with pREP1-derived plasmids to express GST, or GST fused to wild-type and mutant Ryh1 proteins. The transformants were cultured in thiamine-free medium to induce the plasmid genes from the thiamine-repressible nmt1 promoter, and their lysate was analyzed by anti-phospho-Ser546, anti-Gad8 and anti-GST immunoblotting. GST-Ryh1 mutants used were GTP-locked Q70L (“QL”), GDP-locked T25N (“TN”), effector domain defective I44E (“IE”), as well as Q70L I44E and T25N I44E double mutants. (B) Ryh1 regulates TORC2-dependent phosphorylation of Gad8.Δ ste20, Δste20 ryh1Q70L, ryh1Q70L, ryh1Q70L Δsin1, and Δsin1 strains were grown in YES medium at 30°C, and their lysate was analyzed by immunoblotting to detect Gad8 phosphorylated at Ser-546 (left panels). Wild type, ryh1Q70L and ryh1Q70L mip1-310 strains were grown at 28°C and examined for Gad8 phosphorylation at Ser-546 (right panels). (C) Ypt3 GTPase does not stimulate TORC2-dependent phosphorylation of Gad8. Expression of GST, GST-Ryh1Q70L or GST-Ypt3Q69L was induced in wild-type cells using the thiamine-repressive nmt1 promoter, followed by immunoblotting with anti-phospho-Ser546, anti-Gad8 and anti-GST antibodies. (D) TORC2 signaling is compromised in the ypt3-i5 mutant. Δtor1, Δryh1, wild-type, and ypt3-i5 strains grown in YES medium at 25°C were examined for Gad8 phosphorylation at Ser-546. (E) The temperature-sensitive phenotype of the ryh1Q70L mutant is complemented by the gad8S546A mutation. Wild-type, ryh1Q70L, gad8S546A, and ryh1Q70L gad8S546A strains were grown on YES agar medium at 28°C and 36°C.
Figure 4
Figure 4. Physical interaction of Ryh1 GTPase with TORC2
(A) Interaction of Ryh1 with Tor1 in the presence and absence of other TORC2 subunits. In the NTAP:tor1 strain, expression of GST or GST-Ryh1 was induced from the nmt1 promoter in EMM medium without thiamine, and the proteins were affinity-purified onto glutathione (GSH)-Sepharose beads, followed by immunoblotting. The experiment was performed in the wild-type, Δsin1, Δste20 and Δwat1 backgrounds. (B) Co-purification of TORC2 subunits with GST-Ryh1. GST or GST-Ryh1 proteins were expressed in sin1:myc and ste20:myc strains, followed by purification with GSH-beads. Co-purification was detected by immunoblotting with anti-myc antibodies. (C) The I44E mutation to the effector domain of Ryh1 partially compromises the interaction between Ryh1 and Tor1. GST alone or GST-fused to the wild-type or mutant Ryh1 proteins were expressed in the FLAG:tor1 strain and affinity-purified by GSH-beads. Crude cell lysate and proteins bound to the beads were analyzed by immunoblotting. The Ryh1 mutations used were I44E (“IE”), GTP-locked Q70L (“QL”), GDP-locked T25N (“TN”) as well as Q70L I44E and T25N I44E double mutations. (D) The Δryh1 mutation does not impair the integrity of TORC2. TORC2 was affinity-purified onto IgG-Sepharose beads from tor1+ (“+”) and NTAP:tor1 (“NT”) strains carrying the sin1:FLAG, ste20:myc or wat1:FLAG in both ryh1+ (“+”) and Δryh1 (“Δ”) backgrounds. Proteins bound to the beads and those in the cell lysate were detected by immunoblotting.
Figure 5
Figure 5. Ryh1-dependent regulation of the TORC2–Gad8 pathway
(A) The catalytic activity of Tor1 kinase is not altered in strains lacking the Ryh1 GTPase. Wild type, Δryh1, Δsat1 and Δsat4 strains carrying the NTAP:tor1+ allele were grown in YES medium, and the NTAP-Tor1 protein was affinity-purified onto IgG-Sepharose beads, followed by an in vitro kinase assay in the presence of [©-32P]ATP and PHAS-I [31]. Phosphorylated PHAS-I (32P-PHAS-I) and NTAP-Tor1 (32P-NTAP-Tor1) are presented together with immunoblotting of NTAP-Tor1 (NTAP-Tor1). The NTAP:tor1D2137A strain expressing catalytically inactive Tor1 kinase was used as a negative control (“tor1DA”). (B) Cortical localization of Ste20 in S. pombe. Z-axial images of wild-type cells carrying the ste20:3GFP allele were deconvolved, and mid- and top-section images are shown. (C) Cortical localization of Ste20 is dependent on the Tor1 protein. Δ tor1, tor1D2137A (“tor1DA”), Δ sin1 and Δ gad8 mutants carrying the ste20:3GFP allele were observed by fluorescence microscopy. Mid-section images are presented after deconvolution. (D) Cellular localization of TORC2 is not affected by the Δ ryh1, Δ sat1 and Δ sat4 mutations. Deconvolved mid-section images of Δ ryh1 ste20:3GFP, Δ sat1 ste20:3GFP and Δ sat4 ste20:3GFP mutants are shown. See also Figure S5. (E) Gad8 is localized throughout the cytoplasm in wild type and the sat mutants. Wild-type, Δ ryh1, Δ sat1 and Δ sat4 strains carrying the gad8:3GFP allele were observed by fluorescence microscopy. Mid-section images are presented after deconvolution. Exclusion of Gad8-3GFP from vacuoles is apparent in wild-type cells, but not in the Δ ryh1, Δ sat1 and Δ sat4 cells that have much smaller, fragmented vacuoles (see Figure 6A). (F) Expression of GTP-locked Ryh1 promotes physical interaction of TORC2 with Gad8. Unfused GST (“-“), GST-Ryh1Q70L (“QL”), GST-Ryh1T25N (“TN”), GST-Ryh1Q70L/I44E or GST-Ryh1T25N/I44E proteins were expressed using the nmt1 promoter in a gad8:FLAG sin1:myc strain grown in the absence of thiamine. Immunoprecipitation was performed with anti-FLAG affinity gel and co-purified Sin1myc was detected with anti-myc antibodies. Scale bars, 5 μm.
Figure 6
Figure 6. Vacuolar defects in ryh1, TORC2 and gad8 mutants
(A) Cells lacking functional Ryh1 GTPase, TORC2 and Gad8 have smaller, fragmented vacuoles. Vacuolar membrane was visualized in wild type, Δ ryh1, Δ tor1 and Δ gad8 by the fluorescent dye FM4-64, together with the CDCFDA dye that generates fluorescence in acidic vacuolar compartments. Z-axial images were deconvolved, and mid-section images are shown. Scale bar, 5 μm (B) Human Rab6 GTPase can stimulate TORC2–Gad8 signaling in S. pombe. The Δ ryh1 strain was transformed by the empty pREP1 vector, or by the plasmid carrying wild-type Rab6A, GTP-locked Rab6A(Q72L), GDP-locked Rab6A(T27N), effector domain-defective Rab6A(I46E) or Rab6A(Q72L/I46E). The transformants were grown in thiamine-free EMM medium to induce the plasmid genes, and their lysate was analyzed by anti-phospho-Ser546 and anti-Gad8 immunoblotting. (C) Human Rab6 can substitute the Ryh1 function in S. pombe. Serial dilutions of Δ ryh1 cells carrying the empty pREP1 vector, pREP1-ryh1+, or pREP1-HsRab6A were spotted onto a YES plate at 25°C (“-”), 34°C, or those containing either 50 mM CaCl2 or 0.4 M KCl at 25°C.
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