Ivy1 is a negative regulator of Gtr-dependent TORC1 activation

Abstract

The highly conserved TORC1 complex controls cell growth in response to nutrients, especially amino acids. The EGO complex activates TORC1 in response to glutamine and leucine. Here, we demonstrate that the I-BAR domain-containing protein Ivy1 colocalizes with Gtr1 and Gtr2, a heterodimer of small GTPases that are part of the EGO complex. Ivy1 is a negative regulator of Gtr-induced TORC1 activation, and is contained within puncta associated with the vacuolar membrane in cells grown in nutrient-rich medium or after brief nitrogen starvation. Addition of glutamine to nitrogen-starved cells leads to dissipation of Ivy1 puncta and redistribution of Ivy1 throughout the vacuolar membrane. Continued stimulation with glutamine results in concentration of Ivy1 within vacuolar membrane invaginations and its spatial separation from the EGO complex components Gtr1 and Gtr2. Disruption of vacuolar membrane invagination is associated with persistent mislocalization of Ivy1 across the vacuolar membrane and inhibition of TORC1 activity. Together, our findings illustrate a novel negative-feedback pathway that is exerted by Ivy1 on Gtr-dependent TORC1 signaling and provide insight into a potential molecular mechanism underlying TORC1 activation by vacuolar membrane remodeling.

Keywords: EGO complex; Gtr1; Gtr2; Ivy1; Microautophagy; TORC1; Vps1.

Fig. 1.

Changes in Ivy1 vacuolar distribution in response to glutamine. (A) W303A cells expressing Ivy1-EGFP were grown in SC medium. For nitrogen starvation, cells were transferred to SD −N for 3 h. Glutamine (gln, 3 mM) was added for 30 min, where indicated. Vacuoles were labeled with FM 4-64 (10 µM in the appropriate medium) for 1 h prior to imaging. (B) Cells were treated with rapamycin (200 ng/ml in the appropriate medium) for 3 h. Vacuoles were stained and cells were imaged as in A. (C) Quantification of the results shown in A. For each condition and each field of cells, Z-stacks were taken and the number of puncta associated with the vacuole in each stack was determined. Shown is mean±s.d. Two-way ANOVA was conducted to determine the effects of growth medium (SC or SD −N) and absence or presence of glutamine supplementation on the percentage of vacuoles decorated with Ivy1 puncta. There was a significant interaction term (F_1,31=62, hence P<0.0001). Each main effect was also significant: medium (F_1,31=111.08, hence P<0.0001) and absence or presence of glutamine supplementation (F_1,31=224.75, hence P<0.0001). Selected pairs of values significant at the 0.05 (*) and 0.01 (**) levels are indicated. (D) Quantification of the results shown in B. No significant differences were observed among any of the assessed conditions. Scale bar: 5 µm (for A and B).

Fig. 2.

Ivy1 localizes in close proximity to Gtr1 and Gtr2. (A) Controls for the split YFP assay. W303A cells expressing genomically integrated Ivy1, Gtr1 or Gtr2 C-terminally tagged with either the N-terminal (VN) or C-terminal (VC) fragments of YFP. Vacuolar lumens were labeled with CMAC. Cells were grown in SC medium. (B) YFP fluorescence was detected in W303A/α cells co-expressing appropriate pairs of proteins tagged with complementary fragments of YFP. Cells were grown and stained with CMAC as in A. (C,D) As in B but cells were grown in either SD −N for 3 h (C) or SD −N for 3 h followed by glutamine refeeding (3 mM) for 30 min prior to visualization (D). Scale bar: 5 µm (for A-D).

Fig. 3.

Ivy1 concentrates in vacuolar membrane invaginations. (A) W303A cells expressing Ivy1-EGFP were grown in SD −N for 3 h. Glutamine (3 mM) was added for 1 h. Vacuoles were labeled with FM 4-64 for 1 h prior to imaging. Images were collected at the indicated time points. (B) W303A cells expressing Ivy1-EGFP were grown and treated as in A. A time-lapse was taken and selected frames at various time points (indicated), showing the formation of a microautophagic invagination with simultaneous redistribution of Ivy1-EGFP across the vacuolar membrane, are shown. Scale bars: 5 µm.

Fig. 4.

Ivy1 inhibits glutamine-stimulated TORC1 activation. (A,B) Cells expressing Ivy1-mCherry and Gtr1-EGFP (A) or Gtr2-EGFP (B) were visualized using confocal microscopy. Where indicated, cells were treated with rapamycin (200 ng/ml) for 3 h, washed and recovered in YPD for 1 h prior to visualization. Representative cells are shown. Line scans show fluorescence intensity profiles along the indicated path for the EGFP and mCherry signals. The maxima for mCherry and EGFP fluorescence along the path were equalized. (C) W303A, Δivy1 and Δgtr1 Δgtr2 cells were treated with 200 ng/ml rapamycin in YPD for 5 h at 30°C. After washing, cells were plated on YPD and were incubated at 30°C for 2 days. The left-most spot in each case corresponds to 2 µl of a culture with OD₆₀₀ 0.5. Spots to the right of this correspond to 2 µl of sequential 5-fold dilutions. (D) Phosphorylation levels of Rps6 were evaluated under the indicated conditions. Untreated cells were grown in SC medium. Cells were nitrogen-starved by incubation in SD −N for 3 h. For stimulation, cells were treated with SD −N supplemented with either glutamine (Gln, 3 mM) or were re-fed with complete SC medium and were incubated for the indicated times prior to lysis and processing. Pgk1 and total Rps6 were used as loading control. Representative blots are shown. The quantification of the 3–4 replicates of the blots is shown below. Shown are the means of the ratios of phosphorylated Rps6 (phospho-Rps6) to Pgk1 (mean±s.d.) for each condition, in the control case (pCM190, white bars) or with Ivy1 overexpression (pCM190 IVY1, gray bars). Significant differences between pairs of measurements (pCM190 and pCM190 IVY1) are indicated (*P<0.05, **P<0.01). (E) W303A cells expressing the indicated combinations of constructs were nitrogen-starved by incubation in SD −N for 3 h. Where indicated, cells were treated with SD −N supplemented with Gln (3 mM) and were incubated for the indicated times prior to lysis and processing as in D. Representative blots are shown. The quantification of 3 replicates for each condition is depicted below the blots. Shown are the means of the ratios of phosphorylated Rps6 (phospho-Rps6) to Pgk1 (mean±s.d.) under each condition, color-coded according to the combination of constructs expressed in the cells as indicated to the left of the corresponding representative blot (maroon: W303A+pCM190 IVY1+GTR1 Q65L+GTR2 S23L; green: W303A+pCM190 IVY1+GTR1 Q65L; blue: W303A+pCM190 IVY1+GTR2 S23L). For each combination, the means of the untreated, treated and recovery measurements were determined to be significantly heterogeneous one-way ANOVA (maroon: F_4,10=48.09, P=1.7×10⁻⁶; green: F_4,10=59.98, P=5.95×10⁻⁷; blue: F_4,10=49.82, P=1.74×10⁻⁸). The most-relevant significantly different pairs of means, as assessed using the post-hoc Tukey HSD test, are indicated by the appropriate colored bar (**P<0.01). The most-relevant pair of means showing no significant difference is shown with a color-coded bar below the chart. Scale bar: 5 µm (for A and B).

Fig. 5.

Inhibition of vacuole invagination results in Ivy1 mislocalization on the vacuolar membrane. (A) W303A and Δvps1 cells were treated with rapamycin (200 ng/ml) for 3 h, followed by washing and recovery in YPD. Vacuoles were stained with FM 4-64 prior to visualization using confocal microscopy. Representative images are shown. (B) W303A and Δvps1 cells were plated in serial dilutions on YPD (top) or YPD supplemented with 2.5 ng/ml rapamycin (bottom) and were incubated at 30°C for 2 days. The left-most spot in each case corresponds to 2 µl of a culture with OD₆₀₀ 0.5. Spots to the right of this correspond to 2 µl of sequential 5-fold dilutions. (C) W303A or Δvps1 cells expressing Ivy1-EGFP were stained with FM 4-64 and were visualized using confocal microscopy. Where indicated, cells were treated with rapamycin (200 ng/ml) for 3 h, or were treated, washed and recovered in YPD for 1 h prior to visualization. Representative cells are shown. (D) Quantification of untreated vacuoles associated with Ivy1 puncta in W303A and Δvps1 cells. For untreated fields of W303A and Δvps1 cells, Z-stacks were obtained and the number of puncta associated with the vacuole in each stack was determined. The means of the ratios of vacuoles with Ivy1 puncta to total vacuoles were significantly different in W303A and Δvps1 cells (t=12.8; ***P<0.001; n=143 and 152 for W303A and Δvps1 vacuoles, respectively). Scale bars: 5 µm (A and C).

Fig. 6.

Ivy1 regulates Tor1 localization. (A) Cells, as indicated, expressing EGFP-Tor1 were stained with FM 4-64 and visualized using confocal microscopy. Representative fields are shown. Scale bar: 5 µm. (B) Quantification of Tor1 puncta on vacuoles. The means of the ratios of vacuoles with Tor1 puncta to total vacuoles for the indicated strains were significantly heterogeneous (one-way ANOVA, F_5,18=227.83, P=1.3×10⁻¹⁵). Pairs of means not significantly different from one another (Tukey HSD post-hoc test) are indicated by horizontal lines below the chart (P>0.05). Selected pairs of means that are significantly different from one another are indicated by horizontal lines above the graph (**P<0.01). 260–480 vacuoles were quantified for each strain. (C) Cells expressing Tor1 L2134M as indicated were plated on YPD supplemented with 2.5 ng/ml rapamycin and incubated at 30°C for 3 days. The left-most spot in each case corresponds to 2 µl of a culture with OD₆₀₀ 0.5. Spots to the right of this correspond to 2 µl of sequential 5-fold dilutions. (D) Model for the regulation of TORC1 signaling by Ivy1. Glutamine or leucine activate Gtrs that regulate TORC1 activation and distribution. Reactivated TORC1 results in redistribution of Ivy1 on the vacuolar membrane. Ivy1, in turn suppresses activation of Gtrs.