Ait1 regulates TORC1 signaling and localization in budding yeast

  1. Ryan L Wallace
  2. Eric Lu
  3. Xiangxia Luo
  4. Andrew P Capaldi  Is a corresponding author
  1. Department of Molecular and Cellular Biology, University of Arizona, United States

Abstract

The target of rapamycin complex I (TORC1) regulates cell growth and metabolism in eukaryotes. Previous studies have shown that nitrogen and amino acid signals activate TORC1 via the highly conserved small GTPases, Gtr1/2 (RagA/C in humans), and the GTPase activating complex SEAC/GATOR. However, it remains unclear if, and how, other proteins/pathways regulate TORC1 in simple eukaryotes like yeast. Here, we report that the previously unstudied GPCR-like protein, Ait1, binds to TORC1-Gtr1/2 in Saccharomyces cerevisiae and holds TORC1 around the vacuole during log-phase growth. Then, during amino acid starvation, Ait1 inhibits TORC1 via Gtr1/2 using a loop that resembles the RagA/C-binding domain in the human protein SLC38A9. Importantly, Ait1 is only found in the Saccharomycetaceae/codaceae, two closely related families of yeast that have lost the ancient TORC1 regulators Rheb and TSC1/2. Thus, the TORC1 circuit found in the Saccharomycetaceae/codaceae, and likely other simple eukaryotes, has undergone significant rewiring during evolution.

Editor's evaluation

Genetic analysis of the yeast S. cerevisiae identifies a role for the vacuolar GPCR Ait1 in the regulation of TORC1. The mechanism of Ait1 function is mediated by regulation of the small GTPases Gtr1/2. This finding identifies novel signaling modality in yeast for the control of TORC1 function.

https://doi.org/10.7554/eLife.68773.sa0

Introduction

To function correctly, cells have to set their growth rate based on a wide array of nutrient, stress, and hormone signals. In eukaryotes, this fine-tuned control depends—in a large part—on a single, highly conserved, signaling hub called the target of rapamycin kinase complex I (TORC1) (González and Hall, 2017; Loewith and Hall, 2011; Liu and Sabatini, 2020). In the presence of pro-growth hormones and abundant nutrients, TORC1 drives growth by activating protein, ribosome, lipid, and nucleotide synthesis (González and Hall, 2017; Loewith and Hall, 2011; Liu and Sabatini, 2020; Huber et al., 2009; Robitaille et al., 2013; Hsu et al., 2011; Peterson et al., 2011; BenSahra and Manning, 2017; BenSahra et al., 2016; Kim et al., 2011; Kamada et al., 2000). In contrast, when nutrient or hormone levels drop, TORC1 is repressed, causing cells to switch from anabolic to catabolic metabolism, and eventually enter a quiescent state (Düvel et al., 2010; Barbet et al., 1996).

TORC1 is regulated by a sophisticated signaling network that, in humans, includes two well-defined channels:

(1) Growth factor and mitogen signals are transmitted to TORC1 through a GTPase activating protein (GAP) called the tuberous sclerosis complex (TSC) (Manning et al., 2002; Inoki et al., 2002). In the presence of pro-growth hormones (such as insulin), TSC is repressed, triggering accumulation of the active, GTP-bound, form of Rheb (Menon et al., 2014; Dibble and Manning, 2013). GTP-Rheb then binds to TORC1 on the lysosomal membrane, driving a conformational change that increases TORC1 activity (Menon et al., 2014; Yang et al., 2017). The AMP-activated protein kinase (AMPK) also signals to TORC1 via TSC (as well as the TORC1 subunit Kog1/Raptor) to ensure TORC1 is inhibited when ATP levels fall (Inoki et al., 2003b; Inoki et al., 2003a; Gwinn et al., 2008).

(2) Amino acid (and glucose) signals are transmitted to TORC1 via a heterodimeric pair of GTPases, consisting of RagA or B and RagC or D, that are tethered to the lysosomal membrane by the Ragulator complex (Sancak et al., 2010; Sancak et al., 2008; Kim et al., 2008; BarPeled et al., 2012; Efeyan et al., 2013). In the presence of adequate nutrients, RagA/B and C/D are in their GTP- and GDP-bound forms, respectively, and bind tightly to TORC1 to keep it on the lysosomal membrane and near Rheb (Menon et al., 2014; Sancak et al., 2010; Sancak et al., 2008; Kim et al., 2008; Rogala et al., 2019). However, when amino acid levels fall, the large multiprotein GAP, GATOR1/2, drives RagA/B to the GDP-bound form, triggering the release of TORC1 from the lysosome so that it cannot be activated by Rheb (BarPeled et al., 2013).

GATOR1/2, in turn, is regulated by at least three different amino acid-binding proteins to ensure that cell growth halts during starvation: the leucine sensor Sestrin2 (Wolfson et al., 2016; Saxton et al., 2016b); the arginine sensor CASTOR1 (Saxton et al., 2016a; Chantranupong et al., 2016); and the methionine—or more specifically S-adensylmethionine (SAM)—sensor SAMTOR (Gu et al., 2017). Arginine signals are also transmitted to the Rags via SLC38A9, an amino acid transporter in the lysosomal membrane (Wang et al., 2015; Castellano et al., 2017).

Outside of humans, however, much less is known about TORC1 regulation. The amino acid sensors discussed above are only fully conserved in vertebrates (Liu and Sabatini, 2020; Tatebe and Shiozaki, 2017), and while Rheb/TSC and the Rags/GATOR are ancient TORC1 pathway components—likely present in the last common eukaryote—many yeasts, worms, plants, and protists/excavata have lost Rheb and TSC (Liu and Sabatini, 2020; Tatebe and Shiozaki, 2017). It therefore seems likely that: (1) there are conserved TORC1 pathway components that remain to be discovered, and (2) simple eukaryotes have evolved novel, currently unknown, nutrient sensing and TORC1 control mechanisms to replace Rheb/TSC.

One well-studied organism with a TORC1 signaling network that appears to have diverged significantly from that in humans is the budding yeast, Saccharomyces cerevisiae.

S. cerevisiae has two GTPases, Gtr1 and Gtr2, that are homologs of RagA/B and RagC/D, respectively (Binda et al., 2009; Dubouloz et al., 2005). Furthermore, Gtr1/2 are tethered to the vacuole (the yeast equivalent of the lysosome) by a complex that is very similar—but not obviously homologous to—the Ragulator, called Ego1, Ego2, and Ego3 Powis et al., 2015 (Zhang et al., 2019; Zhang et al., 2012). The GATOR1/2 GAP that acts upstream of the Rags is also conserved in yeast, and made up of Npr2, Npr3, and Iml1 (the GATOR1 equivalent, known as SEACIT) and Rtc1, Mtc5, Sea4, Seh1, and Sec13 (the GATOR2 equivalent, known as SEACAT) (Panchaud et al., 2013; Neklesa and Davis, 2009; Chen et al., 2017; Laxman et al., 2014; Algret et al., 2014). However, S. cerevisiae do not have SLC38A9, Sestrins, CASTOR, or SAMTOR, and are also missing TSC1/2 and functional Rheb (González and Hall, 2017; Liu and Sabatini, 2020; Tatebe and Shiozaki, 2017).

In line with the expectation that there are differences between TORC1 signaling in yeast and humans, we recently discovered that glucose and nitrogen starvation cause TORC1 in S. cerevisiae to move from its position distributed around the vacuolar membrane to a single body on the edge of the vacuole (Hughes Hallett et al., 2015; Sullivan et al., 2019). Adding the missing nutrient back to the cell—even in the presence of cycloheximide—then reverses the process (Hughes Hallett et al., 2015). We also found that TORC1-body formation is initiated by inactivation of Gtr1/2 and requires an interaction between TORC1 and the recently identified TORC1 regulator Pib2 (Hughes Hallett et al., 2015; Sullivan et al., 2019; Varlakhanova et al., 2017; Kim and Cunningham, 2015; Ukai et al., 2018; Tanigawa and Maeda, 2017; Michel et al., 2017). TORC1 agglomeration, itself, is then driven by two glutamine-rich, prion-like domains in the TORC1 subunit Kog1/Raptor, and ultimately functions to increase the threshold for TORC1 reactivation (Hughes Hallett et al., 2015). In other words, the formation of TORC1 bodies helps to ensure that cells commit to the quiescent state when they have been starving for a significant period of time. Interestingly, the prion-like domains in Kog1/Raptor are found in yeast species and worms that are missing the TSC genes, but are absent from S. pombe and higher organisms that do carry the TSC genes (Hughes Hallett et al., 2015). This suggests that organisms use either TSC and Rheb, or TORC1-body formation, alongside Gtr1/2 (Rag proteins) to control TORC1 activity (Hughes Hallett et al., 2015).

Here, to learn more about TORC1 regulation in yeast, and other simple eukaryotes, we map the TORC1 interactome in S. cerevisiae in a wide range of stress and starvation conditions. These experiments lead to the identification of numerous new TORC1 regulators, the most notable of which are a putative phosphate channel, Syg1, and a previously unstudied GPCR-like protein, Ydl180w, that we have named Ait1 (Amino acid-dependent Inhibitor of TORC1). In follow-up experiments, we show that Ait1 is required to hold TORC1 in its native position around the vacuolar membrane during log-phase growth. We also show that Ait1 is required for TORC1 inhibition during amino acid starvation in cells expressing Gtr1 and/or Gtr2. Interestingly, Ait1 is only found in the Saccharomycetaceae and Saccharomycodaceae. The yeast species within these related families—which include the pathogen Candida glabrata—are unique in that they (1) have highly divergent Rheb, or no Rheb, (2) are missing TSC2 and/or TSC1, and (3) have prion-like domains in the TORC1 subunit Kog1/Raptor (Tatebe and Shiozaki, 2017). Thus, an ancestor of the Saccharomycetaceae/codaceae gained the novel TORC1 regulator, Ait1, at around the same time it lost functional Rheb and TSC1/2 (approximately 200 million years ago) (Tatebe and Shiozaki, 2017; Shen et al., 2018), to aid in amino acid signaling and appropriate TORC1 localization. We suggest that similar rewiring of the TORC1 pathway likely occurred during the evolution of many other simple eukaryotes, and that Ait1 represents an important new drug target in yeast.

Results

The TORC1 interactome in budding yeast

As a first step toward building a map of the TORC1 regulatory network, we developed an immunopurification protocol that makes it possible to capture and identify TORC1 interactors. Cells carrying Kog1-FLAG, and in parallel cells carrying Kog1-HA, were grown to log-growth phase, or grown to log-growth phase and transferred into stress or starvation medium, rapidly filtered and flash frozen. The cells were then lysed and treated with the short (12 Å) cleavable crosslinker dithiobis(succinimidyl propionate) (DSP) and the nonionic detergent digitonin (Murley et al., 2017). The supernatants from the Kog1-HA and Kog1-FLAG strains were then immunopurified in parallel on anti-FLAG columns, the crosslinkers broken, and the samples analyzed using mass spectrometry. Proteins with at least twofold higher abundance in the true IP (Kog1-FLAG) versus the mock IP (Kog1-HA), and with at least seven peptide spectral maps in the true IP, were scored as potential interactors. In total, we immunopurified Kog1 in 11 different experiments (in nine conditions) and ran a similar experiment to identify Pib2 interactors in two conditions. These purifications led to the identification of over 200 interactors, 138 of which were identified in four or more experiments (Supplementary file 1). At the top of this list are 45 proteins identified in 7 or more experiments, including: (1) all four subunits of TORC1 (blue names, Figure 1); (2) Gtr1, Gtr2, and Ego1, all three subunits of SEACIT, and two subunits of SEACAT (green names, Figure 1); (3) a variety of proteins that have been shown to play some role in TORC1 signaling previously, including several TORC1 substrates (Huber et al., 2009; Liu et al., 2003; Breitkreutz et al., 2010) (black names, Figure 1); and (4) 15 proteins that have, to the best of our knowledge, not been connected to TORC1 signaling previously, or in many cases, studied at all (red names, Figure 1). Several of these new interactors—including Ydl180w/Ait1, Vsb1, and Vtc5—form interactions with TORC1 that are as tight, or tighter, than those between TORC1 and Gtr1/2, as judged by the amount of material captured in the purification (Figure 1).

The target of rapamycin complex I (TORC1) interactome in budding yeast.

Blue circles show the number of background corrected peptide spectral maps (PSMs) from each protein identified in a specific Kog1 or Pib2 immunopurification, while the red to yellow scale shows the average number of PSMs across all experiments. The figure shows data for the top 45 TORC1 interactors (those identified in seven or more immunopurifications). The complete dataset is in Supplementary file 1.

To learn more about the interaction between TORC1 and the novel interactor Ait1 (a previously unstudied GPCR-like protein located in the vacuolar membrane Genome Resources, 2020; UniProt, 2019; Figure 2), we also immunopurified GFP-Ait1 after crosslinking, and mapped the interactors, as described above for Kog1. These experiments showed that the TORC1 subunits Kog1, Tor1, and Tco89 are among the most abundant proteins captured in an Ait1 purification, indicating that there is a close interaction between TORC1 and Ait1 (Figure 3, Figure 3—figure supplement 1).

Ait1 is a putative seven-helical transmembrane (GPCR-like) protein that localizes to the vacuolar membrane.

(A) The predicted topology of Ait1/Ydl180w from Protter 1.0 (Omasits et al., 2014). The two large cytosolic loops in Ait1, both of which are predicted to be intrinsically disordered (C3 and C4), are labeled. (B, C) GFP-Ait1 localizes to the vacuolar membrane, as shown by the overlap between the GFP-Ait1 signal and the vacuolar membrane stain FM4-64 signal and does not relocalize in amino acid starvation (shown), or other starvation conditions (not shown).

Figure 3 with 1 supplement see all
The Ait1 interactome.

Blue circles show the number of background corrected peptide spectral maps (PSMs) for the top 25 proteins identified in the GFP-Ait1 immunopurification (based on the average number of PSMs in the four experiments), along with the data for Gtr1, Gtr2, and Pib2 for comparison.

Impact of TORC1 interactors on TORC1-body formation

To examine the impact that the new TORC1 interactors have on TORC1 signaling, we measured Kog1-YFP localization during nitrogen starvation in a collection of strains, each missing one of the top 50 proteins identified in our immunopurification experiments (excluding interactors that were examined in our previous studies Hughes Hallett et al., 2015; Sullivan et al., 2019). These experiments showed that—as expected (Sullivan et al., 2019) — many of the known TORC1 interactors are important for TORC1-body formation, including the SEACIT/CAT subunits Iml1, Seh1, Sea2/Rtc1, and Npr3 (Figure 4). These experiments also showed that several of the previously unknown TORC1 interactors have a profound impact on TORC1-body formation and/or TORC1 localization. Specifically, deletion of Vnx1 (a vacuolar monovalent cation/proton antiporter; Wilson et al., 2018) or Syg1 (a putative phosphate channel in the vacuolar membrane; Genome Resources, 2020) blocks TORC1-body formation, just like deletion of Npr2, Npr3, Iml1, or Pib2 (Figure 4 and Sullivan et al., 2019). The most striking result, however, was found in the ait1Δ strain: Deletion of Ait1 causes TORC1 to move into a body, even during log-phase growth in nutrient replete medium (Figure 4 and Figure 5A, B).

Target of rapamycin complex I (TORC1)-body formation during nitrogen starvation in strains missing key TORC1 interactors.

Each square on the heat map shows the fraction of cells with a Kog1-YFP focus/body at a specific timepoint, calculated by examining the images of >200 cells, per strain, per timepoint. Replicate experiments confirmed the severe defects in the syg1Δ, vps30Δ, rtg2Δ, and vnx1Δ strains (<15% bodies after 1 hr of nitrogen starvation). These follow-up experiments also revealed dramatic variation in the results for vsb1Δcells (even comparing between colonies) leading us to drop the strain from our analysis.

Figure 5 with 1 supplement see all
Target of rapamycin complex I (TORC1)-body formation in the ait1Δ strain.

(A) Kog1-YFP localization in the wild-type and ait1Δ strains, during log-phase growth in nutrient-rich (SD) medium. The white bar shows 5 μm. (B) Fraction of wild-type and ait1Δ cells that form Kog1-YFP foci during nitrogen starvation. The points and error bars show the average and standard deviation from five replicate experiments, with at least 100 cells examined, per timepoint, per replicate. (C) Impact of the Ait1 deletion on TORC1-body formation in the wild-type and various mutant strains (compare left and right columns). Experiments were carried out in at least duplicate with over 200 cells examined per timepoint, per mutant. Individual timepoints have errors ranging from 0.05 to 0.10.

To learn more about Ait1 function, we next measured the impact that deleting Ait1 has on Kog1-YFP localization in strains carrying mutations that block, or promote, TORC1-body formation (Hughes Hallett et al., 2015; Sullivan et al., 2019; Figure 5C). These experiments revealed that deletion of Ait1 completely overrides the severe defects in TORC1-body formation caused by (1) locking Gtr1 in its active, GTP-bound, conformation (GTR1Q65L or Gtr1on for short), (2) deleting the Gtr1 inhibitor Npr2, or (3) deleting the TORC1-binding protein and regulator Pib2 (Figure 5C). However, deletion of Ait1 does not rescue TORC1-body formation in a strain carrying Q to A mutations in the two prion-like domains of Kog1 (Prm1 + 2, Figure 5C). Thus, Ait1 acts at, or below, the level of Gtr1/2 and Pib2 to hold TORC1 in its native position (distributed around the vacuolar membrane) in nutrient replete conditions. This tethering effect is then lost, or overridden, in starvation conditions.

In contrast to its influence on TORC1, Ait1 does not have a dramatic impact on the localization of the TORC1-binding proteins Gtr1/2 and Pib2, as judged by images of Gtr1-YFP and GFP-Pib2 (Figure 5—figure supplement 1). Instead, Pib2 and Gtr1 remain distributed around the vacuolar membrane in the ait1Δ strain, albeit with additional enrichment in foci on the edge of the vacuole (movement that is likely driven by TORC1 agglomeration; Figure 5—figure supplement 1).

Ait1 inhibits TORC1 during amino acid starvation

To test if Ait1 regulates TORC1 signaling, we followed the phosphorylation of a downstream reporter of TORC1 activity, Rps6 (Chen et al., 2018; Yerlikaya et al., 2016), in wild-type and ait1Δ strains. These experiments showed that deletion of Ait1 almost completely blocks TORC1 repression during amino acid starvation (in a standard lab strain, Figure 6); a phenotype similar to that seen in a strain with Gtr1 locked in its active, GTP-bound, state (Figure 6). In contrast, Ait1 does not impact TORC1 inhibition during complete nitrogen starvation (Figure 6—figure supplement 1).

Figure 6 with 1 supplement see all
Impact of Ait1 on target of rapamycin complex I (TORC1) activity during amino acid starvation.

(B) TORC1 activity during complete amino acid starvation (top), and leucine starvation (bottom), in wild-type, ait1Δ, and GTR1Q65L (Gtr1on) strains, as measured by western blot using an anti phospho-Rps6 antibody. (b) Values show the ratio of the p-Rps6 signal divided by the PGK (loading control) signal in each lane, relative to the value for the wild-type strain at time = 0. Wild-type and mutant strains were grown and processed together and run on the same gel.

Previous studies have shown that leucine is the primary amino acid activating TORC1 via Gtr1/2 (Bonfils et al., 2012). We therefore tested if Ait1 is also required for TORC1 inhibition in cells starved for leucine. This was the case; an ait1Δ strain has over 80% TORC1 activity after 6 hr of leucine starvation, as judged by Rps6 phosphorylation (Figure 6), and nearly 100% TORC1 activity as judged by the phosphorylation of the direct TORC1 substrate (Figure 6—figure supplement 1).

Ait1 acts at, or above, the level of Gtr1/2 to regulate TORC1

The observation that Ait1 and Gtr1/2 both regulate TORC1 during amino acid starvation led us to consider two models of Ait1 function: (1) Ait1 acts at, or above, the level of Gtr1/2 to promote TORC1 inhibition, and (2) Ait1 acts downstream of Gtr1/2 to repress TORC1 activity once Gtr1/2 are inactivated. To distinguish between these models, we measured the impact that Ait1 has on TORC1 signaling in strains with: Gtr1 locked in its GDP-bound, inactive, state (GTR1S20L or Gtr1off for short); Gtr2 locked in its GTP-bound, inactive, state (GTR2Q66L or Gtr2off for short); and Gtr1 and Gtr2 both locked in their inactive states (Gtr1off/Gtr2off) (Panchaud et al., 2013). These experiments showed that Ait1 is still important for TORC1 inhibition in a Gtr1off strain, has limited impact on TORC1 inhibition in a Gtr2off strain, and actually helps activate TORC1 in a Gtr1off/Gtr2off strain (Figure 7). Thus, Ait1 promotes TORC1 repression via Gtr1/2 during amino acid starvation, likely by helping to drive Gtr2 into its inactive, GTP-bound, state. In line with this, deletion of Gtr1, Gtr2, or Gtr1/2 completely bypasses the need for Ait1 in amino acid starvation-dependent TORC1 signaling (Figure 7).

Impact of Ait1 on target of rapamycin complex I (TORC1) activity in strains with mutations in Gtr1/2, or the prion domains in Kog1, during amino acid starvation.

(A) TORC1 activity during complete amino acid starvation in mutant strains with (left column) and without Ait1 (right column), measured using a western blot, as described in Figure 6. (B) Values show the ratio of the p-Rps6 signal divided by the PGK (loading control) signal in each lane, relative to the value for the wild-type strain at time = 0. Mutant and double mutant strains were grown and collected together and run on the same gel.

Role of the C3 loop in Ait1-dependent TORC1 inhibition

To gain insight into the mechanism underlying Ait1-dependent regulation of Gtr1/2, we performed sequence alignments to look for similarity between Ait1 and known Gtr1/2- and RagA/C-binding proteins. These alignments uncovered analogous sequences in the N-terminal region of SLC38A9 and the third cytosolic (C3) loop of Ait1 (Figure 8A). The N-terminal region of SLC38A9 has been shown to dissociate from the pore of the SLC38A9 channel in the presence of arginine, and then bind (via residues 39–97; box Figure 8A) to a cleft at the interface between RagA and RagC (Wang et al., 2015; Wyant et al., 2017; Lei et al., 2018; Fromm et al., 2020). This cleft sits near the GTP-binding pockets in RagA and RagC and faces up and away from TORC1 in the RagA/C-TORC1 complex, at a distance ~80 Å away from the vacuolar surface/membrane (Rogala et al., 2019; Fromm et al., 2020). Thus, it seemed likely that Ait1 inhibits Gtr1/2, at least in part, via its 180 aa long, and intrinsically disordered, C3 loop. In line with this model, there are over 50 amino acids either side of the putative Gtr1/2-binding sequence in the C3 loop (box, Figure 8A)—more than enough unstructured peptide for the C3 loop to extend over TORC1 and interact with Gtr1/2.

Figure 8 with 3 supplements see all
Role of the Ait1 C3 loop in target of rapamycin complex I (TORC1) regulation.

(A) BLAST alignment of the SLC38A9 (top) and Ait1 sequences, showing the entire C3 loop (no other sequences in these proteins align). (B, C) TORC1 activity in Ait1 C3 and C4 loop mutants during leucine starvation, measured as described in Figure 6. Values show the ratio of the p-Rps6 signal divided by the PGK (loading control) signal in each lane, relative to the value for the wild-type strain at time = 0. Mutant and wild-type strains were grown and collected together and run on the same gel.

To test If Ait1 regulates Gtr1/2 and TORC1 via its C3 loop, we first built two mutant versions of Ait1; one in which the C3 loop, and the other in which the C4 loop, is replaced by the short, flexible, linker GGSGSGEGSGSGG (ait1Δc3 and ait1Δc4, respectively). Both mutant proteins fold and are trafficked to the vacuolar membrane, as judged by GFP-AitΔC3 and GFP-AitΔC4 localization (Figure 8—figure supplement 1). However, in line with the C3 loop model, only aitΔc3 cells had a defect in TORC1 inhibition during leucine starvation (Figure 8B, C).

Next, to test the function of the central portion of the C3 loop, we created two strains carrying mutations in the region running from Arg 208 to Tyr 231 since it is highly conserved across the yeasts (Figure 8—figure supplement 2): In the first strain (ait1c3v1) we mutated five residues at the center of the 208–231 stretch to alanine (L213A, C216A, I217A, E218A, and R220A; Figure 8C). In the second strain (ait1c3v2), we added two mutations to ait1c3v1 (I211A and I226A; Figure 8C). We also created a control strain (ait1c3v3) that has nine mutations in a poorly conserved portion of the loop (residues 242–250; Figure 8C). All three mutant proteins fold and are transported to the vacuolar membrane as judged by GFP-Ait1 localization (Figure 8—figure supplement 1).

As expected, ait1c3v1 and ait1c3v2 cells had defects in TORC1 inhibition during leucine and amino acid starvation—a phenotype not seen in the ait1c3v3 strain (Figure 8B, C, Figure 8—figure supplement 3). Importantly, however, the signaling defect was larger in ait1c3v2 cells than in ait1c3v1 cells, indicating that Ile 211 and/or Ile 226 play a key role in the Ait1-dependent regulation of TORC1 (Figure 8B, C). Disruption of the C3 loop also triggered the formation of TORC1 bodies. Specifically, the ait1c3v1, ait1c3v2, and aitΔc3 strains all form more TORC1 foci during nitrogen starvation, and/or log-phase growth, than the wild-type strain (Figure 9A, B). Again here, ait1c3v2 cells had a stronger phenotype than ait1c3v1 cells, consistent with the idea that the mutations in Ait1C3v1 only partially disrupt the C3 loop.

Figure 9 with 1 supplement see all
Impact of AIt1 C3 and C4 loop mutations on target of rapamycin complex I (TORC1) localization and Gtr1/2 binding.

(A) Each square on the heat map shows the fraction of cells with a Kog1-YFP focus/body at a specific timepoint (as labeled), calculated by averaging the data from three replicate experiments (>100 cells analyzed at each timepoint and replicate). Individual timepoints have errors ranging from 0.02 to 0.12 (average 0.06). (B) Bar graph showing the fraction of cells with a TORC1 body during log-phase growth in SD medium. (C) Coimmunoprecipitation showing a strong interaction between GFP-Ait1 and Gtr1-myc, but not GFP-Ait1C3v2 and Gtr1-myc. The graph shows the ratio of the Gtr1 and Ait1 signals in the wild-type (black line) and Ait1C3v2 (blue line) strains, before, and 30 min after, amino acid starvation. The Immunoprecipitation data with a full set of controls are shown in Figure 9—figure supplement 1.

Finally, to see if the C3 loop drives an interaction with Gtr1/2, we immunopurified GFP-Ait1 and GFP-AitC3v2 in nonionic detergent (without a crosslinker), in the presence and absence of amino acids, and then measured the amount of Gtr1-myc that remains associated with Ait1. This experiment showed (1) that Ait1 interacts with Gtr1/2 in both log-phase growth and starvation conditions, and (2) that the Gtr1/2-Ait1 interaction is significantly weaker in the ait1c3v2 strain (Figure 9C, Figure 9—figure supplement 1).

Together, these data show that the C3 loop in Ait1 helps hold TORC1 in place around the vacuolar membrane, and drives TORC1 inhibition during amino acid starvation, likely by binding directly to Gtr1/2. We note, however, that the signaling defects seen in the ait1c3v2 and aitΔc3 strains (Figure 8 and Figure 8—figure supplement 3) are smaller than those seen in the ait1Δ strain (Figure 6), suggesting that Ait1 also regulates TORC1 via regions outside of its C3 loop.

Discussion

In this report, we show that: (1) The previously unstudied GPCR-like protein, Ait1, binds directly or indirectly to TORC1-Gtr1/2 and holds TORC1 around the vacuolar membrane during log-phase growth; (2) Ait1 acts through Gtr1/2 (most likely Gtr2) to inhibit TORC1 during amino acid starvation; and (3) Ait1 regulates TORC1, in part, via a central region in its 180 amino acid C3 loop that resembles the Rag A/C (Gtr1/2)-binding domain in SLC38A9.

Putting our results together with the previously published work showing that the GAP SEAC inactivates Gtr1 during nitrogen and amino acid starvation (BarPeled et al., 2013; Panchaud et al., 2013; Neklesa and Davis, 2009; Chen et al., 2017; Laxman et al., 2014; Algret et al., 2014), we propose the following model: First, at the onset of amino acid starvation, SEAC is activated and triggers GTP hydrolysis in Gtr1. The resulting conformational change in Gtr1, and/or signals transmitted through Ait1, then trigger a change in Ait1 structure/activity. Next, Ait1 drives the release of GDP from Gtr2, completing the conversion of Gtr1/2 from the active GTP/GDP-bound state, to the inactive GDP/GTP-bound state. Finally, the resulting conformational change in Gtr1/2, and subsequent movement of the C3 loop in Ait1, exposes a key surface on TORC1 to initiate TORC1-body formation.

As an extension of this model, it may be that strong signals through SEAC, such as those in complete nitrogen starvation (Figure 6—figure supplement 1), override the need for Ait1 due to coupling between the GTP-binding sites in Gtr1 and Gtr2 (Shen et al., 2017).

It is important to point out, however, that while our working model provides a simple explanation for all of the available data (including the observation that deletion/mutation of Ait1 triggers TORC1-body formation), it remains unclear how Ait1 regulates Gtr1/2 and TORC1; Ait1 could also promote TORC1 inhibition by stabilizing the inactive state of Gtr1/2, controlling access to Gtr1/2 activators/repressors (including SEAC), and/or other mechanisms.

The dual role of Ait1 in holding TORC1 around the vacuolar membrane and helping to regulate TORC1 via Gtr1/2 is especially interesting in the context of yeast evolution. Ait1 is found in species throughout the closely related Saccharomycetaceae and Saccharomycodaceae, but not in other clades (Supplementary file 1). The Saccharomycetaceae/codaceae, which include S. cerevisiae, Ashbya gossypii, Kluyveromyces lactis, and the pathogen C. glabrata, split from other yeasts approximately 200 million years ago (Shen et al., 2018), and are unique in that they have highly divergent Rheb, or no Rheb, and have lost TSC2 and/or TSC1 (Figure 10). They are also unique in that many species in these families have prion-like, glutamine-rich, domains in Kog1/Raptor; domains that at least in S. cerevisiae help control the commitment to quiescence (Hughes Hallett et al., 2015). Thus, a common ancestor of the Saccharomycetaceae/codaceae lost functional Rheb and TSC1/2, gained prion-like domains in Kog1, and gained Ait1.

Evolution of the target of rapamycin complex I (TORC1) circuit in yeast.

Species identified as carrying Ait1 in a BLAST search (p < 0.001 cutoff) are marked with a red circle on a previously constructed map of Rheb and TSC1/2 conservation among the budding yeast, taken from Tatebe and Shiozaki, 2017. The light blue circles denote the presence of a highly divergent (nonfunctional) Rheb in species closely related to S. cerevisiae (Tatebe and Shiozaki, 2017). Ait1 was not detected in any of the yeasts outside the Saccharomycetaceae and Saccharomycodaceae.

We argue that these events were almost certainly linked. First, the data presented here show that Ait1 is required to block TORC1 from agglomerating via its prion-like domains during log-phase growth in nutrient replete conditions. That is, without Ait1-dependent anchoring of TORC1, the addition of prion-like domains in TORC1 would have triggered constitutive body formation, rather than add a reversible, regulatory, transition to the TORC1 circuit. Second, our data show that Ait1 has taken over part of the role of ancestral Rheb (Fukuda et al., 2021) by helping to regulate TORC1 in starvation conditions.

Further work is needed to fully dissect the structure and function of the TORC1 circuit in S. cerevisiae, and other simple eukaryotes, and explore the implications of the rewiring we have uncovered. For example, it still remains uncertain how Gtr1/2 regulate TORC1 in the absence of Rheb. However, what is clear from this, and other recent studies, is that while the core of the TORC1 pathway (including TORC1 itself and Gtr1/2) is highly conserved, other aspects of this ancient growth control circuit are plastic and have changed significantly during evolution. Learning more about these changes will not only shed light on the way that eukaryotes have adapted to different niches, it should also open the door to creating drugs that specifically block the growth of fungal pathogens and a variety of parasites.

Materials and methods

Strain construction

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All strains used in this study were generated in haploid (W303) S. cerevisiae, using standard methods (Storici and Resnick, 2006; Storici et al., 2001), and are listed in Supplementary file 3.

Crosslinking and immunopurification

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Yeast carrying Kog1-FLAG, and separately Kog1-HA, were inoculated into 5 ml of synthetic complete medium containing 2% glucose (SD medium) and grown overnight at 30°C in a 20 ml tube, rotating at 40 rpm. The cells were then: (1) diluted to an OD600 of 0.1 in 250 ml of fresh SD medium, and grown shaking at 200 rpm and 30°C in a 1 l flask, until they reached an OD600 of 0.6; (2) captured by filtration, washed with 2 × 100 ml of the appropriate stress or starvation medium, and transferred into 200 ml of synthetic medium lacking all nitrogen (-N), all glucose (-Glu), SD medium containing 0.4 M KCl or 1 mM H2O2, or SD medium at 42°C; (3) grown again for the indicated period of time (Figure 1), shaking at 200 rpm and 30°C (or 42°C for heat stress) in a 1 l flask; (4) harvested by filtration, and rinsed into 2 ml screw-cap tubes using a small volume of ImmunoPrecipitation Lysis Buffer (IPLB; 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.5, 150 mM potassium acetate, 2 mM magnesium acetate, 1 mM ethylene glycol bis(2-aminoethyl)tetraacetic acid (EGTA), and 0.6 M sorbitol) (Murley et al., 2017); and (5) centrifuged for 30 s at 8000 rpm, the supernatant discarded, and the pellet flash frozen, and stored at −80°C.

To lyse the cells, the frozen pellets were resuspended in approximately 600 µl of IPLB buffer containing protease and phosphatase inhibitors (Roche, Indianapolis, IN; 04693159001 and 04906845001; IPLB++), and 1 ml of glass microbeads, and the slurries subjected to 6 × 1 min of vigorous shaking in a Mini-Beadbeater-24 (BioSpec) at 4°C. The tubes were then punctured using a 23-gauge needle and the lysates eluted into 1.5 ml tubes by centrifugation at 3000 rpm at 4°C, for 5 min. The lysates were then homogenized by gentle vortexing, decanted into a fresh 1.5 ml tube, and treated with 0.25 µM of DSP at 4°C for 30 min (with gentle rotation). At this point crosslinking was then quenched by adding 70 µl of 1 M Tris–HCl, pH 7.5, to each tube and holding the extracts on ice for 30 min. Finally, 1% digitonin was added to each tube, and the extracts incubated at 4°C for 1 hr (with gentle rotation), before they were clarified by centrifugation at 12,000 rpm at 4°C, for 10 min, and the supernatant transferred into a fresh tube.

To purify Kog1 and any crosslinked interactors, 50 µl of µMACS anti-FLAG beads (Miltenyi Biotech, 130-101-591) was added to each clarified extract, and the tubes rotated at 4°C for 1.5 hr. The µMACS columns were then prepared by washing them with 200 µl of the lysis buffer supplied with the purification kit, followed by 200 µl of IPLB++ containing 1% digitonin, before the bead/extract mix was loaded into each column (on a magnet) and allowed to flow through by gravity. The beads were then washed in three steps: (1) four times with 200 µl of IPLB++ containing 0.1% digitonin, (2) two times with 400 µl of IPLB++ containing no digitonin, and (3) once with 200 µl of 20 mM Tris–HCl, pH 7.5. Kog1 and any crosslinked proteins were then eluted by incubating each column with 20 µl of the elution buffer supplied with the kit (heated to 95°C), for 5 min, and then adding of 2 × 40 µl of the same elution buffer containing 50 mM DL-Dithiothreitol (DTT;also at 95°C). The pooled eluate from each column was then loaded into a single lane on a 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel and allowed to migrate until it completely entered the gel. The gels were then stained with colloidal blue, destained, and the lane excised for analysis by mass spectrometry.

Identical procedures were used to identify Pib2 and Ait1 interactors, except that in the these experiments the IP was done using GFP-Pib2 or GFP-Ait1 and anti-GFP beads (Miltenyi Biotech, 130-101-125).

Protein identification by mass spectrometry

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Gel slices were washed with water, 50% acetonitrile/50% water, acetonitrile, ammonium bicarbonate (100 mM), and then 50% acetonitrile/50% ammonium bicarbonate (100 mM). The liquid was then removed from each sample, and the gel slices dried in a speed vac. The gel slices were then: (1) Reduced with dithiothreitol (10 mM in 100 mM ammonium bicarbonate) at 56°C for 45 min, and the solution removed and discarded. (2) Alkylated with iodoacetamide (55 mM in 100 mM ammonium bicarbonate) in the dark at ambient temperature for 30 min. (3) Washed with ammonium bicarbonate (100 mM) for 10 min on a shaker, an equal volume of acetonitrile added, and washed for an additional 10 min on a shaker, and then dried in a speed vac for 45 min. (4) Cooled on ice and a treated with a cold solution of 12.5 ng/μl trypsin (Promega, Madison, WI) in ammonium bicarbonate (100 mM). After 45 min, the trypsin solution was removed and discarded, and an equal amount of ammonium bicarbonate (50 mM) was added, and the sample incubated overnight at 37°C with mixing. The samples were then spun down in a microfuge and the supernatants collected. Peptides were further extracted from the gel slices by adding 0.1% trifluoroacetic acid (TFA; enough to cover the gel slices) and mixed at ambient temperature for 30 min. An equal amount of acetonitrile was then added, and the samples were mixed for an additional 30 min. The samples were then spun on a microfuge and the supernatants pooled and concentrated in a speed vac. Finally, all samples were desalted using ZipTip C18 (Millipore, Billerica, MA) and eluted with 70% acetonitrile/0.1% TFA, and concentrated in a speed vac.

For analysis, the peptide samples were brought up in 2% acetonitrile and 0.1% formic acid (10 μl) and analyzed (8 μl) by LC/ESI MS/MS with a Thermo Scientific Easy1000 nLC (Thermo Scientific, Waltham, MA) coupled to a hybrid Orbitrap Fusion (Thermo Scientific, Waltham, MA) mass spectrometer. Inline desalting was accomplished using a reversed-phase trap column (100 μm × 20 mm) packed with Magic C18AQ (5 μm 200 Å resin; Michrom Bioresources, Auburn, CA) followed by peptide separations on a reversed-phase column (75 μm × 250 mm) packed with Magic C18AQ (5 μm 100 Å resin; Michrom Bioresources, Auburn, CA) directly mounted on the electrospray ion source. A 90 min gradient from 2% to 35% acetonitrile in 0.1% formic acid at a flow rate of 300 nl/min was used for chromatographic separations. A spray voltage of 2000 V was applied to the electrospray tip and the Orbitrap Fusion instrument was operated in the data-dependent mode, MS survey scans were in the Orbitrap (AGC target value 500,000, resolution 120,000, and injection time 50 ms) with a 3-s cycle time and MS/MS spectra acquisition were detected in the linear ion trap (AGC target value of 10,000 and injection time 35 ms) using HCD activation with a normalized collision energy of 27%. Selected ions were dynamically excluded for 45 s after a repeat count of 1.

Data analysis was performed using Proteome Discoverer 2.2 (Thermo Scientific, San Jose, CA). The data were searched against an SGD yeast database that included common contaminants. Searches were performed with settings for the proteolytic enzyme trypsin. Maximum missed cleavages were set to 2. The precursor ion tolerance was set to 10 ppm and the fragment ion tolerance was set to 0.6 Da. Variable modifications included oxidation on methionine (+15.995 Da) and carbamidomethyl (57.021). Sequest HT was used for database searching. All search results were run through Percolator for scoring.

Fluorescence microscopy

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TORC1-body formation was measured as described previously (Hughes Hallett et al., 2015; Sullivan et al., 2019). Briefly, stains carrying Kog1-YFP were patched from their glycerol stocks onto fresh YEPD plates and grown overnight at 30°C. The patches were then used to inoculate 5 ml of SD medium, and the tubes grown at 30°C in a 20 ml tube, rotating at 40 rpm, until they reached an OD600 of 0.1. These starter cultures were then used to inoculate 20 ml of SD medium in a 150-ml Erlenmeyer flask (to an OD600 below 0.01) and grown at 30°C and shaking at 200 rpm, until they reached an and OD600 between 0.5 and 0.7. 300 µl of each culture was then pipetted into one chamber in an 8-well microslide (Ibidi, 80826) that had been pretreated with concanavalin A. The chambers were then washed three times with SD -nitrogen, and images acquired using a Nikon Eclipse Ti-E microscope equipped with a ×100 objective, a Photometrics Prime 95B camera, and λEX 510/25 and λEM 540/21 filters, every 10 min for an hour. Each image consisted of a z-stack of sixteen 200 ms images, spaced 0.4 µm apart, to ensure that the bodies in all planes were detected, and was compressed into a maximum projection stack in ImageJ for analysis.

Imaging of GFP-Ait1, Gtr1-YFP, and GFP-Pib2 was done in an identical manner except that GFP images were acquired λEX 470 and λEM 515/30 filters.

Rps6 phosphorylation assays

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Cultures were grown in conical flasks shaking at 200 rpm and 30°C until mid-log phase (OD600 between 0.55 and 0.6). At this point, a 47 ml sample was collected, mixed with 3 ml 100% trichloroacetic acid (TCA), and held on ice for at least 30 min (and up to 6 hr). The remaining culture was then collected by filtration, and transferred to SD -N, SD -aa, or SD -leu medium after two washes with 100 ml of the same medium, and further samples collected in TCA, as described above. The samples were then centrifuged at 4000 rpm for 5 min at 4°C, washed twice with 4°C water, twice with acetone, and disrupted by sonication at 15% amplitude for 5 s before centrifugation at 8000 rpm for 30 s. The cell pellets were then dried in a speedvac for 10 min at room temperature, and frozen until required at −80°C.

Protein extraction was performed by bead beating (6 × 1 min, full speed) in urea buffer (6 M urea, 50 mM Tris–HCl pH 7.5, 5 mM Ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM NaF, 5 mM NaN3, 5 mM NaH2PO4, 5 mM p-nitrophenylphosphate, 5 mM β-glycerophosphate, and 1% SDS) supplemented with complete protease and phosphatase inhibitor tablets (Roche, Indianapolis, IN; 04693159001 and 04906845001). The lysate was then harvested by centrifugation for 5 min at 3000 rpm, resuspended into a homogenous slurry, and heated at 65°C for 10 min. The soluble proteins were then separated from insoluble cell debris by centrifugation at 12,000 rpm for 10 min, and the lysate stored at −80°C until required.

For protein phosphorylation analysis, the protein extracts were run on a 12% acrylamide gel and transferred to a nitrocellulose membrane. Western blotting was then carried out using anti-pRPS6 antibody (Cell Signaling, 4858) at a 1/2500 dilution, and anti-PGK1 antibody (Invitrogen, 459250) at a 1/10,000 dilution, and anti-mouse and anti-rabbit secondaries, labeled with a IRDye 700CW and IRDye 800CW (LiCor), both at a 1/10,000 dilution, and the blots scanned using a LiCor Odyssey Scanner (LiCor, Lincoln, NE). Band intensities were quantified using the LiCor Image Studio Software.

Sch9 bandshift experiments

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Sch9 bandshift measurements were performed as described previously (Urban et al., 2007; Hughes Hallett et al., 2014), and using the same procedure listed above for the Rps6 Western, except that lysates were subjected to cleavage by 2-nitro-5-thiocyanatobenzoic acid (NTCB) for 12–16 hr at room temperature (1 mM NTCB and 100 mM N-Cyclohexyl-2-aminoethanesulfonic acid (CHES, pH 10.5)) prior to analysis, and the Western was done using an anti-HA (12CA5) antibody.

Coimmunoprecipitation experiments

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Figure 3—figure supplement 1. Kog1-FLAG was immunopurified, as described above, the eluate run on a 9% SDS–polyacrylamide gel and transferred to a nitrocellulose membrane. Western blotting was then carried out using a rabbit Anti-GFP polyclonal antibody (enQuirebio, Cat# QAB10298) at a 1:2500 dilution, a mouse Anti-FLAG monoclonal antibody (Sigma Cat# F1804) at a 1:1000 dilution, and the same secondary antibodies used in the Rps6 assay. A sample of each extract (collected prior to immunopurification) was also run on a separate 12% gel and probed with anti-PGK1 antibody (Invitrogen, 459250) at a 1/10,000 dilution, as described in the Rps6 assay. Ait1 levels were monitored in the same cell lines and conditions, but after immunopurification (following the protocol described below).

Figure 9 and Figure 9—figure supplement 1. GFP-Ait1 was immunopurified as described above for Kog1-Flag, but with a few modifications: (1) no crosslinker was used; (2) the beads were washed three times with 200 µl of IPLB++ containing 0.1% digitonin, and once with 400 µl of IPLB++ containing no digitonin; (3) Anti-GFP MicroBeads (Miltenyi Biotec Cat# 130-091-125) were used instead of Anti-FLAG beads. Once the immunopurification was complete, the eluted material was run on a 12% SDS–polyacrylamide gel and transferred to a nitrocellulose membrane. Western blotting was then carried out using a rabbit Anti-GFP polyclonal (enQuirebio, Cat# QAB10298) at a 1:2500 dilution, a mouse Anti-Myc (9E10) (Roche Cat# 1257900), and the same secondary antibodies listed in the Rps6 assay. Gtr1-myc was immunoprecipitated using an anti-Myc antibody (Roche cat# 1257900) and protein A/G beads (Santa Cruz cat# sc-2003).

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

References

Decision letter

  1. Roger J Davis
    Reviewing Editor; University of Massachusetts Medical School, United States
  2. Anna Akhmanova
    Senior Editor; Utrecht University, Netherlands

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

Thank you for submitting your article "Ait1 regulates TORC1 signaling and localization in budding yeast" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Philip Cole as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

This study uses a proteomics approach to identify proteins that interact directly or indirectly with the Torc1 component Kog1 and microscopy to determine whether these proteins influence the formation of TORC1 bodies, which have been established previously to form on vacuoles in budding yeast upon nitrogen starvation. The authors focus on an interesting new GPCR-like protein identified that they call Ait1, which when deleted results in constitutive formation of TORC1 bodies concomitant with resistance of TORC1 to suppression upon amino acid starvation. Genetic approaches suggest that Ait1 acts upstream of the Gtr1/Gtr2 proteins, known to directly engage TORC1 at the vacuole, for TORC1 suppression and that this suppression might involve sequences within a cytosolic loop of Ait1. The data are clear that Ait1 represents an important new component of TORC1 regulation by amino acids in S. cerevisiae that is not present in most eukaryotic species. The authors make rather specific conclusions regarding the function of Ait1 in the regulation of Gtr1/2 and TORC1. However, its molecular function(s) is incompletely characterized in the study, leaving the conclusions supported by ambiguous data, with several fundamental questions unanswered regarding its placement in the known regulatory network upstream of yeast TORC1.

Essential revisions:

Revisions required to address the stated conclusions:

1) At the heart of the authors' conclusions on the function of Ait1 is its genetic placement amongst core regulators of TORC1, especially the highly conserved Gtr1/2 proteins. Based on Figure 7, the authors conclude that Ait1 acts upstream of Gtr1/2 for TORC1 regulation (Gtr1/2 are genetically epistatic to Ait1). However, the data in Figure 5 clearly show that the ability of the Gtr1-GTP (GTR1on) and npr2-δ mutants to inhibit TORC1-body formation during nitrogen starvation is dependent on Ait1, thereby placing Ait1 downstream of Gtr1/2 for this regulation.

2) Page 5-6. The authors suggest that there is a direct interaction between Ait1 and TORC1, citing the evidence that they mutually pull down each other in IP-MS experiments. The following experiments are required to support this conclusion: a) Identify residues or mapping critical domain/motif(s) on Ait1 that required for interaction with TORC1; b) Native gel or gel-filtration analysis to show the co-migration of Ait1 and TORC1; c) in vitro binding assay with purified proteins is preferred and thus recommended; d) Co-IP experiment to show the interaction between Ait1 and TORC1 is regulated by nitrogen/amino acid availability.

3). Figure 8C describes a key experiment suggesting that the ait1C3v1 mutant is a potential separation-of-function mutant, defective in suppressing TORC1 upon leucine starvation but maintaining its ability to prevent TORC1 body formation. However, this effect is never properly examined under nutrient deprivation time courses. Kog1-YFP foci formation over a time course of nitrogen, leucine, and amino acid starvation in this mutant versus wild-type and ait1-δ should be quantified to better demonstrate the effects of this mutant on these two putative functions of Ait1. Effects on pRps6 under nitrogen and amino acid starvation should also be shown.

4) Page 8: The authors suggest that Ait1 and SLC38A9 share sequence homology so they likely bind to Rag/Gtr in a similar way. The following experiments are needed to support this claim: a) A critical motif in SLC38A9 that mediates the binding with the Rags is an α helix (H60-Y71). In the sequence alignment of Figure 8A, the corresponding region on Ait1 contains no feature of an α helix because multiple serine residues will likely introduce flexibility to prevent α helix formation. The authors show provide stronger evidence that this loop directly contacts the Rags; b) Based on the cryo-EM structure of SLC38A9-Rag, the triple mutant that the authors generated (Ait1c3v1) is not appropriate. None of the three residues directly contact the Rags in the structure, so its defect is difficult to explain; c) What is the molecular mechanism of Ait1 function? Does it change the GTP/GDP state of Gtr? Is it a GEF, GDI, or GAP?

5) What signal does Ait1 respond to? Does it bind amino acids? Does Ait1 mediate the effects of leucine or other amino acids?

In the possible absence of the requested studies, the conclusions presented should be modified to include only those that are directly supported by experiment.

Reviewer #1:

This is an interesting study that identifies a novel component of the amino acid signaling pathway that regulates TORC1 activity in yeast. It is proposed that the 3rd cytoplasmic loop of GPCR Ait1 recruits TORC1 to the lysosomal surface in response to amino acids by a mechanism analogous to SLC38A9 in humans. The importance of this study is that it highlights the rewiring of the TORC1 signaling pathway in yeast compared to humans.

The major strength of this study is the rigor of the mass spec analysis used to identify novel TORC1 signaling components coupled with yeast genetic analysis. This leads to high confidence that the conclusions of this study are correct. However, there are a number of key issues that are not addressed by the authors, including the direct demonstration of a role for amino acids in Ait1 function. The study will be appropriate for publication in eLife after these issues have been addressed in a revised manuscript.

Reviewer #2:

This paper identified a potentially key mediator of nitrogen/amino acids signals in yeast TORC1 pathway. If confirmed, these results would significantly advance our understanding of the TORC1 pathway in yeast and its evolutionary path.

Strength: The authors performed comprehensive IP-MS experiments to probe the interactome of TORC1 in budding yeast, and the resulting datasets are of great interest to researchers in the field of mTOR.

Weakness: The molecular mechanism proposed here is not sufficiently supported by the experiments.

Specifically, the authors propose that there is a direct interaction between Ait1 and TORC1, citing the evidence that they mutually pull down each other in IP-MS experiments. However, additional support for this claim would be needed, such as the identification of residues or mapping of critical domain/motif(s) on Ait1 that physically touch TORC1, native gel or gel-filtration analysis to show the co-migration of Ait1 and TORC1, in vitro binding assays with purified proteins and co-IP experiment to show the interaction between Ait1 and TORC1 is regulated by nitrogen/amino acid availability.

Furthermore, the authors propose that Ait1 and SLC38A9 share sequence homology and therefore they likely bind to Rag/Gtr in a similar way. Additional support for this claim would be needed. For example, it is known that a critical motif in SLC38A9 that mediates the binding with the Rags is an α helix (H60-Y71). In the sequence alignment of Figure 8A, the corresponding region on Ait1 contains no feature of an α helix because multiple serine residues will likely introduce flexibility to prevent α helix formation. The authors would need to provide stronger evidence that this loop directly contacts the Rags. Based on the cryo-EM structure of SLC38A9-Rag, the triple mutant that the authors generated (Ait1c3v1) does not seem appropriate, because none of the three residues directly contact the Rags in the structure, so its defect is difficult to explain.

Reviewer #3:

This study uses a proteomics approach to identify proteins that interact directly or indirectly with the Torc1 component Kog1 and microscopy to determine whether these proteins influence the formation of TORC1 bodies, which have been established previously to form on vacuoles in budding yeast upon nitrogen starvation. The authors focus on an interesting new GPCR-like protein identified that they call Ait1, which when deleted results in constitutive formation of TORC1 bodies concomitant with resistance of TORC1 to suppression upon amino acid starvation. Genetic approaches suggest that Ait1 acts upstream of the Gtr1/Gtr2 proteins, known to directly engage TORC1 at the vacuole, for TORC1 suppression and that this suppression might involve sequences within a cytosolic loop of Ait1.

The data are clear that Ait1 represents an important new component of TORC1 regulation by amino acids in S. cerevisiae that is not present in most eukaryotic species. The authors make rather specific conclusions regarding the function of Ait1 in the regulation of Gtr1/2 and TORC1. However, its molecular function(s) is incompletely characterized in the study, leaving the conclusions supported by ambiguous data, with several fundamental questions unanswered regarding its placement in the known regulatory network upstream of yeast TORC1.

At the heart of the authors' conclusions on the function of Ait1 is its genetic placement amongst core regulators of TORC1, especially the highly conserved Gtr1/2 proteins. Based on Figure 7, the authors conclude that Ait1 acts upstream of Gtr1/2 for TORC1 regulation (Gtr1/2 are genetically epistatic to Ait1). However, the data in Figure 5 clearly show that the ability of the Gtr1-GTP (GTR1on) and npr2-δ mutants to inhibit TORC1-body formation during nitrogen starvation is dependent on Ait1, thereby placing Ait1 downstream of Gtr1/2 for this regulation.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Ait1 regulates TORC1 signaling and localization in budding yeast" for further consideration by eLife. Your revised article has been evaluated by Anna Akhmanova (Senior Editor), the Reviewing Editor, and two expert reviewers.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1) Controls for the co-ip study presented in Figure 3S1 are required, as noted by reviewer 32.

2) The function of Ait1 remains unclear, including the position of this protein in the amino acid sensing pathway, as noted by reviewer #2. The authors should provide a concise summary of their conclusions that is consistent with the data presented that can be used to generate testable hypotheses for future studies.

Reviewer #1 (Recommendations for the authors):

The revised manuscript has been improved by removing some of the previously included analysis that was weakly supported by the data presented. The manuscript now describes a better study. However, the biochemical function of Ait1 remains unclear. Nevertheless, the publication of this study will be useful to the field because of the genetic analysis presented.

Reviewer #2 (Recommendations for the authors):

In the revised manuscript, Wallace et al. partially addressed some of my previous concerns. However, several new questions arose from this revision.

1. In Figure 3—figure supplement 1, the authors performed a co-IP experiment between Kog and Ait1 in the presence of a crosslinker. First, the authors should show an input panel for Kog and Ait1, as well as a loading control. Second, why does Kog1 show up in three to five bands?

2. The authors claimed that "Ait1 acts at, or above, the level of Gtr1/2 to regulate TORC1", while at the same time, "Ait1 acts at, or below, the level of Gtr1/2 and Pib2 to hold TORC1 in its native position". While some experimental evidence is provided, I am very confused by two claims here. What is the sequence of events upon amino acid supplementation and amino acid deprivation?

https://doi.org/10.7554/eLife.68773.sa1

Author response

Essential revisions:

Revisions required to address the stated conclusions:

1) At the heart of the authors' conclusions on the function of Ait1 is its genetic placement amongst core regulators of TORC1, especially the highly conserved Gtr1/2 proteins. Based on Figure 7, the authors conclude that Ait1 acts upstream of Gtr1/2 for TORC1 regulation (Gtr1/2 are genetically epistatic to Ait1). However, the data in Figure 5 clearly show that the ability of the Gtr1-GTP (GTR1on) and npr2-δ mutants to inhibit TORC1-body formation during nitrogen starvation is dependent on Ait1, thereby placing Ait1 downstream of Gtr1/2 for this regulation.

We are sorry that this part of the paper was unclear and believe the confusion stems from the fact that the genetics in Figure 7 shows that Ait1 acts at or above the level of Gtr1/2 and the data in Figure 5 shows that Ait1 acts at or below the level of Gtr1/2, not simply above Gtr1/2 in Figure 7 and below Gtr1/2 in Figure 5 (we should have used more precise language and have fixed this in the revised manuscript).

To be specific, in Figure 5 we show that deletion of Ait1 leads to the formation of TORC1 foci/bodies, even in nutrient replete conditions. Deletion of Ait1 also completely overrides the severe body formation defects found in npr2, Gtr1on and Pib2 deletion mutants. This places Ait1 as acting at, or below, the level of Gtr1/2 and Pib2 in TORC1 body formation. In contrast, Ait1 does not override the body formation defect seen in a strain carrying mutations in the prion domains of Kog1, indicating that Ait1 acts upstream of the TORC1 agglomeration process itself.

Given that Ait1 interacts with TORC1 (figures 1-3) and Gtr1/2 (new figure 9) it is very likely that Ait1 limits TORC1 body formation by acting at the level of TORC1-Gtr1/2. The simplest model is that Ait1 binds to TORC1-Gtr1/2 holding it in place around the vacuolar membrane, and/or by shielding regions of the complex that ultimately drive body formation (such as the prion domains). Once you disrupt the tethering/shielding interaction between Ait1 and TORC1-Gtr1/2, the regulatory events that promote TORC1 body formation during starvation (such as inactivation of Gtr1/2 and TORC1 binding to Pib2) become superfluous.

In Figures 6 and 7 we show that Ait1 is required for TORC1 inactivation in amino acid and leucine starvation in a wild-type strain, but not in strains where Gtr1/2 are locked in the Gtr1-GDP and Gtr2-GTP bound off states or with Gtr1/2 deleted. This places Ait1 as acting at, or above, the level of Gtr1/2 in TORC1 regulation. Again here, given that Ait1 interacts with TORC1-Gtr1/2 and regulates/binds to the complex via a loop that resembles the RagA/C binding domain of SLC38A9, it almost certain that Ait1 regulates Gtr1/2-TORC1 at the level of Gtr1/2.

Thus, all the genetic (and interaction) data fits with a model where Ait1 (i) is bound to TORC1-Gtr1/2 and acts to block its movement into TORC1 bodies until the cell experiences starvation and (ii) helps inactivate TORC1 via Gtr1/2 when cells run short of amino acids. That is, Ait1 acts at the level of TORC1-Gtr1/2 to regulate TORC1 body formation and TORC1 activity.

To clarify this, we have altered the text around Figures 5 and 7 to point out that the data shows that Ait1 acts at or below the level of Gtr1/2 to regulate TORC1 body formation, and at or above the level of Gtr1/2 to regulate TORC1 activity.

2) Page 5-6. The authors suggest that there is a direct interaction between Ait1 and TORC1, citing the evidence that they mutually pull down each other in IP-MS experiments. The following experiments are required to support this conclusion: a) Identify residues or mapping critical domain/motif(s) on Ait1 that required for interaction with TORC1; b) Native gel or gel-filtration analysis to show the co-migration of Ait1 and TORC1; c) in vitro binding assay with purified proteins is preferred and thus recommended; d) Co-IP experiment to show the interaction between Ait1 and TORC1 is regulated by nitrogen/amino acid availability.

We have added Co-IP experiments examining the interaction between Ait1 and TORC1 (Kog1) in detail. These experiments show (i) that the interaction between TORC1 and Ait1 is maintained in starvation conditions (Figure 3s) and (ii) that the interaction between TORC1 and Ait1 can only be detected in extracts treated with cross-linkers (legend Figure 3s)—suggesting the off rate is relatively fast (although this may be caused by the partial degradation of the very large protein Kog1 in extracts seen by us and others). Importantly, however, we have now shown that Ait1 interacts with Gtr1/2 in an extract, even in the absence of a crosslinker (Figure 9 and Figure 9 supplement 1). The Ait1-Gtr1/2 interaction is also constitutive but disrupted by mutations in the C3 loop of Ait1 (Figure 9). More specifically, we see that mutations in the tip of the C3 loop weaken the interaction between Ait1 and Gtr1/2 during log growth conditions, and almost completely block the interaction between Ait1 and Gtr1/2 in amino acid starvation conditions. These data (and signaling data discussed below) strengthen the argument that the interaction between Ait1 and TORC1 (or more accurately the TORC1-Gtr1/2 complex) is close/direct. They also indicate that the interaction between Gtr1/2 and Ait1 involves both the C3 loop and other regions of Ait1.

We also agree that in vitro binding assays are an important next step. However, at this stage we have overwhelming evidence that Ait1 is an important new regulator of Gtr1/2-TORC1 that acts via a very close if not direct interaction with TORC1-Gtr1/2 and have provided clear insight into of Ait1 function. Therefore, given that experiments examining Ait1-Gtr1/2, Ait1-TORC1 and Ait1-TORC1-Gtr1/2 interactions are almost going to be complex since there are multiple proteins and domains involved, the interactions normally occur on a membrane, and Ait1 (and fragments of Ait1) are proving difficult to overexpress and purify, we believe that the work is best left to follow up papers.

3). Figure 8C describes a key experiment suggesting that the ait1C3v1 mutant is a potential separation-of-function mutant, defective in suppressing TORC1 upon leucine starvation but maintaining its ability to prevent TORC1 body formation. However, this effect is never properly examined under nutrient deprivation time courses. Kog1-YFP foci formation over a time course of nitrogen, leucine, and amino acid starvation in this mutant versus wild-type and ait1-δ should be quantified to better demonstrate the effects of this mutant on these two putative functions of Ait1. Effects on pRps6 under nitrogen and amino acid starvation should also be shown.

We have now completely characterized the C3 mutants presented in the paper (Figures 8, 9 and associated supplements). This includes full timecourses of body formation for all C3/C4 mutants (in triplicate), TORC1 activity timecourses in leucine starvation for all C3/C4 mutants, Ait1 localization data for all C3 mutants, and analysis of the impact that the most important C3 mutant (c3v2) has in leucine, complete amino acid, and nitrogen starvation, as well as on the interaction between Gtr1/2 and TORC1.

4) Page 8: The authors suggest that Ait1 and SLC38A9 share sequence homology so they likely bind to Rag/Gtr in a similar way. The following experiments are needed to support this claim: a) A critical motif in SLC38A9 that mediates the binding with the Rags is an α helix (H60-Y71). In the sequence alignment of Figure 8A, the corresponding region on Ait1 contains no feature of an α helix because multiple serine residues will likely introduce flexibility to prevent α helix formation. The authors show provide stronger evidence that this loop directly contacts the Rags; b) Based on the cryo-EM structure of SLC38A9-Rag, the triple mutant that the authors generated (Ait1c3v1) is not appropriate. None of the three residues directly contact the Rags in the structure, so its defect is difficult to explain; c) What is the molecular mechanism of Ait1 function? Does it change the GTP/GDP state of Gtr? Is it a GEF, GDI, or GAP?

These comments were prescient. As described above, the C3v1 mutant presented in the original paper turned out to have a frame shift mutation in its last helix that we missed in the initial round of sequencing. When we remade the mutant (correctly) it had no impact on TORC1 signaling. We therefore went back and looked at the sequences of SLC38A9, Ait1, RagA/C and Gtr1/2, and the RagAC-SLC38A9 structure in more detail. We also examined the conservation of the C3 loop sequence across yeast species. This showed that the residues in the N-terminus of SLC38A9 that align with a highly conserved region of Ait1 around the CIEV sequence (centered at 216-220 in Ait1) make clear contacts with RagC at residues that are conserved in Gtr2 (on a strand that helps form the GTP binding pocket). Other regions are less well conserved. We therefore created three new loop variants, the first with five 5 mutations in the CIEV region, the second with 7 mutations in the CIEV region, and the third introducing nine mutations into a poorly conserved region of the loop. As expected, the first two mutants both had defects in TORC1 regulation (Figure 8). We also showed that these mutations disrupt the interaction between Ait1 and Gtr1/2 (Figure 9).

5) What signal does Ait1 respond to? Does it bind amino acids? Does Ait1 mediate the effects of leucine or other amino acids?

These are very important questions, but we believe best suited for a follow up study. Amino acid metabolism in yeast is highly interconnected and thus strong conclusions about the signals Ait1 responds to have to rely (at least in part) on biochemical studies. It is likely that Ait1 directly binds to leucine, and/or other amino acids, but at least some of this probably occurs via the membrane spanning domains of this GPCR-like protein and thus we have to get this protein correctly overexpressed and folded in a membrane. We will need to identify the binding site (sites), so they are specific since they likely have low affinity for amino acids (which can accumulate up to 1mM concentration in a vacuole) mutate them, and then carry out studies of these mutants in vivo. We are working on this, but due to the complexity of the interactions and system it is going to take a lot of time.

In the possible absence of the requested studies, the conclusions presented should be modified to include only those that are directly supported by experiment.

We have made additional edits to ensure that it is clear that, while we know that Ait1 interacts with TORC1-Gtr1/2 and regulates the complex, we have not proven that this is a direct interaction (and now emphasize binding to TORC1-Gtr1/2 rather than just TORC1 due to the data outlined above). The most important edit is the first sentence of the discussion “In this report, we show that: (i) The previously unstudied GPCR-like protein, Ait1, binds directly or indirectly to TORC1-Gtr1/2 and holds TORC1 around the vacuolar membrane during log-phase growth …” Moreover, in the discussion we start by listing conclusions that are worded to ensure they are directly supported by data. We then present an overall model of Ait1 function that is not fully proven yet (although strongly supported by the data) but then explicitly state that other models can still hold.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Reviewer #2 (Recommendations for the authors):

In the revised manuscript, Wallace et al. partially addressed some of my previous concerns. However, several new questions arose from this revision.

1. In Figure 3—figure supplement 1, the authors performed a co-IP experiment between Kog and Ait1 in the presence of a crosslinker. First, the authors should show an input panel for Kog and Ait1, as well as a loading control.

This is a good point, we should have included these controls in the original figure and have now added both a PGK loading control and an Ait1 input panel to Figure 3—figure supplement 1a and b. Note that Ait1 is difficult/impossible to detect in an extract and so the Ait1 input controls are from GFP-Ait1 IPs (carried out in the appropriate cells/conditions) as described in the Methods section. Kog1 is also very difficult to detect in an extract and so the IP material serves as the input control.

Second, why does Kog1 show up in three to five bands?

The 3-5 bands observed are Kog1 (top band) and its degradation products (lower bands). Kog1 is a large (approx. 180kDa) protein with multiple disordered loops and is therefore very susceptible to degradation. We have seen similar patterns in the numerous Kog1-IPs we have carried out over the years and, despite our best efforts, have been unable to block its degradation. For example, as shown in panel c in Figure 3—figure supplement 1, Kog1 is partially degraded even when we increase the concentration of protease inhibitor cocktail to 4x that used in our other experiments. We have made this clear in the figure legend in the line: “Note (i) that Kog1 partially degrades during the IP and thus shows up as multiple bands…”

2. The authors claimed that "Ait1 acts at, or above, the level of Gtr1/2 to regulate TORC1", while at the same time, "Ait1 acts at, or below, the level of Gtr1/2 and Pib2 to hold TORC1 in its native position". While some experimental evidence is provided, I am very confused by two claims here. What is the sequence of events upon amino acid supplementation and amino acid deprivation?

We thank the reviewer for this feedback. Indeed, while we outlined the steps involved in TORC1 inhibition in the discussion we did not integrate the Ait1 dependent regulation of TORC1 localization (TORC1-body formation) into our model. We have therefore edited the section to describe all the steps in TORC1 inhibition/localization. The new section reads:

“Putting our results together with the previously published work showing that the GAP SEAC inactivates Gtr1 during nitrogen and amino acid starvation28,42-46, we propose the following model:

First, at the onset of amino acid starvation, SEAC is activated and triggers GTP hydrolysis in Gtr1. The resulting conformational change in Gtr1, and/or signals transmitted through Ait1, then trigger a change in Ait1 structure/activity. Next, Ait1 drives the release of GDP from Gtr2, completing the conversion of Gtr1/2 from the active GTP/GDP-bound state, to the inactive GDP/GTP-bound state. Finally, the resulting conformational change in Gtr1/2, and subsequent movement of the C3 loop in Ait1, exposes a key surface on TORC1 to initiate TORC1-body formation.

As an extension of this model, it may be that strong signals through SEAC, such as those in complete nitrogen starvation (Figure 6—figure supplement 1), override the need for Ait1 due to coupling between the GTP binding sites in Gtr1 and Gtr267.

It is important to point out, however, that while our working model provides a simple explanation for all of the available data (including the observation that deletion/mutation of Ait1 triggers TORC1-body formation), it remains unclear how Ait1 regulates Gtr1/2 and TORC1; Ait1 could also promote TORC1 inhibition by stabilizing the inactive state of Gtr1/2, controlling access to Gtr1/2 activators/repressors (including SEAC), and/or other mechanisms.”

To clarify further, we know (i) that Ait1 binds Gtr1/2, and forms a close interaction with TORC1, in both log growth and starvation conditions and (ii) that deletion or mutation of Ait1 causes TORC1 to move into bodies or aggregates (an event that normally only occurs during starvation) in nutrient rich medium, and blocks TORC1 inhibition during amino acid starvation. Thus, the most parsimonious model holds that once SEAC and Ait1 turn Gtr1/2 off, the resulting conformational change in Gtr1/2 moves the C3 loop in Ait1 to expose a surface of TORC1 that promotes aggregation/body formation. In this model, deletion of Ait1, or mutation of the C3 loop, short circuits the system by constitutively exposing the aggregation domains of TORC1. Following on from the reviewer’s question, this places Ait1 at or below the level of Gtr1/2 (as we see in Figure 5c) since even if you lock Gtr1/2 in their active forms, deleting Ait1 will still remove the C3 loop and expose the TORC1 surfaces that promote aggregation. Our model also places Ait1 as acting at, or above, the level of, Gtr1/2 in the regulation of TORC1 kinase activity (as shown in Figure 7) since locking Gtr1/2 in an off state, or deleting Gtr1 and or 2, bypasses the need for Ait1 in driving TORC1 repression.

It is also important to note that Reviewer 2 specifically asks us to outline the steps that occur in both amino acid supplementation and depletion. However, we only present a model outlining the steps that occur during amino acid depletion since (i) that is what we measure/study in this paper and (ii) the De Virgilio lab has established that TORC1 activation (in amino supplementation) occurs via a distinct mechanism/pathway where the GAP for Gtr2 (Lst4/7) binds the vacuole to initiate reactivation of the Gtr1/2 complex (Pelli-Gulli, 2015 and 2017) and is then driven off the vacuolar membrane once TORC1 is active. In other words, TORC1 inhibition is not simply the reverse of TORC1 activation. In line with this, we recently discovered that the C3 loop of Ait1 is heavily phosphorylated during long term starvation and that this limits Ait1 activity. Thus, further experiments are required to resolve the role of Ait1 in TORC1 reactivation (which appear to be very limited).

https://doi.org/10.7554/eLife.68773.sa2

Article and author information

Author details

  1. Ryan L Wallace

    Department of Molecular and Cellular Biology, University of Arizona, Tucson, United States
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Eric Lu
    Competing interests
    No competing interests declared
  2. Eric Lu

    Department of Molecular and Cellular Biology, University of Arizona, Tucson, United States
    Contribution
    Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Ryan L Wallace
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3144-3563
  3. Xiangxia Luo

    Department of Molecular and Cellular Biology, University of Arizona, Tucson, United States
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Andrew P Capaldi

    Department of Molecular and Cellular Biology, University of Arizona, Tucson, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    capaldi@email.arizona.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7902-2477

Funding

National Institute of General Medical Sciences (R01GM097329)

  • Andrew P Capaldi

National Institute of General Medical Sciences (T32GM136536)

  • Ryan L Wallace
  • Andrew P Capaldi

University of Arizona

  • Andrew P Capaldi

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Claudio De Virgilio for sharing GTR1 and 2 mutant plasmids, and Kyle Cunningham for sharing the GFP-Pib2 plasmid, used to make mutant strains. We also thank Phil Gafken and Lisa Jones of the Fred Hutchinson Cancer Research Center’s Proteomics Facility, and Paul Langlais at the University of Arizona for carrying out the peptide mapping experiments. This work was supported by the National Institutes of Health (NIH) grants R01GM097329 and T32GM136536.

Senior Editor

  1. Anna Akhmanova, Utrecht University, Netherlands

Reviewing Editor

  1. Roger J Davis, University of Massachusetts Medical School, United States

Publication history

  1. Received: March 25, 2021
  2. Accepted: August 31, 2022
  3. Accepted Manuscript published: September 1, 2022 (version 1)
  4. Version of Record published: September 22, 2022 (version 2)

Copyright

© 2022, Wallace, Lu et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Ryan L Wallace
  2. Eric Lu
  3. Xiangxia Luo
  4. Andrew P Capaldi
(2022)
Ait1 regulates TORC1 signaling and localization in budding yeast
eLife 11:e68773.
https://doi.org/10.7554/eLife.68773

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