RESEARCH ARTICLE

Pib2 and the EGO complex are both required for activation ofTORC1Natalia V. Varlakhanova1, Michael J. Mihalevic2, Kara A. Bernstein2 and Marijn G. J. Ford1,*

ABSTRACTThe TORC1 complex is a key regulator of cell growth and metabolisminSaccharomyces cerevisiae. The vacuole-associated EGO complexcouples activation of TORC1 to the availability of amino acids,specifically glutamine and leucine. The EGO complex is alsoessential for reactivation of TORC1 following rapamycin-inducedgrowth arrest and for its distribution on the vacuolar membrane. Pib2,a FYVE-containing phosphatidylinositol 3-phosphate (PI3P)-bindingprotein, is a newly discovered and poorly characterized activator ofTORC1. Here, we show that Pib2 is required for reactivation ofTORC1 following rapamycin-induced growth arrest. Pib2 is requiredfor EGO complex-mediated activation of TORC1 by glutamine andleucine as well as for redistribution of Tor1 on the vacuolarmembrane. Therefore, Pib2 and the EGO complex cooperate toactivate TORC1 and connect phosphoinositide 3-kinase (PI3K)signaling and TORC1 activity.

KEY WORDS: EGO complex, Gtr1, Gtr2, Pib2, TORC1

INTRODUCTIONThe target of rapamycin complex I (TORC1) couples multiplenutritional cues to orchestrate an appropriate cellular growthresponse. Nutrients, in particular amino acids, activate TORC1signaling, which results in a multi-pronged anabolic response,including ribosome and protein synthesis, increase of biomass andgrowth. On nutrient starvation, TORC1 is inactivated, which leadsto a coordinated starvation response, including amino acid permeasesynthesis and transport, amino acid biosynthesis and induction ofmacroautophagy (Broach, 2012; Loewith et al., 2002; Neufeld,2010).TORC1 is a multisubunit complex of ∼2 mDa and consists of

Tor1 or Tor2, a PIK-like kinase and the accessory subunits Kog1,Lst8 and the non-essential Tco89 (Loewith et al., 2002; Wedamanet al., 2003). TORC1 appears to be constitutively associated with thevacuolar membrane, independently of nutrient status, althoughsome sequestration to peri-vacuolar foci has been observed (Kiraet al., 2014; Sturgill et al., 2008). TORC1 exerts its growth effectsvia several downstream signaling branches that together constitutethe anabolic or catabolic response. TORC1 stimulates protein andribosome synthesis through several downstream effector kinases

including Sch9 and Ypk3 (González et al., 2015; Urban et al.,2007). Simultaneously, active TORC1 inhibits the PP2A (Pph3,Pph21 and Pph22) and PP2A-related (Ppg1 and Sit4) phosphatases,whose downstream effects include responses to nitrogen starvation(Loewith and Hall, 2011). Furthermore, TORC1 inhibitsmacroautophagy (Kamada et al., 2010). In addition to these maineffector branches, TORC1 directly interacts with an extensive arrayof kinases and phosphatases (Breitkreutz et al., 2010). Theseinclude Npr1, a kinase involved in regulating trafficking andlocalization of amino acid permeases (MacGurn et al., 2011; Merhiand Andre, 2012; Schmidt et al., 1998) and Nnk1, which has beenimplicated in nitrogen metabolism (Breitkreutz et al., 2010).

Amino acids regulate TORC1 via several mechanisms thatlargely depend on the ‘escape from rapamycin-induced growtharrest’ complex (the EGO complex) (Peli-Gulli et al., 2015). TheEGO complex consists of two small GTPases, Gtr1 and Gtr2, whichare recruited to the vacuolar membrane by a scaffold subcomplex(Powis et al., 2015) consisting of Meh1 (also known as Ego1), Ego2and Slm4 (also known as Ego3). The EGO complex is highlyconserved, and the Gtrs have homologs in higher eukaryotes knownas the Rag GTPases. The Gtrs form a constitutive heterodimerwhose activity depends on their nucleotide-binding status: theheterodimer is active when Gtr1 is GTP-bound and Gtr2 is GDP-bound (Binda et al., 2009; Jeong et al., 2012; Nakashima et al.,1999) and inactive in the opposite configuration. The nucleotidestatus of the Gtrs is regulated by several complexes that impinge onGTP hydrolysis, loading or dissociation: Vam6, a component of theHOPS complex involved in vacuolar fusion, was demonstrated to bea GEF for Gtr1 (Binda et al., 2009); Lst4–Lst7, which is a GTPase-activating complex (GAP) for Gtr2, which results in activation ofTORC1 (Peli-Gulli et al., 2015), and the SEA complex, which is aGAP for Gtr1 that inactivates it (Neklesa and Davis, 2009;Panchaud et al., 2013).

Particularly potent activators of TORC1 via the EGO complex arethe amino acids leucine and glutamine. Leucine promotesinteraction between GTP-loaded Gtr1 (Gtr1GTP) and Meh1 (Bindaet al., 2009), and the leucyl tRNA synthetase Cdc60 was shown todirectly interact with Gtr1 in a leucine-dependent manner (Bonfilset al., 2012). Glutamine stimulates interaction of the GAP Lst4–Lst7with Gtr2, thereby promoting formation of Gtr2-GDP, the activeform that can activate TORC1 (Peli-Gulli et al., 2015). Active Gtrsstimulate TORC1 via direct physical interactions: Gtr1GTP interactswith Tco89 (Binda et al., 2009), and the active heterodimer itselfinteracts with Kog1 (Sekiguchi et al., 2014).

In addition to GTPases, TORC1 is also regulated by signaling viathe phosphoinositide 3-kinase (PI3K) Vps34 and its productphosphatidylinositol 3-phosphate (PI3P) in both yeast andmammalian cells. In mammalian cells, signaling dependent onVps34 (also known as PIK3C3) is well characterized: amino acidsactivate Vps34, which results in an elevation of PI3P levels(Nobukuni et al., 2005), which, in turn, leads to activation ofReceived 30 June 2017; Accepted 3 October 2017

1Department of Cell Biology and Physiology, University of Pittsburgh School ofMedicine, 3500 Terrace Street, Pittsburgh, PA 15261, USA. 2Department ofMicrobiology and Molecular Genetics, University of Pittsburgh School of Medicine,5117 Centre Avenue, Pittsburgh, PA 15213, USA.

*Author for correspondence (marijn@pitt.edu)

M.G.J.F., 0000-0002-9115-4413

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mTORC1 (Byfield et al., 2005; Yoon et al., 2011). Importantly, theVps34 pathway is also necessary for the activation of mTORC1 bythe mammalian homologs of the Gtr GTPases (the Rag GTPases).Hence, amino acids activate mTORC1 via two necessary mutuallyinterdependent pathways: Rag GTPases and Vps34. In yeast,deletion of Vps34 also results in a strong inhibition of TORC1(Bridges et al., 2012) but the downstream effectors of Vps34 inactivation of TORC1 are unknown. It is also currently unknownhow Vps34-dependent and Gtr-dependent activation of TORC1 areintegrated.Recent work has identified Pib2 (phosphatidylinositol-3-

phosphate-binding 2) as an additional activator of TORC1 (Kimand Cunningham, 2015; Michel et al., 2017; Tanigawa and Maeda,2017). Pib2 was initially identified, together with severalcomponents of the EGO complex, as a hit in a screen for factorsunable to recover from rapamycin exposure (Dubouloz et al., 2005).Later, Pib2 was reported to be required for TORC1 activation andlysosomal membrane permeabilization in the presence of ER stress(Kim and Cunningham, 2015). It has a FYVE domain, a conservedC-terminal tail motif and a series of conserved stretches of aminoacids in a region otherwise predicted to be unstructured. The N-terminal region harbors a TORC1 inhibitory function whereas theC-terminal region is important for activation of TORC1 (Michelet al., 2017). Pib2 interacts with vacuoles via its FYVE domain in aPI3P-dependent manner and this depends on Vps34 (Kim andCunningham, 2015). Thus, we hypothesize that Pib2 integratesVps34 signaling into Gtr-dependent activation of TORC1.Here, we report that Pib2 indeed genetically interacts with

components of the EGO complex and TORC1 signaling. Pib2deletion phenocopies simultaneous loss of Gtr1 and Gtr2 in TORC1reactivation after rapamycin exposure, microautophagy and Gtr-dependent relocalization of Tor1 to perivacuolar foci. Furthermore,Pib2 and the Gtrs are reciprocally required for activation of TORC1by glutamine and leucine. Our data suggest that Pib2 and the EGOcomplex function in the same molecular pathway that leads toactivation of TORC1. Therefore, our findings provide evidence for arole for Pib2, together with the EGO complex, in the reactivation ofTORC1, thus offering insight into how PI3P signaling might becoupled with Gtr-dependent activation of TORC1.

RESULTSPIB2 genetically interacts with components of the EGOcomplex and TORC1Recent studies have identified Pib2 as a regulator of TORC1 but themechanism of Pib2 action remains unclear (Michel et al., 2017;Tanigawa and Maeda, 2017). To identify functional interactionpartners of Pib2 at the genomic level, we performed a syntheticdosage lethality (SDL) screen by overexpressing Pib2 in eachmember of the non-essential yeast gene deletion collection (Giaeverand Nislow, 2014). The premise of SDL is that overexpression of agene of interest, when combined with a mutant of a functionalinteraction partner, results in a measurable fitness defect, or, in theextreme case, lethality (Kroll et al., 1996). In contrast,overexpression of the same gene of interest in a wild-typebackground may result in no observable phenotype. SDL hasbeen used to screen the non-essential deletion collection for novelparticipants in various cellular processes (Measday et al., 2005). Weused selective ploidy ablation (SPA) to efficiently introduce thePib2 overexpression plasmid, or appropriate controls, into eachhaploid member of the non-essential gene deletion collection (Reidet al., 2011). The result is rapid introduction of overexpressionplasmids into haploid members of the deletion collection. We

obtained several strong SDL hits (P<0.0001, when the deletionstrain overexpressing Pib2 is compared to the same deletion strainexpressing an empty vector or overexpressing EGFP), whichincluded Δmeh1 (Δego1) and Δtor1 (Fig. 1A; Table S1). SinceMeh1 (Ego1) is a vacuolar membrane anchor for both Gtr1 andGtr2, these newly uncovered genetic interactions demonstrate thatPib2 is functionally related to the EGO complex. An additionalstrong hit (P<7.8×10−7) was Δpar32, a component of the PP2Asignaling branch downstream of TORC1, as well as the deletion ofYDL172C, which overlaps with the coding sequence of PAR32(Fig. 1A; Table S1). We also identified a set of genes enriched inendosomal structure and function (for example Δvps30, Δvps27 andΔvps28) (Fig. 1A; Table S1). These results are consistent with anenrichment of Pib2 in PI3P-containing endosomal/vacuolarmembranes (Burd and Emr, 1998; Kim and Cunningham, 2015).We also identified several hits in genes known to be involved in theregulation of the cell cycle and amino acid biosynthesis. In thiswork, we pursued further characterization of the connectionsbetween Pib2, the EGO complex and TORC1.

Pib2 is required for reactivation of TORC1 after treatmentwith rapamycinPib2 was initially identified as a hit in a screen for cells that wereimpaired in recovery from rapamycin, together with constituents ofthe EGO complex (Dubouloz et al., 2005). It has been shown thatthe EGO complex is required for reactivation of TORC1 afterinactivation by rapamycin (Binda et al., 2009) as well as for a poorlyunderstood subclass of autophagy known as microautophagy(Dubouloz et al., 2005). Given that Pib2 genetically interacts withthe EGO complex and Tor1, we compared the phenotypes of cellslacking Pib2 with those lacking components of the EGO complex,specifically the Rag family GTPases Gtr1 and Gtr2. Like cellslacking Gtr1 or Gtr2 (Fig. 1B; Fig. S1), Δpib2 cells do not recoverfrom exposure to rapamycin and fail to resume growth afterrapamycin-induced growth arrest. By contrast, cells lacking Atg7,which have a defect downstream of TORC1 (cannot undergomacroautophagy; Xie and Klionsky, 2007), recover from exposureto rapamycin like W303A cells (Fig. 1B).

To assess TORC1 activity, we monitored the phosphorylationstatus of a well-characterized target, ribosomal protein S6 (Rps6).Yeast Rps6 is phosphorylated at two serine residues at its C-terminus (S232 and S233) in a TORC1-dependent manner(González et al., 2015). Hence, the phosphorylation status ofRps6 at these sites can be used as a faithful readout of TORC1activity. Rapamycin treatment virtually eliminated phosphorylationof Rps6 at these sites in both wild-type and Δpib2 cells, as expected,upon TORC1 inactivation (Fig. 1C). Following recovery fromrapamycin exposure, an increase in Rps6 phosphorylation wasobserved in wild-type cells, to levels comparable to those seen inuntreated cells. By contrast, Rps6 remained dephosphorylated atS232 and S233 in Δpib2 cells, even after 24 h of recovery (Fig. 1C,D; recovering to ∼3.5% of the value seen in the wild-type untreatedcontrol, P<0.01). This suggests that cells lacking Pib2 fail toreactivate TORC1 during recovery.

To determine whether the growth defect of Δpib2 cells onrecovery from rapamycin exposure is due to a defect in Gtractivation, we introduced constitutively active forms of both Gtr1and Gtr2 (Gtr1 Q65L, constitutively GTP-bound, and Gtr2 S23L,constitutively GDP-bound) (Gao and Kaiser, 2006) into Δpib2 cells.Cells lacking Pib2 could not be rescued by introduction ofconstitutively active Gtrs (Fig. 1B). As a control, cells lackingGtr1 or Gtr2 were fully rescued by introduction of active Gtrs

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(Fig. S1). Therefore, activation of Gtrs is not the underlying cause ofthe defect in Δpib2 cells. To eliminate the possibility that Pib2 isrequired for the recruitment of Gtrs to the vacuolar membrane, orthat the Gtrs are mislocalized away from the vacuolar membrane in

Δpib2 cells and thus cannot activate TORC1, we compared thelocalization of Gtr1, Gtr2 and Meh1 (Ego1) in wild-type (W303A)and Δpib2 cells. The cellular distribution of Gtr1, Gtr2 and Meh1(Ego1) was identical between Δpib2 andW303A cells (Fig. S2; data

Fig. 1. See next page for legend.

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not shown). Hence, Pib2 is not required for vacuolar localization ofthe Gtrs.The recovery defect in Δpib2 cells was TORC1 dependent, as

introduction of a TOR1 mutant allele (L2134M, within the kinasedomain), previously shown to render Tor1 hyperactive regardless ofGtr activation (Kingsbury et al., 2014; Takahara and Maeda, 2012),into Δpib2 cells resulted in recovery and growth similar to that seenin wild-type cells (Fig. 1B). Vector alone controls are provided inFig. S3A. Since Δpib2 cells could not be rescued by constitutivelyactive Gtrs, the defect in Δpib2 is not due to a defect in activation ofGtrs. This result also suggests that activated Gtrs require Pib2 foractivation of TORC1.Mutants in components of the EGO complex display a striking

vacuolar phenotype after exposure to rapamycin: grossly enlargedvacuoles that cannot return to their pre-exposure size after removalof rapamycin (Dubouloz et al., 2005). This was proposed to be dueto a defect in microautophagy. We next asked whether Δpib2 cellsdisplay a similar vacuolar morphology defect. We evaluated the sizeof vacuoles in W303A, Δpib2 and Δgtr1 Δgtr2 cells before, duringand after rapamycin treatment. On rapamycin exposure, vacuoles ofwild-type cells increased in size, as expected, as a consequence ofincreased macroautophagy (Chan and Marshall, 2014) (Fig. 1E).During recovery, the vacuolar size returned to pre-exposure levelsafter 48 h. By contrast, vacuoles of Δgtr1 Δgtr2 cells enlarged onrapamycin exposure and did not recover. Vacuoles of Δpib2 cells

likewise enlarged on rapamycin treatment but continued expanding,even during recovery from rapamycin exposure, similar to what wasseen in Δgtr1 Δgtr2 cells (Fig. 1E). We quantified theseobservations by calculating the ratio of the maximal vacuolarcross-sectional area to the maximal cellular cross-sectional area(vac:cell area), to normalize to cell size (Fig. 1F). UntreatedW303Acells had a vac:cell area ratio of 0.23±0.05 (mean±s.d.), whichincreased to 0.47±0.11 after rapamycin treatment (P<0.01), beforerecovering to 0.21±0.03 after 48 h. Δgtr1 Δgtr2 cells had anuntreated vac:cell ratio of 0.36±0.12. Rapamycin treatmentincreased this to 0.54±0.07 (P<0.01), which increased further to0.70±0.08 after 48 h recovery (P<0.01). Similarly, Δpib2 cells hadan untreated vac:cell ratio of 0.30±0.12, which increased to 0.52±0.06 after rapamycin exposure (P<0.01). As was the case for cellslacking Gtrs, this ratio increased to 0.80±0.07 after 48 h recovery(P<0.01). Hence, vacuolar size and the cell:vac scaling ratio doesnot recover after rapamycin treatment in Δgtr1 Δgtr2 or Δpib2 cells.These results demonstrate that loss of Pib2 phenocopies loss of theGtrs. Pib2 is therefore, like components of the EGO complex,involved in vacuolar dynamics and microautophagy.

Pib2 and Gtrs are both required for activation of TORC1 byglutamine and leucineGlutamine and leucine are known to be the most potent activators ofTORC1 (Bonfils et al., 2012; Peli-Gulli et al., 2015), and theseactivating stimuli require the EGO complex for relay to TORC1(Binda et al., 2009; Kim et al., 2008; Sancak et al., 2008). If Pib2indeed acts within the same pathway as the Gtrs, we predict that wewould observe a defect in stimulation of TORC1 by glutamine andleucine in cells lacking Pib2. We therefore compared TORC1reactivation by glutamine and leucine in cells lacking either Pib2 orboth Gtr1 and Gtr2. When grown in nutrient-rich medium, bothΔpib2 and Δgtr1 Δgtr2 double mutant cells exhibit basal TORC1activity, as determined by assessing the phosphorylation state ofRps6 (Figs 2A,B and 3A,B, left-most column, no significantdifferences). Nitrogen starvation resulted in loss of detectableTORC1 activity, as expected (P<0.01 in all cases). Addition ofeither glutamine (3 mM) or leucine (3 mM) for the indicated times(Figs 2A and 3A) evoked reactivation of TORC1 in wild-type cells(5 min, P<0.01; 30 min, P<0.05) but not in Δpib2 or Δgtr1 Δgtr2cells. Importantly, expressing activated Gtrs in Δpib2 cells did notrescue the glutamine- or leucine-dependent activation of TORC1(Figs 2C,D and 3C,D). Pib2 is therefore not required for activationof Gtrs. Active Gtrs cannot overcome the requirement for Pib2 inactivation of TORC1. These observations are quantified in Figs 2B,D,F and 3B,D,F, for glutamine and leucine, respectively. We,therefore, conclude that Pib2 and the Gtrs are both required to relaythe glutamine and leucine signals to TORC1.

Of note, refeeding nitrogen-starved Δpib2 or Δgtr1 Δgtr2 cellswith a mixture of all amino acids results in a robust and fullphosphorylation of Rps6 and thus activation of TORC1 (Fig.S3D). The degree of phosphorylation of Rps6 was comparable ineach strain and directly comparable to that in W303A cells. Thissuggests the existence of an additional amino acid signal thatstimulates TORC1 in a Gtr1/2- and/or Pib2-independent manner.This serves as a positive control for our readout that demonstratesthat the extent of the potential response in Δpib2 or Δgtr1 Δgtr2cells is comparable to the response in W303A cells when thestimulus is not glutamine or leucine. Therefore, the defect inTORC1 activation in Δpib2 or Δgtr1 Δgtr2 cells is stimulusspecific and the activation of TORC1 by leucine and glutamine isdependent on both Pib2 and Gtr1/2.

Fig. 1. Pib2 is required for exit from rapamycin-induced growth arrest.(A) Representative quartets from matched control and Pib2-overexpressingstrains in the SDL screen. Overexpression of Pib2 results in synthetic lethalitywith Δmeh1 (Δego1), Δtor1, Δpar32, Δydl172c and Δvps30 but not with Δavo2,which is shown here as a non-interacting control. (B) Growth of W303A, Δatg7,Δpib2 and Δgtr1 expressing the indicated constructs on YPD during recoveryfrom exposure to rapamycin. Exponentially growing cells (OD600 0.6–0.8) weretreated with 200 ng/ml rapamycin in YPD at 30°C for 5 h. After washing, cellswere plated on YPD and were incubated for 3 days at 30°C. The left-most spotin each case corresponds to 2 µl of a culture with an OD600 of 0.5. Spots to theright of this correspond to 2 µl of sequential 1:5 serial dilutions. (C) Evaluationof the phosphorylation levels of S232 and S233 of Rps6 in W303A and Δpib2cells. Cells as indicated were treated with rapamycin as in B. Total Rps6 andPgk1 levels are shown as loading controls. (D) Quantification of the datapresented in C. Ratios of phosphorylated Rps6 to Pgk1 for each measurement(mean±s.d.; n=3 in each case) were normalized to the mean ratio ofphosphorylated Rps6 to Pgk1 for untreated W303A cells (set at 1). A two-wayANOVA was conducted to determine the effects of genetic background(W303A and Δpib2) and treatment (untreated, rapamycin treated and recovery)on Rps6 phosphorylation levels. There was a significant interaction effect ofbackground and treatment on Rps6 phosphorylation levels (F2,12=9.46, henceP=0.0034). Selected pairs of values significant by the post-hoc Tukey honestsignificant difference (HSD) test (**P<0.01) are shown. (E) W303A or theindicated knockout strains were stained with FM 4-64 for 45 min, and thenwashed and chased in YPD for 1 h prior to visualization. Where indicated, cellswere treated with rapamycin (200 ng/ml) for 3 h. For recovery, cells werethoroughly washed and were incubated for 48 h in YPD. Scale bar: 5 µm. (F)Quantification (mean±s.d.) of the increase in vacuolar scaling for the cellsshown in E. The maximal vacuolar cross-sectional area was divided by themaximal cellular cross-sectional area. For cells where more than one vacuolarlobe existed (usually only W303A untreated or at 48 h recovery), the maximalcross-sectional area of each lobe was determined. A total of 10–14 vacuolesand cells were measured for untreated and rapamycin-treated cells and 5–10for cells after recovery. For W303A and the knockout strains, the means of theuntreated, treated and recovery measurements were determined to besignificantly heterogeneous (one-way ANOVA: W303A F2,31=45.25, henceP<6.39×10−10; Δgtr1 Δgtr2 F2,34=36.62, hence P<7.26×10−9; Δpib2F2,26=55.40, hence P<1.01×10−9). Significantly different pairs of means, asassessed by the post-hoc Tukey HSD test, are indicated (**P<0.01). Non-significantly different means are indicated below the W303A chart (P=0.90).

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Expression of activated Gtrs (Gtr1 Q65L and Gtr2 S23L) in Δgtr1Δgtr2 cells resulted in sustained activity of TORC1, even understarvation conditions, whereas TORC1 remains inhibited bynitrogen starvation in wild-type cells overexpressing active Gtrs(Figs 2C,D and 3C,D, compare W303A with overexpressed Gtrs toΔgtr1 Δgtr2with overexpressed Gtrs, P<0.01). Sincewild-type cellsstill express endogenous Gtr1 and Gtr2, we conclude that inactiveforms of Gtrs (i.e. Gtr1-GDP and Gtr2-GTP) are therefore requiredfor inhibition of TORC1 by nitrogen starvation, as previouslyobserved (Kira et al., 2014).To further confirm the interdependence of Pib2 and Gtrs in

TORC1 activation by glutamine and leucine, we also evaluatedthe effect of overexpression of Pib2 in Δgtr1 Δgtr2 cells.Overexpression of Pib2 in Δgtr1 Δgtr2 cells did not rescueTORC1 activity, whereas it rescued TORC1 activity in Δpib2cells (Figs 2E,F and 3E,F; 5 min, P<0.05). These observationsagain suggest that Pib2-dependent TORC1 activation byglutamine or leucine requires Gtrs. Pib2 overexpression in

Δgtr1 Δgtr2 cells repressed even TORC1 basal activity (P<0.01for both Pib2 overexpressed in W303A versus Δgtr1 Δgtr2, andPib2 overexpressed in Δpib2 versus Δgtr1 Δgtr2), confirmingthe existence of a previously reported Gtr-independentinhibitory function of Pib2 on TORC1 (Michel et al., 2017).Repression of TORC1 basal activity is only observed in cellslacking Gtrs and not wild-type cells. We further examined theeffects of overexpression of a truncated Pib2 construct lackingits N-terminal 164 amino acids (Pib2 ΔN-term) on TORC1activation. The N-terminal 164 amino acids of Pib2 werepreviously reported to harbor an inhibitory function on TORC1(Michel et al., 2017). Indeed, the Pib2 ΔN-term did not inhibitbasal activity of TORC1 in Δgtr1 Δgtr2 cells, confirming theimportance of this domain for the observed inhibitory functionof Pib2 (Fig. S3C). Taken together, these data strongly suggesta novel dual mode of action of Pib2 on TORC1 activity: in thepresence of Gtrs, Pib2 is an activator of TORC1, whereas intheir absence it is an inhibitor.

Fig. 2. Pib2 is required for stimulation of TORC1 activity by glutamine. Phosphorylation levels of Rps6 were evaluated under the indicated conditions.Untreated cells were grown in SC medium. Cells were nitrogen-starved by incubating in SD –N medium for 3 h. For stimulation, cells were treated with SD –Nsupplemented with glutamine (Gln, 3 mM) andwere incubated for the indicated times prior to lysis and processing. Both total Rps6 andPgk1 are shown as loadingcontrols. (A)W303A, Δpib2, Δgtr1 Δgtr2. (B) Quantification mean of the data shown in A. Gray lines: selected statistically significant differences betweenmeans ofphospho-Rps6 (Tukey HSD; *P<0.05; **P<0.01). For each cell type, differences in means of phospho-Rps6 were evaluated by one-way ANOVA for each of thetreatment conditions. Black lines: selected statistically significant differences between means of phospho-Rps6 (Tukey HSD; *P<0.05; **P<0.01). For eachtreatment shown, the means of phospho-Rps6 were compared for W303A, Δpib2 and Δgtr1 Δgtr2 by one-way ANOVA. For quantification, the phospho-Rps6signal was normalized to the corresponding Pgk1 loading control. (C) Strains as in A but expressing Gtr1 Q65L and Gtr2 S23L from their native promoters oncentromeric plasmids. (D) Quantification of the data shown in C. (E) Strains as in A but overexpressing Pib2 from an episomal Tet-Off plasmid. Cells were grown inappropriate medium containing 5 µg/ml doxycycline. Cells were diluted and inoculated into doxycycline-freemedium for 12 h to allow overexpression of Pib2. Thenitrogen starvation and amino acid stimulation were then performed as in A. (F) Quantification of the data shown in E. Results in B, D and F are mean±s.d. (n=3).

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Pib2 regulates Tor1 localization on the vacuolar membranePreviously, it has been reported that the nucleotide state of Gtr1affects localization of Tor1 at the vacuolar membrane. Gtr1-GTPappears to be required for dispersion of Tor1 throughout thevacuolar membrane; in its absence, Tor1 accumulates inperivacuolar foci (Kira et al., 2016). Tor1 also localizes toperivacuolar foci in the absence of both Gtrs (Fig. 4A) (Kiraet al., 2016). The identity of the puncta remains unknown – previouswork has demonstrated that they do not colocalize with Snf7 orApe1, and, hence, are not endosomal or phagophore assembly sites,respectively (Kira et al., 2014). As our data suggest that Pib2 isrequired to relay a signal from activated Gtrs to TORC1, we soughtto evaluate the role of Pib2 in the localization of Tor1. Ourprediction was that Tor1 will redistribute to puncta in Δpib2 cells ifPib2 indeed relays signals from activated Gtrs.In W303A cells grown in nutrient-rich medium, GFP–Tor1,

expressed under control of its native promoter from a centromericplasmid, had a diffuse vacuolar membrane distribution with somefoci associated with the vacuolar membrane (Fig. 4A), as has beenobserved previously with an integrated genomic copy of GFP–Tor1(Kira et al., 2014). Simultaneous loss of Gtr1 and Gtr2 resulted in a

marked redistribution of GFP–Tor1 into puncta associated with thevacuole: the number of vacuoles with Tor1 puncta increased from18.6±2.9% (mean±s.d.) in W303A cells to 65.6±5.0% in Δgtr1Δgtr2 cells (P<0.01). Similarly, loss of Tco89, a component ofTORC1 required for relay of the Gtr signal (Reinke et al., 2004),resulted in a redistribution of GFP–Tor1 into the vacuole-associatedpuncta (63.3±2.4% of vacuoles were associated with puncta,P<0.01 compared to the result for W303A cells). Loss of Pib2also resulted in an increase in vacuoles associated with GFP–Tor1puncta (41.5±3.4% of vacuoles associated with puncta; P<0.01)(Fig. 4A,B). Of note, expressing constitutively active forms of Gtr1and Gtr2 in Δpib2 cells did not change the number of vacuolesassociated with Tor1 foci (Fig. S3D). This observation may beexplained by two scenarios: either Pib2 and Gtr1/Gtr2 actindependently to regulate Tor1 localization, or Pib2 acts directlydownstream of the Gtrs in regulating Tor1 localization. Furtherstudies are required to distinguish between these possibilities.Currently, the function of Tor1 foci formation is unknown. Todetermine whether Tor1 foci formation impinges on TORC1activity, we analyzed foci formation after nitrogen starvation,when TORC1 activity is known to be repressed. No significant

Fig. 3. Pib2 is required for stimulation of TORC1 activity by leucine. This work was performed as in Fig. 2, but with leucine (Leu) stimulation (3 mM) instead ofglutamine. (A) W303A, Δpib2, Δgtr1 Δgtr2. (B) Quantification of the data shown in A. (C) Strains as in A but expressing Gtr1 Q65L and Gtr2 S23L from their nativepromoters on centromeric plasmids. (D) Quantification of the data shown in C. (E) Strains as in A but overexpressing Tet-Off PIB2 from an episomal Tet-Offplasmid. (F) Quantification of the data shown in E. Results in B, D and F are mean±s.d. (n=3).

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changes in foci formation were observed in W303A, Δpib2 andΔgtr1 Δgtr2 cells (Fig. S4A,B; compare Fig. 4B and Fig. S4B). Thissuggests that Tor1 foci formation does not correlate with the activity

of TORC1. Strikingly, exposure to rapamycin for 3 h, which is alsoknown to inhibit TORC1 activity, resulted in a complete loss ofTor1 foci in all strains, even Δgtr1 Δgtr2 cells (Fig. S4C,D).

Fig. 4. Pib2 regulates localization of Tor1 on vacuoles. (A) GFP–Tor1 localization in W303A, Δgtr1 Δgtr2, Δtco89 and Δpib2 cells as indicated. The indicatedstrains expressedGFP–Tor1 from its native promoter on a centromeric plasmid. Cells were grown in SCmedium until they reached anOD600 of 0.6–0.8.W303A orthe indicated knockout strains were stained with FM 4-64 for 45 min, then washed and chased in YPD for 1 h prior to visualization. (B) Quantification (mean±s.d.)of the numbers of vacuoles displaying GFP–Tor1 foci in each of the indicated strains. Foci were counted on z-stacks collected for each of the strains (from 250 to400 vacuoles were assessed for each strain). Means of numbers of vacuoles displaying foci were significantly heterogeneous (one-way ANOVA, F4,15=150.45;P<8.77×10−10). A post-hoc Tukey HSD test for significance was performed between each of the means. Selected significant differences between means(**P<0.01) are indicated on the plot and the means showing a non-significant difference (P=0.80) are indicated below the plot. (C) As in A, but with strains asindicated expressing GFP–Pib2. (D) Quantification (mean±s.d.) of the data shown in C. Foci were counted on z-stacks collected for each of the strains (∼250vacuoles were assessed in each strain). The means of vacuoles displaying foci were significantly different for the two strains (***P<0.001; two-tail t-test with sixdegrees of freedom; t=7.23, hence, P=0.0003). Scale bars: 5 μm.

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A mechanistic explanation of this observation awaits furtherexperimentation.Pib2 has been reported to directly interact with Tor1 and Kog1

(Michel et al., 2017; Tanigawa and Maeda, 2017). We asked,therefore, whether Pib2 changes its localization in response to lossof the Gtrs, as observed for components of TORC1. Indeed, inW303A cells, Pib2 is associated with the vacuolar membrane withsome foci. In the absence of Gtrs (Fig. 4C) or Tco89 (data notshown), Pib2 distribution alters with an increased number ofvacuoles containing foci (Fig. 4D; in W303A cells 15.8±4.7% ofvacuoles had foci compared to 47.5±7.4% for vacuoles in Δgtr1Δgtr2 cells, P<0.001). These data indicate that Pib2 is likely tofollow the Gtr-dependent distribution of TORC1.

Pib2 is not required for and does not regulatemacroautophagyPI3P is required for macroautophagy, and removing Vps34, whichis the sole PI3K in yeast, results in inhibition of macroautophagy(Burman and Ktistakis, 2010). Since Pib2 is a PI3P-binding proteinand since its recruitment is Vps34 dependent (Kim andCunningham, 2015), we sought to determine whether Pib2 was aneffector of PI3P in regulating autophagy. We therefore used thewell-established GFP–Atg8 processing and flux assay in bothW303A and Δpib2 cells expressing GFP–Atg8 from its nativepromoter (Kirisako et al., 1999; Shintani and Klionsky, 2004).Basal GFP–Atg8 expression levels were directly comparable inW303A and Δpib2 cells (Fig. S5). On rapamycin exposure, similarincreased expression levels of GFP–Atg8 were observed in boththe W303A and Δpib2 cells, a consequence of enhancedmicroautophagic flux (Fig. S5). Likewise, comparable elevatedamounts of free GFP, reflecting processed GFP–Atg8, wereobserved in both W303A and Δpib2 cells (Fig. S5). Hence, Pib2is not required for GFP–Atg expression or processing, and cellslacking Pib2 are not impaired in macroautophagy.

Npr1 is constitutively active in Δpib2 cellsTORC1 directly interacts with, and phosphorylates, Npr1, whichinhibits it (Breitkreutz et al., 2010; MacGurn et al., 2011; Schmidtet al., 1998). Inhibition of TORC1 activity, through rapamycintreatment or nitrogen starvation, therefore leads to activation ofNpr1 that results in a number of downstream effects, includinginhibition of Ldb19 (Art1) (MacGurn et al., 2011), phosphorylationof Bul1 and Bul2 (Merhi and Andre, 2012) and trafficking of thetryptophan permease Tat2 from the surface of the cell to the vacuolefor degradation (Schmidt et al., 1998). One additional target ofactive Npr1 is the poorly characterized protein Par32. Active Npr1results in extensive phosphorylation of Par32 at multiple sites,which leads to a significant change in migration rate (Boeckstaenset al., 2015). Therefore, the migration rate of Par32 can be used as areadout to evaluate the activity of Npr1.Δpar32 was a hit in our SDL screen using overexpressed Pib2

(P<7.82×10−7). We, therefore, evaluated phosphorylation of Par32as a readout of Npr1 activity in W303A and Δpib2 cells. Asexpected, we observe that all of the hemagglutinin-tagged Par32(Par32–3xHA) expressed in W303A cells from the native PAR32promoter is shifted to a slower-migrating form on rapamycintreatment or nitrogen starvation, which would be consistent withextensive post-translational modification (Fig. 5A–C). Essentiallythis shift in migration depends on the presence of Npr1. In Δpib2cells, the steady-state distribution of Par32 is more shifted towardsthe slower-migrating species than in W303A cells, indicating thatNpr1 is more active than in controls. Treatment with rapamycin

maximally shifted Par32–3xHA in Δpib2 cells, indicating furtheractivation of Npr1 (Fig. 5C). All of the shifts in Par32–3xHAmigration, in W303A or in Δpib2 cells, were dependent on thepresence of Npr1 (Fig. 5C; data not shown). In summary, at steadystate, Npr1 is partially active in Δpib2 cells, but not in W303A cells,and can be further activated by additional inhibition of TORC1. Theincreased phosphorylation of Par32 observed in Δpib2 cells couldnot be completely reversed by expression of the hyperactive mutantallele of Tor1 (L2134M) (Fig. 5D). Note that in both W303A cellsand Δpib2 cells, the extent of enhancement in TORC1 activity onexpression of Tor1 L2134M is directly comparable. Thus, Pib2 hasan additional function of repressing Npr1 activity independently ofTORC1.

Deletion of Npr1 has been reported to suppress the defect in exitfrom the rapamycin-induced growth arrest of various EGO mutants,including Meh1 (Ego1), Slm4 (Ego3) and Gtr2 (Dubouloz et al.,2005). We therefore tested whether deletion of Npr1 also suppressesthe defect in recovery from rapamycin of Δpib2 cells. Δnpr1 cellsdisplayed enhanced growth compared to W303A cells on recoveryfrom rapamycin (Fig. 5E). As before, Δpib2 cells did not recoverfrom treatment with rapamycin. However, simultaneous deletion ofPib2 and Npr1 resulted in recovery from rapamycin (Fig. 5E).Hence, activated Npr1 after rapamycin exposure contributes to thelack of growth of Δpib2 cells, in the same way as was previouslyobserved in Δmeh1, Δslm4 and Δgtr2 cells. Taken together, thesefindings indicate that Pib2 has a function in downregulation of Npr1activity, which negatively affects recovery of growth after rapamycintreatment (Fig. 6).

DISCUSSIONIn this work, we provide a detailed characterization of Pib2 and acomparison of its function to that of the EGO complex. Wedemonstrate that Pib2, whose mechanism of action was ill defined,is required, together with the Gtrs, for activation of TORC1. Weidentified strong genetic interactions in an SDL screen between Pib2and components of the EGO complex–TORC1 network. Δpib2 cellsbehaved identically to cells lacking both Gtrs in many aspects,including recovery from exposure to rapamycin, vacuolar dynamics,response to amino acids and distribution of GFP-Tor1 on thevacuolar surface. We, therefore, conclude that these responses ofTORC1 require both Pib2 and the EGO complex (Fig. 6).

Previous reports demonstrated a reduced response to glutamine incells lacking Pib2 (Michel et al., 2017; Tanigawa andMaeda, 2017).Our data showed that cells lacking Pib2 are unable to activateTORC1 in response to glutamine or leucine. Importantly, thepresence of mutants of Gtr1 and Gtr2 that are restricted to activatedstates did not override the requirement for Pib2, suggesting that therole of Pib2 is not activation of Gtrs. In mammalian cells, leucine issensed by leucyl tRNA-synthetase (LRS, also known as LARS),which activates mTORC1 via two mutually necessary mechanisms:LRS has GAP activity for RagD (also known as RRAGD, amammalian homolog of Gtr2) (Han et al., 2012) and LRS directlyinteracts with and activates Vps34, thus mediating TORC1activation via the Vps34–PLD1 branch (Yoon et al., 2016).Thus, leucine-dependent activation of TORC1 integrates PI3P-and Rag-dependent signaling pathways. The yeast homolog ofLRS, Cdc60, has been reported to regulate the activities of the Gtrsin response to amino acids (Bonfils et al., 2012). However, aconnection between PI3P signaling and leucine has not yet beenestablished. We speculate that Pib2 is an integral part of the PI3Psignaling pathway that connects leucine stimulation to TORC1activation.

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Overexpression of Pib2 in Δgtr1 Δgtr2 cells did not rescue theresponse to glutamine or leucine, further highlighting the co-dependence of Pib2 and the Gtrs in activation of TORC1. Previousmodels of Pib2 function suggested a Gtr-independent role inactivation of TORC1 (Kim and Cunningham, 2015; Tanigawa and

Maeda, 2017; Stracka et al., 2014), based on the observations ofknockouts of Gtr1 alone and synthetic lethality between PIB2 andcomponents of the EGO complex. In these reports, residualactivation of TORC1 was detected in Δgtr1 cells. This residualactivity was attributed to Pib2, since it is a known activator ofTORC1. Based on the fact that we do not detect residual TORC1activation in Δgtr1 Δgtr2 cells, it may be that the residual activationdetected in the single knockout stems from the action of theremaining component of the Gtr dimer. Gtr1 and Gtr2, and their Raghomologs in higher eukaryotes, form heterodimers that, whenasymmetrically loaded with GTP and GDP respectively, activateTORC1 (Hatakeyama and De Virgilio, 2016). It is possible that inΔgtr1 cells the presence of Gtr2, combined with endogenous Pib2,and/or the absence of Gtr1-GDP, could have a residual activity onTORC1. It is known that Gtr1 can form homodimers (Nakashimaet al., 1999) and it would be interesting to see whether the same istrue of Gtr2, especially in cells lacking Gtr1.

If the pathways mediated by Pib2 and Gtr1/2 to activate TORC1are independent of each other, there should be an intermediateTORC1 response to glutamine or leucine in cells lacking either Pib2or Gtr1/2. Under our conditions, we do not observe this and weobserve activation only in the presence of both Pib2 and Gtr1/Gtr2.

Fig. 6. Proposedmodel for control of TORC1 signaling by Pib2 andGtr1/2.See the Discussion for further details.

Fig. 5. Npr1 is active and is the underlying cause of the defect in recovery from rapamycin exposure in Δpib2 cells. (A) W303A and Δnpr1 cells expressingPar32–3xHA were treated with rapamycin (200 ng/ml) for 3 h as indicated. Par32–3xHA was visualized using an anti-HA monoclonal antibody. (B) W303A andΔpib2 cells expressing Par32–3xHA were nitrogen starved for 3 h. Par32–3xHA was then visualized as in A. (C) The strains as indicated were treated withrapamycin as inA. (D)W303AorΔpib2 cells expressingPar32-3xHA and Tor1 L2134M, as indicated, were grown in SCmedium. Par32-3xHAwas then visualized asinA. Relative TORC1activity was calculated based on the phosphorylation levels of Rps6, normalized to a Pgk1 loading control.W303Awas set at 100%. (E)Growthof W303A and isogenic strains containing the indicated knockout on YPD during recovery from exposure to rapamycin. Exponentially growing cells (OD600 0.6–0.8)were treated with 200 ng/ml rapamycin in YPD at 30°C for 5 h. After washing, cells were plated on YPD and were incubated for 3 days at 30°C. The left-most spot ineach case corresponds to 2 µl of a culture with OD600 0.5. Spots to the right of this correspond to 2 µl of sequential 1:5 serial dilutions.

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One possibility is that TORC1 is generally impaired in either Δpib2or Δgtr1 Δgtr2 cells, which may dampen an intermediate TORC1activation response below detection thresholds. Our data suggestsotherwise for two reasons. First, basal TORC1 activity is notimpaired in either Δpib2 or Δgtr1 Δgtr2 cells (Figs 2, 3 and 5D).Second, we show that Δpib2 or Δgtr1 Δgtr2 cells can activateTORC1 to the same extent as wild-type cells (using a different,Pib2- and Gtr1/2-independent stimulus as reported in Fig. S3B).This serves as a positive control for our readout that demonstratesthat the extent of the potential response in Δpib2 or Δgtr1 Δgtr2 cellsis comparable to the response of the wild-type (W303A) cells whenthe stimulus is different. This argues against a generally reduced/impaired TORC1 activity in the knockout strains, either at the basallevel or on activation by different stimuli.The mechanism of TORC1 activation by Pib2 and the Gtr1/2 may

be explained by two overarching models: dependent andindependent (Fig. S6). The prediction for the independent model(model C in Fig. S6) is that intermediate levels of TORC1 activationwould be detected.Model A (dependent hierarchical) postulates thatactivation of TORC1 requires both Pib2 and the Gtrs that act in somehierarchical manner (upstream/downstream of each other). Aprediction of this model is that no intermediate activation ofTORC1 will be detected when Pib2 or the Gtrs are missing. A smallmodification of model A is model B (dependent threshold). In thiscase, activation of TORC1 depends, again, on both Pib2 and theGtrs. However, the extent of activation by either Pib2 alone or theGtrs alone either does not exist or is so low that it cannot be detectedby multiple assays. However, a potentiation occurs between Pib2and the Gtrs, which would result in a full response, and thispotentiation implies dependence. Based on our results of activationof TORC1 by glutamine or leucine after nitrogen starvation, wesuggest that models A or B are most plausible. Model C might besupported by the observed synthetic lethality between PIB2 andcomponents of the EGO complex. However, synthetic lethality isnot necessarily inconsistent with a dependent mechanism of actionof Pib2 and Gtr1/2 on TORC1 activation (models A and B). Here,we report that Pib2 has an additional TORC1-independentinhibitory function on Npr1 (Fig. 5). This could provide analternative explanation for the observed synthetic lethality betweenPIB2 and components of the EGO complex. Cells lacking both Pib2and components of the EGO complex will have constitutive Npr1activity that is toxic (Schmidt et al., 1998). Taken together, the lackof an intermediate response to glutamine and leucine in Δpib2 orΔgtr1 Δgtr2 cells, as well as our detection of an additional functionof Pib2 in regulating Npr1, which is an alternative explanation forsynthetic lethality, favors a dependent model of Pib2 action onTORC1 (models A or B).Overexpression of Pib2 in Δgtr1 Δgtr2 cells not only failed to

rescue TORC1 activity in response to amino acids but alsosignificantly dampened the basal response of TORC1, suggestingthat Pib2 has an additional inhibitory function on TORC1 that isunmasked in the absence of the Gtrs. This inhibitory function is Gtrindependent. This supports previous work that identified aninhibitory region at the N-terminus of Pib2 (Michel et al., 2017).Taken together, our observations suggest that Pib2 has twoantagonistic functions: activation of TORC1 in a manner that isco-dependent on the Gtrs, and Gtr-independent inhibition ofTORC1. Intriguingly, mammalian cells have two PI3P-bindinghomologs of Pib2 that are yet to be implicated in the regulation ofmTORC1: Phafin-1 and Phafin-2 (also known as PLEKHF1 andPLEKHF2, respectively). These both lack the N-terminalsupposedly inhibitory regions present in Pib2 and instead have a

PH domain. Currently, a link between the Phafins and mTORC1 hasnot yet been established and thus it is of immediate interest todetermine whether indeed Phafins play a role in mTORC1 signalingand, if so, how their mechanism of action differs from that of Pib2.

Although the vacuolar localization of Tor1 in yeast isindependent of the nutritional status of the cell, the distribution ofthe TORC1 complex is dynamically regulated by the nucleotide-bound state of Gtr1 and Gtr2 (Kira et al., 2016). Absence of theactive form of Gtr1 or of the Gtrs altogether leads to theaccumulation of Tor1 in perivacuolar foci. In Δpib2 cells, weobserve a similar accumulation of Tor1 in foci, suggesting that Pib2also plays a role in Tor1 localization on the vacuolar membrane. InΔgtr1 Δgtr2 cells, Pib2 similarly accumulates in perivacuolar foci.Pib2 physically interacts with Tor1 and Kog1 (Michel et al., 2017;Tanigawa and Maeda, 2017). Pib2 likely therefore associates withTORC1 and follows its distribution in response to signalingvia Gtrs.

We observed that cells lacking Pib2 have partially activated Npr1,as assessed by monitoring the phosphorylation status of the directNpr1 effector Par32. We also observed that Pib2 inactivates Npr1 inparallel to TORC1. Furthermore, loss of Npr1 resulted in growthresumption in cells lacking Pib2 after rapamycin exposure, as hasbeen previously observed for cells lacking components of the EGOcomplex (Dubouloz et al., 2005). Thus, loss of Npr1 overrides therequirement for Pib2 or the EGO complex in reactivation of TORC1after rapamycin exposure. This suggests that sustained Npr1 activityduring recovery from rapamycin makes reactivation of TORC1completely dependent on the EGO complex and Pib2. One potentialmechanism for this is that Npr1 directly phosphorylates TORC1components or regulators, preventing activation by all otheractivators except for activated Gtrs and Pib2. In this context, it isof interest that Npr1 interacts with multiple components of TORC1(Breitkreutz et al., 2010). Alternatively, the mechanism for Npr1-mediated suppression of the phenotypes of loss of Pib2 and EGOcomponents could be more complex and indirect. Npr1 is a knownregulator of the stability, localization and transport of severalpermeases, including the tryptophan permease Tat2 (Schmidt et al.,1998), the arginine and uracil transporters Can1 and Fur4 (MacGurnet al., 2011), and the general amino acid permease Gap1 (Merhi andAndre, 2012; O’Donnell et al., 2010; Shimobayashi et al., 2013).Hence, Npr1 may regulate the stability or activity of a permease thatsupplies an amino acid or other nutrient that is capable of activatingTORC1 in an EGO complex- and Pib2-independent manner afterrapamycin treatment.

In summary, we establish a function for Pib2, a FYVE domain-containing PI3P-binding protein, in Gtr-dependent activation ofTORC1, identifying a molecular bridge between PI3P signaling andthe EGO complex. Future work will focus on the conservation offunction for the Pib2 homologs in mammalian cells.

MATERIALS AND METHODSYeast genetic manipulation and molecular biologyStrains used in this work are listed in Table S2. Gene deletions weregenerated in W303a/α diploids by homologous recombination andcomplete replacement of the target open reading frame with cassettesamplified from pFA6a-kanMX6, pFA6a-His3MX6 (Longtine et al., 1998)or pFA6-natMX4 (Goldstein and McCusker, 1999) flanked with sequence(30 nt) proximal to the coding sequence of the target gene. Diploids weresubsequently sporulated through starvation in SPO medium. Followingmanual tetrad dissection, knockout haploids were validated by colony PCR,microscopy and, in some cases, sequencing. Strains harboring more thanone genomic modification were generated by mating and sporulation ofappropriate parental strains, followed by extensive revalidation. The

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standard PEG 3350/lithium acetate/single-stranded carrier DNA protocolwas used for yeast transformation (Gietz and Schiestl, 2007).

MediaYPD (2% yeast extract, 1% peptone, 2% glucose, supplemented with L-tryptophan and adenine) was used for routine growth. Synthetic Complete(SC; yeast nitrogen base, ammonium sulfate, 2% glucose, amino acids) orSynthetic Defined (SD; yeast nitrogen base, ammonium sulfate, 2%glucose, appropriate amino acid dropout) media were used prior tomicroscopy or to maintain plasmid selection as indicated. For sporulation,cells were successively cultured in YPA (2% potassium acetate, 2%peptone, 1% yeast extract) and SPO (1% potassium acetate, 0.1% yeastextract, 0.05% glucose). For starvation, cells were grown in SD –N (0.17%yeast nitrogen base without amino acids and ammonium sulfate, 2%glucose). For stimulation, cells were treated with SD –N supplemented withglutamine (Gln, 3 mM) or leucine (Leu, 3 mM), or supplemented with acomplete dropout mix, and were incubated for the indicated times prior tolysis and processing.

Cloning and plasmidsPlasmids used in this work are listed in Table S3. GFP-S cer. PIB2 wasgenerated by amplifying the PIB2 promoter and a fragment containing thePIB2 coding sequence and terminator from genomic DNA, prepared fromW303a/α diploids by using a yeast DNA extraction kit (Thermo FisherScientific, Pittsburgh) and appropriate primers. The fragments wereassembled with an additional fragment encoding EGFP by an overlapextension PCR. The resulting construct was introduced into pRS316,previously linearized with SacI and ClaI, by Gibson assembly. S cer.PAR32-3xHA and GFP-TOR1were amplified from genomic DNA and werecloned through a similar approach.

GTR1 Q65L, GTR2 S23L, and TOR1 L2134M, with their respectivepromoters and terminators, were cloned by overlap extension and Gibsonassembly after amplification from W303a/α genomic DNA. All pointmutants described in this work were constructed by overlap extension PCRat the site of the mutation using appropriate primers followed by Gibsonassembly into the linearized target vector. All primer sequences used in thiswork are available on request.

Dosage lethality screeningSelective ploidy ablation was used to introduce a control or Pib2overexpression plasmid into each strain in the non-essential haploiddeletion collection (Thermo Fisher Scientific) (Reid et al., 2011). In brief,the plasmid of interest (PGAL1-S cer. PIB2 or the control PGAL1) isintroduced into a universal donor strain (UDS), where all chromosomes areconditionally unstable, by standard transformation. Each chromosome in theUDS has both a galactose-inducible promoter and a URA3 counter-selectable marker adjacent to its centromere. The UDS containing theplasmid of interest is mated to each member of the non-essential deletioncollection. UDS chromosomes are subsequently eliminated from thediploids by centromere destabilization followed by counter selection(Reid et al., 2011). Destabilization and Pib2 overexpression aresimultaneously induced by switching to galactose as a carbon source.After induction of Pib2 overexpression, colony sizes are measured andcompared to those in the same strain containing either of two controlplasmids (PGAL1, containing only the galatose promoter, or PGAL1-EGFP)and subjected to further analysis.

Yeast colony manipulations were performed by using a BM3 colonyprocessing robot (S&P Robotics Inc., Toronto). The non-essential haploiddeletion collection was reformatted into a density of 4×384 colonies per plate,as 32×48 grids, such that each member of the deletion array was present as atetrad of four colonies. The MATα UDS, containing the plasmid of interest,was pinned into grids of 32×48 colonies per plate on SC –LEU, followed byovernight growth at 30°C. UDS colonies were pinned onto deletion arraycolonies, followed by 24 h incubation at 30°C, to allow thorough mating.

The diploids were repinned onto SC –LEU+galactose to induceoverexpression of Pib2, or the EGFP control, and simultaneousdestabilization of the UDS chromosomes. After ∼48 h, the colonies wererepinned onto SC –LEU+galactose+5-fluoro-orotic acid (5-FOA, Toronto

Research Chemicals Inc., Toronto) and incubated at 30°C. After 72 h, thecolonies were repinned onto SC –LEU+galactose+5-FOA (TorontoResearch Chemicals Inc., Toronto), followed by an additional ∼72 hincubation at 30°C, prior to colony size measurement.

SDL data analysisColony sizes from high-resolution photographs of plates were measured byusing SGAtools (Wagih et al., 2013). Colony size data were then visualizedusing the web interface of the ‘Data Review Engine’ in ScreenMill (Dittmaret al., 2010), to enable manual checking of colonies flagged for attention dueto potential pinning errors or those colonies within individual 2×2 arrays thatmay be suspect. The ‘Data Review Engine’ was also used to normalizecolony sizes to the plate median for every plate analyzed, to allow directcomparison of colony sizes between control and experimental plates.Subsequently, the normalized growth values were used to calculate Z-scoresand P-values for each member of the deletion collection overexpressingeither Pib2 or containing the control plasmid. The results were then analyzedusing the ‘Statistics Visualization Engine’ of ScreenMill. All experimentalstrains with a growth difference compared to the control strains with animplied P-value of <0.0001 were examined further.

Analysis of growth by serial dilutionFollowing overnight growth in YPD, target cells were diluted and regrownto mid-logarithmic phase in YPD at 30°C [optical density at 600 nm (OD600)of 0.6–0.8]. Cells were then diluted to 0.5 OD600/ml and 1:5 serial dilutionswere made in water. 2 µl of each dilution was spotted onto YPD or YPD+2.5 ng/ml rapamycin plates. Where relevant, cells were incubated for theindicated times with YPD supplemented with 200 ng/ml rapamycin at 30°C.After extensive washing, cells were resuspended in fresh YPD and allowedto recover at 30°C for the indicated time prior to plating on YPD. Plates werethen incubated at 30°C for 3 days prior to imaging.

Preparation of yeast for microscopyCells were grown overnight in YPD or SD medium appropriatelysupplemented to maintain plasmid selection. Cells were then diluted inYPD and grown to mid-logarithmic phase. Vacuolar membranes were stainedwith 10 µM FM 4-64 (Thermo Fisher Scientific) for 45 min, followed bywashing and incubation in YPD medium without dye for 1 h. For rapamycintreatment, cells in YPD were treated for the indicated time with a finalconcentration of 200 ng/ml rapamycin (Thermo Fisher Scientific). Forrecovery from rapamycin exposure, cells were extensively washed andresuspended in fresh YPD and incubated as indicated. Cells were plated ontoNo. 1.5 glass-bottomed coverdishes (MatTek Corporation, Ashland)previously treated with 15 µl 2 mg/ml concanavalin-A (Sigma-Aldrich).

Western blottingProtein extracts for western blotting were obtained as described previously(Millen et al., 2009). Briefly, cells were lysed on ice by resuspension in 1 mlice-cold H2O supplemented with 150 µl 1.85 M NaOH and 7.5% (v/v)β-mercaptoethanol. Protein was precipitated by addition of 150 µl 50% (w/v)trichloracetic acid. Pellets were washed twice with acetone, resuspended in150 µl 1× SDS-PAGE buffer and incubated for 30 min at 30°C followed by2 min at 95°C. Antibodies used were as follows: anti-Rps6 (1:1000, ab40820,Abcam, Cambridge), anti-PGK1 (1:1000, ab113687, Abcam), anti-EGFP(1:1000, ab290, Abcam), anti-phospho-Rps6 (1:1000, 4858, Cell SignalingTechnology, Danvers) and anti-HA (1:1000, ab9110, Abcam) antibodies.Labeled secondary antibodies were IRDye 680RD-conjugated goat anti-rabbit-IgG antibody (926-68171, Li-Cor, Lincoln) and IRDye 680RD-conjugated goat anti-mouse-IgG (926-68070, Li-Cor). These were detectedusing the Odyssey system (Li-Cor). Bands were integrated and quantifiedusing the Fiji distribution of ImageJ (Schindelin et al., 2012).

Confocal microscopy and image analysisConfocal images were acquired on a Nikon (Melville, NY) A1 confocalmicroscope, with a 100× Plan Apo 100× oil objective. NIS ElementsImaging software was used to control acquisition. Images were furtherprocessed using Fiji or NIS Elements software.

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AcknowledgementsThe authors would like to thank Suzanne Hoppins and Jeff Brodsky for extensivediscussion, and John Dittmar for assistance with ScreenMill.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: N.V.V., M.G.J.F.; Methodology: M.G.J.F.; Validation: N.V.V.,M.G.J.F.; Formal analysis: N.V.V., M.G.J.F.; Investigation: N.V.V., M.J.M., M.G.J.F.;Resources: K.A.B., M.G.J.F.; Writing - original draft: N.V.V., M.G.J.F.; Visualization:N.V.V.; Supervision: M.G.J.F.; Project administration: M.G.J.F.; Funding acquisition:K.A.B., M.G.J.F.

FundingThis work was supported by the National Institutes of Health [grants GM120102(M.G.J.F) and ES024872 (K.A.B.)]. Deposited in PMC for release after 12 months.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.207910.supplemental

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