EGOC inhibits TOROID polymerization by structurally activating TORC1

Target Of Rapamycin Complex 1 (TORC1) is a protein kinase controlling cell homeostasis and growth in response to nutrients and stresses. Glucose depletion triggers a pronounced redistribution of TORC1 from a dispersed localisation over the vacuole surface into a large, inactive condensate called a TOROID (TORC1 Organised in Inhibited Domain). However, the molecular mechanisms governing this transition were unclear. Here, we show that acute depletion and repletion of EGO Complex (EGOC) activity is sucient to control TOROID distribution, independently of other nutrient-signalling pathways. A 3.8 Å-resolution 3D reconstruction of TORC1 from TOROID cryo-EM data together with interrogation of key interactions in vivo provided structural insights into TORC1-TORC1’ and TORC1-EGOC interaction interfaces. These data support a model in which glucose-dependent activation of EGOC triggers binding to TORC1 at an interface required for TOROID assembly, resulting in inhibition of TORC1 polymerisation and release of active TORC1 dimers. S758


Introduction
The Target Of Rapamycin, TOR, is a Phosphatidylinositol Kinase related protein Kinase (PIKK) family member 1,2 as it resembles lipid kinases but is rather a Ser/Thr protein kinase. Similarly to other PIKKs, TOR functions as the catalytic subunit of distinct multiprotein complexes, TORC1 and TORC2. These complexes are broadly conserved across eukaryote evolution, however, the rapamycin-sensitivity of TORC1 has facilitated studies of this complex and consequently TORC1 signalling is relatively better characterized compared to rapamycin-insensitive TORC2 3,4 . Both complexes are essential regulators of cell homeostasis: in yeast, TORC1 controls cell mass and volume according to nutrient availabilities and abiotic stresses while TORC2 maintains biophysical properties of the plasma membrane 5 .
Yeast TORC1 is composed of four proteins: either of the two TOR paralogs Tor1 or Tor2, Kog1, Lst8, and Tco89 6,7 . In humans, mTORC1 (mammalian/mechanistic Target Of Rapamycin Complex 1) respectively contains mTOR, Raptor and mLst8; a Tco89 orthologue is not apparent. TORC1 phosphorylates the AGCfamily kinase Sch9 which served as a convenient reporter to demonstrate that TORC1 is active in the presence of good carbon (glucose), nitrogen (glutamine), and phosphate sources and inactivated upon nutrient deprivation as well as exposure to osmotic, redox or thermal stresses 8 . Sch9 and other TORC1 effectors subsequently regulate a plethora of molecular processes ranging from ribosome biogenesis to autophagy; from lipid biosynthesis to membrane tra cking [9][10][11][12] . mTORC1, which also phosphorylates an AGC-family kinase, S6K1, is additionally regulated downstream of growth-factor signalling and loss of mTORC1 regulation has been implicated in a large range of illnesses including cancers, neurodegenerative diseases and metabolic disorders 13 . Given its central role in physiological and pathophysiological growth control, molecular dissection of (m)TORC1 signalling is of considerable interest.
Structures of mTORC1 have been recently solved by combinations of X-ray crystallography and electron cryo-microscopy (cryo-EM) [14][15][16] . These studies demonstrated that mTORC1 is dimeric and, when bound, the macrolide rapamycin recruits the 12 kDa proline isomerase FKBP12 such that substrate access to the kinase active site is sterically occluded. Under physiological conditions, mTORC1 activity is regulated through the actions of multiple GTPases. The Rheb GTPase couples growth factor and energy cues to mTORC1 with the binding of GTP-loaded Rheb leading to allosteric rearrangements that increase the kinase activity of mTORC1 16 . In contrast, the RAG GTPases have a more nuanced role in mTORC1 regulation, primarily downstream of nutrient cues.
There are two sets of RAG paralogues in mammalian cells: RAGA and RAGB, and RAGC and RAGD. These proteins form obligate heterodimers with GTP-loading status determining the distance and relative orientation between the two G-domains 17,18 . Unlike other GTPases, the RAGs are not modi ed with lipid but are instead anchored to the lysosome membrane via the heteropentameric Ragulator complex [19][20][21] .
Recent structures of the Raptor•RAG•Ragulator complex suggest that active RagA GTP /RagC GDP is able to bind the Armadillo (ARM) domain of Raptor and grasp onto a speci c short α−helical fragment termed the 'Claw' 22,23 . This binding recruits mTORC1 from a mysterious localization in the cytoplasm to the lysosome where it can be subsequently activated by Rheb GTP .
The regulation of TORC1 in Saccharomyces cerevisiae (budding yeast) does not completely parallel the situation in mammalian cells. In contrast to mammalian cells and Schizosaccharomyces pombe 24 , there are no reported roles for the budding yeast Rheb orthologue, Rhb1, in TORC1 regulation. Furthermore, yeast TORC1 appears to be constitutively localized to vacuole and/or endosome membranes, regardless of its activity status 25,26 . We recently reported that acute glucose starvation triggers the condensation of TORC1 into a large, vacuole-associated helical polymer we named a TOROID (TORC1 organised in Inhibited Domains; 27 . TOROID formation tracked with, and was necessary for, Sch9 dephosphorylation, implying that this polymerization was mechanistically responsible for TORC1 inactivation. Consistently, a low-resolution TOROID structure suggested that stacking of TORC1 protomers (i.e. dimers) into the TOROID polymer would sterically occlude access to the kinase active site analogously to rapamycin-FKBP12 binding 27 . TOROID formation was found to be dependent upon the two yeast RAG orthologues GTR1 (RAGA/B) and GTR2 (RAGC/D): Δgtr1 Δgtr2 cells present constitutive TOROIDs even in the presence of glucose and are impaired in their ability to downregulate Sch9 phosphorylation upon glucose starvation 27 .
Gtr1 and Gtr2, like the RAGs, are obligate heterodimers anchored to the vacuolar membrane via the EGO ternary complex (EGO-TC, the functional equivalent of Ragulator) composed of Ego1, Ego2 and Ego3 28 . Ego1 is myristoylated and palmitoylated on its N-terminal extremity, which serves to tether the complex to the vacuolar membrane 29 . The remainder of Ego1, together with Ego2 and Ego3, cradle the C-terminal dimerization domains of Gtr1 and Gtr2 28 . This orientation exposes the N-terminal G-domains of Gtr1 and Gtr2 to upstream regulators as well as their effector, TORC1. The GTPase activating Proteins (GAPs) that act on Gtr1 (RagA/B) and Gtr2 (RagC/D) are SEACIT (GATOR1) and Lst4/7 (FNIP-FLCN) respectively 30,31 . In contrast to the relatively well-understood and accepted GAPs, numerous Guanine nucleotide Exchange Factors (GEFs) that act on Gtr1 (RagA/B) have been reported 18,25,31 . A GEF for Gtr2 (RagC/D) may not be necessary as this GTPase appears to possess an intrinsically high dissociation constant for GDP 32 . Similar, but not identical to the situation in mammalian cells, guanine nucleotide loading status of the Gtrs appears to have a direct role on TORC1 localization in yeast. Indeed, expressing mutants that lock the Gtrs in an inactive, Gtr1 GDP /Gtr2 GTP conformation, TORC1 is concentrated in a TOROID punctum on the vacuolar membrane whereas in cells expressing active, Gtr1 GTP /Gtr2 GDP , TORC1 is diffusely localized over the surface of the vacuole 27 . Molecular details explaining how the EGO-TC / Gtrs (collectively known as the EGO Complex, EGOC) govern TOROID assembly, alone or in conjunction with other nutrient-regulated signalling pathways, remain unknown.
To speci cally assess the contribution of the EGOC to TORC1 localization dynamics, independently of the multitude of other glucose-responsive signalling pathways 33 , we developed orthogonal approaches by which we could acutely abrogate or reintroduce EGOC activity. We observed that proteolytic shaving of the EGOC off of the vacuole membrane triggered TORC1 puncta (TOROID) formation while reintroduction of the EGOC to the vacuole membrane triggered puncta disassembly. We additionally observed that the EGOC co-localizes with TORC1 puncta in a Gtr-dependent fashion. To better characterize this interaction, we used cryo-EM to obtain a 3.8 Å-resolution structure of TORC1 from TOROID cryo-EM maps. We found that a central hub of interactions locks the Kog1 Armadillo (ARM) domain on top of the kinase domain of Tor2' and sterically occludes the kinase active site. CrispR/Cas9 mutagenesis and pull-down assays were used to verify these core features and additionally revealed that this same hub is engaged by the EGOC to mediate glucose-dependent regulation of TORC1 signalling. Based on these data, we propose that the EGOC is necessary and su cient for TOROID regulation through competition for a common binding hub in Kog1; trans binding of this hub to Tor1'/2' sequesters TORC1 into an inactive TOROID, while binding to active EGOC liberates TORC1 dimers able to signal to downstream targets.

Results
Acute abrogation or restoration of EGOC activity on the vacuole membrane impacts TOROID dynamics independently of glucose changes We previously proposed that TOROID regulation downstream of glucose-derived signals was mediated via the EGOC 27 . This was based on constitutive presence of TOROIDs in Δgtr1 Δgtr2 cells and an inability of cells expressing GTP/GDP-locked Gtr1/2 mutants to regulate TOROID assembly/disassembly according to glucose cues. However, knowing that glucose signals regulate TORC1 activity via additional, parallel pathways including Snf1/AMPK and pH 34,35 , we wished to determine if orthogonal, glucoseindependent manipulation of EGOC function alone would be su cient to alter TOROID formation.
To rapidly abrogate EGOC signalling, we introduced a Tobacco Etch Virus (TEV) Protease cleavage site into the amino-terminal region of Ego1, downstream of the lipidation sites (Ego1 TEV ; Figure 1A and S1A) 28,36 . TEV protease was expressed downstream of the CTH2 promoter, whose tight repression by Fe 2+ is quickly reversed by addition of the iron chelator Bathophenanthrolinedisulfonic acid (BPS; 37 . Neither acute BPS treatment nor TEV protease induction affected TORC1 activity in otherwise WT cells ( Figure  S1B).
While the EGO1 TEV allele per se does not display a phenotype, expression of the TEV protease renders these, but not WT cells, hypersensitive to rapamycin demonstrating a loss of EGOC function similar to cells lacking functional EGOC ( Figure S1C; 25,38 . In EGO1 TEV cells, prior to TEV induction, the EGOC was diffusely localized around the vacuole membrane; this signal was diminished by 50% after 30 minutes of TEV induction ( Figure 1B and S1D). After 75 minutes, the EGOC was almost completely shaved off of the vacuole similarly to Δego1 strain ( Figure 1C). TORC1 puncta formation tracked with EGOC removal from the vacuolar membrane displayed twice more cells presenting TOROIDs after ~60 minutes of BPS treatment ( Figure 1C, 1D and Figure S1D). Measurements from single-cell estimate the half-time of TOROID formation upon EGOC depletion to be about 7 minutes ( Figure 1E and 1F). Taken together, these data demonstrate that acute depletion of EGOC from the vacuole is su cient to induce TOROID formation.
To rapidly re-establish EGOC signalling, we exploited the vacuole fusion that occurs during zygote formation 39 . Speci cally, we crossed a MATa Δgtr1 Δgtr2 strain, which constitutively displays a TOROID marked with GFP-Tor1, with a MATα partner containing Vph1-mCherry to delineate the vacuolar membrane ( Figure 1G). As a control, we also used a MATα Δgtr1 Δgtr2 VPH1-mCherry partner. After mixing of the mating partners, budding zygotes were staged as either early, intermediate, or late ( Figure   1G). Early zygotes possessed a small bud and fragmented vacuoles that remained in the MATα parent. Intermediate zygotes possessed larger buds in which some Vph1-mCherry signal was observed. Late zygotes possessed a bud roughly the size of the parent cells and vacuole exchange between the parents in addition to the bud was observed. In crosses between Δgtr1 Δgtr2 parents, a TOROID, initially present in the MATa parent, started to be also detected in about 50% of buds and some MATα parents, already at the intermediate zygote stage (Figure 1G, and 1H). This pattern was not observed in crosses with MATα GTR1 GTR2 VPH1-mCherry. In this case, TORC1 puncta were observed in only very few buds, and, at late stages even disappear from the MATa parent ( Figure 1H). Live, single-cell imaging also demonstrated that TOROIDs from this mating dissolve promptly upon entry into the bud, upon mixing with vacuolar membrane possessing functional EGOC ( Figure 1I). The half-time to complete punctum dissolution was approximately 2.5 minutes ( Figure 1J). These data indicate that acute loss or re-establishment of EGOC activity, in the absence of potentially confounding inputs from other nutrient-regulated signalling pathways, is su cient to trigger or dissolve TOROIDs, respectively. This supports the notion that the EGOC regulates TOROID formation, potentially through direct physical interaction with TORC1 and/or TOROIDs.
TOROIDs partially co-localize with EGOC puncta Previous work demonstrated that Gtr1/2 can physically interact with TORC1 25,40 and our results suggested that this interaction is necessary for regulating TOROID formation. To probe this idea, we assessed how the EGOC and TORC1 localize with respect to one another. At the outset, we ensured that introduction of uorescent tags did not compromise protein function. This was critical as we frequently encountered rapamycin hyper-sensitivity in singly, and especially doubly tagged strains that correlated with aberrant protein localizations. For example, using a GFP-Tor1, Ego3-3xmCherry strain, it has recently been shown that TORC1 and EGOC co-localize in discrete puncta 41,42 . However, in this background there appears to be a synthetic interaction between these TOR1 and EGO3 alleles as TORC1 puncta are present even in glucose-replete conditions ( Figure S2A). We noted similar, synthetic loss-of-function phenotypes between GFP-TOR1 and EGO3-mScarlet, EGO2-mScarlet, and EGO1-mCherry (data not shown). Importantly, when assessed separately, both TORC1 (marked with GFP-Kog1) and the EGOC (marked with EGO3-GFP) localized diffusely around the vacuole membrane in glucose-replete cells but collapsed into a punctum within 10 minutes of acute glucose depletion. Both puncta dissolved again within minutes of glucose replenishment (Figure 2A-B). We note that failure to appreciate the hypomorphic nature of tagged alleles may lead to incorrect conclusions and likely underlies discrepancies found in the literature.
A strain expressing GFP-Kog1 and Ego3-3xmCherry appeared to be relatively uncompromised with nearly WT levels of TOROIDs in glucose-replete and post-diauxic shift (PDS) cultures ( Figure 2C and S2A). When these cells were cultured PDS, co-localization between TORC1 and EGOC was moderate, as re ected by a Pearson's coe cient of 0.44 ( Figure S2B). This co-localization also appeared to by asymmetric: while the EGOC signal overlapped strongly with the TORC1 signal (Mander's coe cient (MC): 0.71; Figure S2B), the TORC1 signal overlap with the EGOC signal was less robust (MC: 0.45; Figure S2B). Further characterization showed that small EGOC puncta did not co-localize with the TORC1 signal (Pearson's coe cient (PC <500nm ): -0.004 ± 0.095; Figure S2C) while large EGOC puncta did (PC >500nm : 0.525 ± 0.175). In contrast, large and small TORC1 puncta displayed similar overlaps with the EGOC signal (PC <500nm : 0.315 ± 0.023; PC >500nm : 0.361 ± 0.095; Figure S2C). Together, these data show that both EGOC and TORC1 form condensates upon acute glucose depletion or in PDS cells, and that large EGOC puncta seem to require a TOROID but not vice versa.
We next wished to characterize how guanine nucleotide loading status of the Gtrs affects EGOC puncta formation. Although deletion of GTR1 and GTR2 promotes TORC1 puncta formation (Prouteau et al., 2017, Figure 1), EGO-TC puncta were absent in these strains ( Figure 2D). Reintroduction of "active" GTR alleles (Gtr1 GTP /Gtr2 GDP ) antagonized both TORC1 and EGOC puncta formation regardless of the growth state while reintroduction of "inactive" GTR alleles (Gtr1 GDP /Gtr2 GTP ) enhanced formation of TOROIDs but, curiously, not EGOC puncta ( Figure 2E-F). In Δgtr1 Δgtr2 and Gtr1 GDP /Gtr2 GTP strains which contain TOROIDs, TORC1 activity is reduced as evidenced by their rapamycin hypersensitivity ( Figure 2G). As a complementary means to assess the effects of nucleotide loading status of the Gtrs on EGOC and TORC1 puncta formation we also used mutant strains in which Gtr GAP activities had been altered.
Deletion of IML1/SEA1, which compromises SEACIT activity leading to Gtr1 GTP accumulation, antagonized EGOC and TORC1 puncta formation ( Figure S2D-F). In contrast, deletion of SEA3, a proposed SEACAT component, augmented EGOC and TORC1 puncta formation ( Figure S2D-F). Finally, deletion of LST4 or LST7, which leads to Gtr2 GTP accumulation, promoted EGOC and TORC1 puncta formation ( Figure S2D-F). Collectively, these results con rm and extend our previous observations 27 that the nucleotide loading status of the Gtrs dictate TOROID formation: whether generated by nutrient levels, mutations in the GAPs or mutations in the GTRs themselves, Gtr1 GTP /Gtr2 GDP antagonize, while Gtr1 GDP /Gtr2 GTP promote, TOROID formation. The EGOC also forms a punctum, which in most, but not all cases, correlates with the presence of a TOROID, the notable exceptions being the lack of EGOC puncta in Δgtr1 Δgtr2 cells and in exponentially growing cells expressing "inactive" GTRs. These last results demonstrate that EGOC puncta are not necessary for TOROID formation but can assemble with TOROIDs in a Gtr-dependent manner. In contrast, active EGOC is needed to prevent TOROID formation.
Wishing to further characterize the molecular mechanism by which the EGOC interacts with TORC1 to regulate its supramolecular organization we rst performed in vitro binding assays between EGOC variants and free TORC1 dimers puri ed from exponentially growing cells or TOROIDs from PDS cells. These assays demonstrated that puri ed "active" EGOC interacts preferentially, albeit weakly, with TORC1 dimers ( Figure 2H-I). In contrast, puri ed "inactive" EGOC interacts robustly with TORC1 dimers and even more so with TOROIDs ( Figure 2H-I). Thus, in contrast to the situation reported for mammalian cells 21 , in yeast, the EGOC binds TORC1 regardless of which guanine nucleotides are bound to the Gtrs. This is consistent with prior genetic observations that the EGOC can both positively and negatively regulate TORC1 activity 25 , see discussion).

High resolution Cryo-EM TOROID Structure
To better understand the molecular regulation of TOROID (dis)assembly, we puri ed TORC1 from KOG1-TAP ∆tor1 cells grown PDS and acquired a high-resolution TOROID structure by cryo EM. We hypothesized that TOROIDs containing only Tor2, rather than a mix of Tor1 and Tor2, could yield a higher resolution structure. Initial attempts to determine the TOROID structure using helical reconstruction resulted in 3D reconstructions at rather low resolution (FSC 0.143 : 9.1 Å), despite the apparent high quality of the obtained data and ensuing 2D class averages ( Figure 3A, Figure S3A-B). The relatively poor precision of helical symmetry determination on the un-symmetrized map ( Figure S3A) suggested that instead of being a rigid helix, TOROID laments resemble a exible, slinky-like spring with varying pitch. Therefore, helical reconstruction most probably results in averaging out of TORC1 units along the helical assembly. To overcome this issue, we employed signal subtraction protocols 43,44 , using the known positions of TORC1 units in the helical reconstruction, to obtain a set of signal subtracted particles that could be further analyzed using a regular single particle analysis (SPA) pipeline ( Figure S3A). We used a broad 3D mask including parts of neighboring TORC1 units surrounding a central TORC1 in the TOROID helix to ensure that information on the TORC1-TORC1' interfaces remained intact.
3D reconstruction using signal-subtracted TORC1 particles and employing D1 symmetry and non-uniform re nement in CryoSPARC 45 resulted in a 3.85 Å map according to the gold standard FSC=0.143 criterion, which enabled us to build a nearly complete structural model of yeast TORC1 in the TOROID ( Figure 3B, Figure S3A-C). While the nal map displays heterogeneity in terms of resolution ( Figure S3B-C), the symmetric nature of the TOROID assembly could be exploited to unambiguously build the majority of the TORC1 structure, with many regions showing density for amino acid side chains ( Figure 3D). Moreover, it is apparent from the 3D reconstruction and local resolution estimation that the interactions between adjacent TORC1 units along the TOROID helix (intra-coil) are far more prominent than interactions along the helix z-direction (inter-coil; Figure 3A, S3B-C), which is also re ected by the resolution difference at the intra-coil and inter-coil interfaces.
The overall yeast TORC1 structure extracted from TOROIDs is very similar to its mammalian counterpart (mTORC1, Figure 4A), as shown by a structural alignment of individual Tor2/mTOR ( Figure 4B), Kog1/RAPTOR ( Figure 4C) and Lst8 subunits ( Figure S4A, B). While yeast and human Lst8 are virtually identical (RMSD= 0.937 Å over 252 aligned main-chain atoms), the main differences between Tor2/mTOR (RMSD= 3.311 over 1608 aligned main-chain atoms) and Kog1/RAPTOR (RMSD= 2.371 over 866 aligned main-chain atoms), can be traced to a conformational difference of Tor2, and the presence of previously uncharacterized additional α-helical regions for Kog1 ( Figure 4B-C). We compared our structure of yeast TORC1 present in TOROIDs with structures of inactive mTORC1 and active mTORC1 bound to Rheb 16 . Tor2 in TORC1 in TOROIDs is structurally more similar to mTOR in inactive mTORC1 (RMSD inactive = 3.311 Å) than in Rheb bound mTORC1 (RMSD active = 10.388 Å; Figure 4B). Indeed, TORC1 in TOROIDs has an even more "open" conformation than inactive human mTORC1 ( Figure 4A): the HORN region of Tor2 makes a 7° or 23° outward shift when compared to inactive or Rheb-bound mTORC1 respectively ( Figure 4B).
Comparison of yeast Kog1 and human RAPTOR reveals a 'Twix' region contributed by two α-helices connected by a poly-glutamine linker that is present in Kog1 but completely absent from human RAPTOR.
Alignment of S. cerevisiae Kog1 with orthologous sequences from H. sapiens, S. Pombe and C. thermophilum indicated that the Twix is uniquely present in budding yeast ( Figure S4C-D). The Twix region is situated between α-helices 26 and 29 of Kog1 ( Figure S4C-D), and interacts with the FRB' domain of Tor2' in an adjacent TORC1' subunit with residues Gln2039, Asp2042 and Tyr2045 of Tor2' respectively interacting with Glu865, Tyr829/Asn872 and Asn 876 of Kog1 ( Figure 4D). Adjacent to the Twix region, we observed density for an α-helix not directly connected to surrounding parts of the structure, yet in close proximity to Lys1016 of Kog1 which terminates α-helix 34 (residues 1005 -1016, Figure S4D). Careful inspection of the sequence succeeding Lys1016 and ensuing model building in Coot led to the identi cation of the region as being a helix with sequence 'P 1069 MRTSLAKLFQSLGFSES 1086 ' ( Figures 3D, 4D, S4E), which we termed the 'Tack' region. Sequence alignments demonstrated that this region precedes the sequence that aligns with the RAPTOR 'Claw' which was shown to interact with RagC in RAPTOR-Rag-Ragulator reconstructions 22,23 . In this vicinity, the structure displays a number of prominent molecular interactions leading to a robust TORC1-TORC1' binding interface that we term the hub of interactions. Phe1083 and Ser1086, at the very tip of the Tack α-helix, make Van der Waals interactions with Pro2201 of the KD' region of Tor2' and Arg884' of Kog1' respectively ( Figure 4D). Adjacent to Pro2201, Trp2207 sits in between Arg884, Lys885 and Arg888 of Kog1, forming a prominent cation-pi interaction with Arg884 situated near the tip of the Tack α-helix and the base of the Twix region ( Figure 4D). The Twix and the Tack shield the kinase active site and thereby block substrate access, providing, together with the large allosteric change described above, a structural explanation for why TORC1 in TOROIDs is inactive ( Figure 4D, Figure S4C).

Mutations con rm observed TORC1/TORC1' interfaces
To interrogate the structural insights obtained from the observed interfaces, we selected sets of residues in the inter-and intra-coil interfaces for mutation using CrispR/Cas9 ( Figure 5A-B and S5). The impact of these mutations was assessed by determining the relative size of TORC1 puncta in cells grown PDS ( Figure S5E). In parallel, all mutants were tested in spot assays on rapamycin plates to identify mutations that compromise TORC1 activity ( Figure S5F). In these analyses, we rst assessed what we considered to be control mutations -mutation of exposed residues in the vicinity of the intra-and inter-coil interfaces, but not obviously engaged in TORC1-TORC1' interactions. As expected, these mutations (Kog1 K1383A and Kog1 Δ1544−end ) did not signi cantly affect TOROID size or rapamycin sensitivity ( Figure S5A, C and F). In contrast, mutation of W1279 of Tor1, which is not predicted to mediate contacts within the helix but is predicted to be important to stabilize the FAT domain, yielded a reduction in TOROID size as well as rapamycin hypersensitivity, consistent with a loss of TORC1 particle integrity ( Figure S5A, C and F).
Inter-coil interactions, although clearly visible in the helical reconstruction, appear almost completely absent in the signal subtracted map, and only become apparent when using a higher contour threshold level during visualization. Based on our helical reconstruction, the inter-coil interface is predominantly formed between the Tor2 HORN and the Kog1' WD domain ( Figure 3). To validate this, two exposed loops in the HORN, Tor2 337-345 (Tor1 326-334 ) and Tor2 379-381 (Tor1 368-370 ), were substituted by poly alanineglycine stretches. These strains displayed a signi cant reduction in TOROID size as well as an increase in rapamycin resistance ( Figure S5B and S5D-F). An additional protruding region of the Tor2 kinase domain also appeared to interact with Kog1'. Although attempts to generate mutations in this region of Tor2 were unsuccessful, mutation of the equivalent region of Tor1 (Tor1 1449/54/56A ) similarly resulted in a reduced TOROID size and increased rapamycin resistance ( Figure S5B and S5D-F).
Given the higher resolution of this region in the signal subtracted map, amino acids involved in intra-coil interactions were easier to trace ( Figure 5 and S5A, S5C and S5E-F). Intra-coil interactions are plentiful, and mainly contributed by Kog1 interacting with Kog1', Lst8', and the KD' and FAT' regions of Tor2' from an adjacent TORC1' unit. Glu784 and Ser896 of Kog1 interacts with His292 of Lst8' and alanine substitution of this His reduces TOROID formation (Lst8 H292A ; Figure 5A Striking amongst the intra-coil interactions, and as alluded to above, is the TORC1-TORC1' hub of interactions, formed through extensive contacts between Kog1 and Tor2'. These contacts include interactions between the Twix helices of Kog1 and the FRB domain of Tor2' (Figure S4C-D), the tip of the Tack helix of Kog1 and the KD of Tor2' (Figure 4D, S4C and 5), and a basic pocket of Kog1 that hosts Trp2207 from the Lst8 Binding Element (LBE) of Tor2'. The apparent cation-pi interaction between Tor2 W2207 and Kog1 R884 where particularly intriguing as mutations in the corresponding residues of mTOR and RAPTOR (mTOR R2266P and Raptor D635N ) have been identi ed in human carcinomas (https://cancer.sanger.ac.uk/cosmic). To test if this cation-pi interaction is important for TORC1 regulation, we assessed the consequences of combined mutations of Trp2207 of Tor2 and the analogous site in Tor1 (Tor1 W2203R , Tor2 W2207R ), anticipating to create a charge repulsion with Kog1'. Consistently, in this strain, TOROID formation was absent ( Figure 5C and S5E), and TORC1 was hyperactive as shown by increased rapamycin resistance ( Figure S5F). We attempted to rescue these phenotypes by reverting Arg884 of Kog1 to Asp, the corresponding amino acid found in RAPTOR. However, KOG1 R884D neither blocked TOROID formation on its own, nor did it rescue the increased rapamycin resistance and lack of TOROIDs of TOR1 W2203R TOR2 W2207R cells (data not shown), perhaps owing to the remaining positively charged residues surrounding Arg884 (see below). The potential signi cance of these cancer associated mutations is discussed below.
TOROID-like structures have not been reported in other systems, including mammals and we wondered if the yeast-speci c Twix helices of Kog1 might be a reason why. To test if the Twix region is necessary for TOROID formation, and considering the poor local sequence conservation, we initially, and rather conservatively, replaced a region from α26 to α29 (including the Twix helices: α27 α28), with the corresponding, smaller region of human Raptor to generate an allele that we named kog1 Humanized Chimera (kog1 HC ; Figure 5C). Cells expressing this allele of KOG1 cultured PDS formed TOROIDs indistinguishably from WT cells ( Figure 5B, C and S5E) suggesting that the Twix region is not required for TOROID formation (see also below).

kog1 HC allele unveils the TORC1/EGOC interaction domain
The kog1 HC allele also replaces helix α29 of yeast Kog1 which contains the three basic residues (Kog1 R884 , Kog1 K885 and Kog1 R888 ) thought to interact with Trp2207 of Tor2 with the equivalent helix α29 of Raptor which contains acidic amino acids instead of these arginines ( Figure S4D). Although KOG1 R884D did not suppress the inability of TOR1 W2203R TOR2 W2207R cells to form TOROIDs we wondered if kog1 HC might. Indeed, we found this to be the case ( Figure S5E) but, as presented below, not necessarily through charge complementation of Tor1 W2203R Tor2 W2207R .
As expected, the TOR1 W2203R TOR2 W2207R kog1 HC strain presented a strong reduction in rapamycin resistance compared to the TOR1 W2203R TOR2 W2207R strain ( Figure S5F). However, we unexpectedly noticed that kog1 HC cells displayed hypersensitivity to rapamycin, to a level comparable to ∆gtr1 ∆gtr2 cells ( Figure S5F). One explanation for the rapamycin hypersensitivity of kog1 HC cells would be if they, like ∆gtr1 ∆gtr2 cells, constitutively from TOROIDs even in the presence of glucose. Thus, we decided to re-assess TORC1 localization in kog1 HC cells, as well as in all of the other mutant strains that we had created, in glucose-replete conditions. Amongst all of these strains, the kog1 HC strain alone displayed TOROIDs in glucose-replete conditions similarly to ∆gtr1 ∆gtr2 cells ( Figure 6A-D and data not shown).
Guessing that these phenotypes might be due to the human sequences (helices α28 and/or α29 of Raptor) that were introduced during the construction of kog1 HC , we generated a new kog1 allele in which the Twix helices α27 and α28 of Kog1 were "cleanly" replaced with a short (AG) 4 linker ( Figure 6A). We named this allele, kog1 ΔTwix . kog1 ΔTwix , like kog1 HC , formed TOROIDs normally when cells were cultured PDS ( Figure 6B), but, unlike kog1 HC , do not form TOROIDs in exponential phase and displayed nearly normal sensitivity to rapamycin ( Figure 6C). These observations con rm that the Twix helices are not a prerequisite for TOROID formation or normal TORC1 activity but that Kog1 helix α26 and/or α29 is important for basal TORC1 activity in a manner that cannot be complemented by the corresponding Raptor sequence.
We hypothesized that the phenotypes observed in the kog1 HC strain could be explained if TORC1 in these cells was unable to engage the EGOC. Several additional observations support this model. Similarly to ∆gtr1 ∆gtr2 cells, in kog1 HC cells, EGOC puncta were not observed ( Figure 6D-E), and TORC1 activity, as assessed by Sch9 phosphorylation, was reduced and largely insensitive to acute changes in glucose levels ( Figure 6G). Furthermore, in pulldown experiments, unlike WT TORC1, TORC1 containing Kog1 HC failed to robustly interact with puri ed EGOC ( Figure 6H-I, S6A). Collectively, these results suggest that a key binding site for EGOC lies within helix α26 and/or α29 of Kog1, i.e. within the TOROID hub of interactions. They also demonstrate that TOROID assembly is necessary, but not su cient for EGOC puncta formation (see Figure 2E, F; cells expressing inactive Gtr1 GDP /Gtr2 GTP , do not e ciently form EGOC puncta in glucose-replete conditions despite the presence of TOROIDs).
Based on these observations we wondered if kog1 HC suppressed the inability of TOR1 W2203R TOR2 W2207R cells to form TOROIDs because of its own inability to bind the EGOC rather than, or in addition to, charge complementation. Consistent with this idea we observed that the ∆gtr1 ∆gtr2 double deletion was epistatic to TOR1 W2203R TOR2 W2207R : ∆gtr1 ∆gtr2 TOR1 W2203R TOR2 W2207R cells presented constitutive TOROIDs ( Figure S6B). The implications of this observation are discussed below.
CrispR-based random mutagenesis con rms that the EGOC-TORC1 interaction interface overlaps with the TORC1-TORC1' hub of interactions Using the strains generated thus far, we observed a strong anti-correlation between TOROID size in PDS and rapamycin sensitivity (Pearson=-0.75; Figure S6C). Mutations compromising TORC1-TORC1' interactions have small TOROIDs and are resistant to rapamycin while mutations abrogating TORC1-EGOC binding have large TOROIDs and are sensitive to rapamycin. Thus it appears that sensitivity to rapamycin depends on the proportion of free and active TORC1 dimers ( Figure S6C). Exploited this idea we set up a CrispR-based PCR random mutagenesis screen to further de ne the TORC1-EGOC interface. A portion of the ARM domain of Kog1 surrounding α26 -α29 (residues 696-922) was chosen for mutagenesis ( Figure 7A). The rst selection step was rapamycin-hypersensitivity, expecting that mutants of interest would present a hypersensitive phenotype similar to Δgtr1/2 and kog1 HC strains. The second step was to select for TOROID formation in glucose-replete cultures. A total of 3500 mutants were screened on 2.5 and 4 nM rapamycin plates. While the latter concentration appeared to be too stringent, selection with 2.5 nM rapamycin yielded 42, rapamycin-sensitive clones. Among these, 13 presented TOROIDs in glucose-replete media and these were retained and sequenced ( Figure 7A and S7A-D). 17point mutations were identi ed, of which 7 (marked in bold below) were the only mutation present in the clone and could thus be deemed causative ( Figure 7B, S7B-D). Remarkably, while the Twix-encoding region was included in this screen, no mutation within this sequence was recovered, supporting the idea that it does not participate in EGOC-dependent regulation of TOROIDs.
Several mutations, alone (bold) or in combination, altered leucine residues involved in hydrophobic interactions between α-helices (L762P, L765P, L766P, L900P, L912Q; Figure 7B and S7C-D) suggesting that local perturbation of domain packing may affect EGOC binding. L737Q (found in combination with L762P), at the junction of α21-22 helices, C777R and M778T in helix α24, I802N and A804E in helix α26, and I891N in helix α29 were recovered ( Figure 7B and S7B-D) and we note that many of these map to a region which in Raptor is important for interaction with RagA (Raptor residues N557, C594 and 634-36; Figure 7B and alignment Figure S4D); 23 ). Building on this observation we found that Kog1 helix α21 (which corresponds to α23 in Raptor, known to be important for RagA binding) is essential for proper TOROID dynamics since its substitution to an Alanine-Glycine stretch triggers constitutive TOROID formation ( Figure 7C). Finally, we found that puri ed TORC1 harboring Kog1 C777R or AG-substituted Kog1 helix α21 respectively presented much reduced or no EGOC binding activity ( Figure 7D). Thus, Kog1 appears to interact with the GTRs similarly to how Raptor interacts with the RAGs.
Remarkably, the Tack helix appears to correspond to the Claw helical fragment of Raptor described to interact with RagC. To probe the importance of the Tack helix in TOROID regulation we made three poly AG substitutions. Replacement of the proximal α-helix 34 (Kog1 1004-1022 ) had no effect on TOROID regulation, while replacement of the entire Tack helix (Kog1 1121-1131 ) leads to a mild, but signi cant, impairment of TOROID formation ( Figure S7E, F). Replacement of the putative yeast Claw sequence (Kog1 1121−1131 ; 23 presented no signi cant effect on either TOROID formation or regulation ( Figure S7E-F), whereas equivalent mutations in Raptor prevent Rag-binding 23 which we predict would lead to constitutive TOROID formation. From these observations we conclude that, although similarly localized, the Tack helix does not function equivalently to the Raptor Claw. Instead, it provides structural elements (Kog1 F1083 binding to Tor2' P2201 ) needed to stabilize the TOROID.
Based on these collective observations, we propose that, analogous to the binding observed in mammalian cells, EGOC binds to helices α21, α24, α26 and α29 of Kog1. This binding ( Figure 7G) competes with a critical interface needed for TOROID formation, and would thus release/sequester free TORC1 dimers that are competent to signal to downstream effectors.

Discussion
We previously reported 27 that glucose withdrawal triggers a rapid, Gtr1/2-dependent reorganization of TORC1 from a disperse localization over the surface of the vacuole to a hollow helical assembly that we named a TOROID. Our, at this time, low-resolution, 3D reconstruction revealed that TOROID-sequestered TORC1 is inactive due, at least in part, to rapamycin-FKBP12-like occlusion of the kinase active site. This unexpected mechanism underlying TORC1 regulation differs from the widely accepted model of mammalian TORC1 regulation and raised some important questions, some of which we have addressed in the present study.
We show here that the EGOC alone is su cient to regulate TORC1/TOROID dynamics. Although we conclude that EGOC-mediated TOROID formation represents an important mechanism for the cell to regulate TORC1 signaling output, we do not wish to imply that this is the only mechanism through which glucose or other stimuli regulate TORC1. Indeed, in the absence of TORC1-EGOC interactions (e.g. ∆gtr1 ∆gtr2 or kog1 HC cells) TORC1 is still partially regulated by glucose withdrawal/repletion ( Figure 6G, 27 ) and by osmotic shock (data not shown). This clearly indicates the existence of EGOC-independent TORC1 regulators. How such regulators (e.g. Snf1 and Pib2 46,47 interface with TORC1/TOROID-based regulation remains an important unknown. The observation that some TORC1 activity remains in ∆gtr1 ∆gtr2 cells -and mTORC1 activity in ragA ragB ragC ragD cells 48 -implies that not all TORC1 is sequestered into TOROIDs in this genetic background. Indeed, we favor a model in which there is a dynamic equilibrium between active, free TORC2 dimers and inactive TOROID polymers in a cell. Mutations or stimuli that shift the equilibrium towards free dimers would stimulate TORC1 signaling output, and vice versa. This hypothesis is supported by the strong anti-correlation that we see between TORC1 puncta size and rapamycin resistance (Supplementary Figure 6B). In such a model, one could envision that non-EGOC TORC1 regulators independently act on the pool of free TORC1 dimers. Additionally, distinct pools of TORC1 (e.g. endosomal TORC1; 26 could be subjected to distinct upstream regulatory mechanisms. The major advance of our present work, however, is the molecular insights we provide to explain how binding of EGOC liberates free TORC1 from TOROID polymers. Our new, high-resolution TORC1 map reveals that TOROIDs are rather spring-like with loose inter-coil interactions and robust intra-coil interactions. The weak inter-coil interactions bestow exibility upon the TOROID structure for an as of yet unknown purpose. In contrast, the strong intra-coil interactions serve to occlude access to the active site, which, together with allosteric changes to the catalytic spine, ensures that TOROID-associated TORC1 is inactive. Closer inspection of the TORC1-TORC1' interface proximal to the kinase active site revealed an intriguing hub of interactions primarily involving Kog1 and Tor2'. Within this hub lies the budding-yeastspeci c Twix helices (Kog1 α27 and α 28), that we posited might underlie TOROID formation which, to date, has also only been observed in budding yeast. While performing experiments from which we ultimately concluded that the Twix per se does not play an essential role in TOROID formation, we discovered that the region anking the Twix does. Speci cally, using a CrispR/cas9-supported random mutagenesis screen, we found that α-helices 21-26 and 29 of Kog1 serve as a binding platform for Gtr1 and Gtr2 of the EGOC. This observation was satisfying but not obvious: satisfying because the corresponding region of Raptor (α23-α29) was previously found to bind to RagA and RagC (Rogala et al., 2019); not obvious because the positioning of the Twix helices should occlude GTPase binding. We hypothesize that the Twix helices are exible -potentially more so at the TOROID termini -and that they are displaced upon EGOC binding. The molecular details of this displacement await a TORC1-EGOC structure. Nevertheless, these observations clearly demonstrate that EGOC binding to this region of Kog1 disrupts the hub of interactions needed to sustain TOROID assembly, providing a mechanism to explain how the EGOC can liberate and/or maintain free, active TORC1 dimers -the major conclusion of this paper.
Although this model of TORC1 regulation is appealing, it is oversimpli ed, as it does not take into account the nucleotide loading status of Gtr1 and Gtr2. A priori, one could posit that only "active" EGOC binds TORC1, preventing its association into a TOROID. However, compelling evidence suggests that inactive EGOC also binds TORC1: i/ In in vitro pulldowns we observe that TORC1 interacts with "inactive" EGOC even more robustly than "active" EGOC ( Figure 2H).
ii/ Expression of "inactive" gtr alleles produce phenotypes that are more severe than simple gtr1/2 deletion 25 . Consistently, deletion of LST4 or LST7 (Subunits of the Gtr2 GAP; LST = Lethal with Sec Thirteen) shows synthetic lethality with hypomorphic alleles of sec13 49 which encodes a SEACAT component.
The RAG GTPases do not solely interact with TORC1; they also interact with their respective GAPs.
Interestingly, recent evidence suggests that with both GATOR1 and Folliculin-Fnip, the RAGs have multiple binding modes 18,50,51 . Based on this previous literature and the arguments presented above, we speculate that the EGOC may also have multiple TORC1-interaction modes.
Is mammalian TOR similarly regulated via a TOROID like structure? We do not yet have an answer for this important question. As far as we are aware, comparison of yeast and mammalian TORC1 structures does not present any obvious structural argument to preclude the existence of a mammalian TOROID. mTORC1 resides on lysosomes, which range in diameter from 0.1 to 1 µm 52 . In contrast, a yeast vacuole is around 2 -3 µm in diameter. Thus, there are likely far fewer mTORC1s per lysosome compared to the 500-1000 yeast TORC1s per vacuole. This implies that if mTOROIDs do exist their appearance by light microscopy would be far less striking than what we observe in yeast. Furthermore, the mTOR eld almost exclusively uses a single antibody -Cell Signaling 7C10 -for IHC studies of mTOR. This antibody recognizes a phospho-epitope that may not be accessible or even present in a putative mTOROID helix. Thus, it is very possible that mTORC1 in mTOROIDs would not be visible using this reagent. We note that the RagA GDP RagC GTP conformation of the RAGs, often considered to be inactive, in some studies has been shown to potently inhibit mTORC1 function 53 . This observation is not consistent with the current model that active RAGs simply recruit mTORC1 to the lysosome for subsequent activation by Rheb. In conclusion, we believe that it is not yet appropriate to rule out the existence of mTOROIDs.
The presence of mTOROIDs could explain why mTOR R2266P , and possibly Raptor D635N , are associated with carcinoma based on our nding that substitution of the analogous residue in Tor1 W2203R Tor2 W2207R generated cells that did not form TOROIDs and appeared to possess hyperactive TORC1 signaling (i.e. were resistant to rapamycin). This double mutant is intriguing as it still requires the Gtrs to prevent TOROID formation. Perhaps this mutant can use the binding of "inactive" EGOC to prevent TOROID formation. Alternatively, the mutant may possess a higher a nity for low levels of "active" EGOC in PDS cells. Further characterization of this mutant is clearly warranted.

DECLARATION OF INTERESTS
The authors declare no con icts of interest.

Glucose deprivation and repletion
At OD 600 0.6 cells growing exponentially at 30 °C in liquid CSM were ltered, washed once with starvation medium (CSM-glucose) and then transferred to CSM-glucose. 2% Glucose was subsequently added after 30 min of starvation. Confocal Z-stack images were acquired on LSM800 confocal microscope and/or aliquots for western blot analyses were taken at the relevant time points.

Yeast Mating experiments
Experiments were performed by co-spotting equivalent amounts of exponentially growing MATa and MATalpha cells, onto a YPD plate and leaving it at 30°C for 2.5 hours. The yeast mixture was then subsequently resuspended in liquid CSM at 30 °C and deposited on μ-slides VI 0.4 channels (ibidi) coated with Concanavalin A (0.5mg/mL) to be subsequently imaged by Z-stacks or time-series (1 image/ 30 seconds) Z-stacks using TCS SP5 confocal microscope (Plan-Apochromat 63x/1.4 Oil Objective, Leica).

Light Microscopy and analyses
All confocal images presented in the main and extended data are maximum projections of 3-5 images within a Z-stack (ImageJ). Z slices were initially separated by 500 nm. Image deconvolution was performed with Huygens Software. Analysis of TORC1 foci into size/volume classes followed 3D reconstructions of the stack image and intensity measurements using Imaris software 27 . The vacuolar enrichment of EGO3-GFP, EGO1 TEV subjected to BPS treatment was measured with ImageJ. The mean intensity value in the cytosol was subtracted from the vacuolar membrane signal. The time of TOROID formation or dissolution upon EGOC depletion or replenishment was analysed with imageJ by measuring the intensity of TORC1 focus across the Z-stacks and over the duration of the acquisition. To quantify the degree of colocalization between TORC1 and EGOC, the intensity based colocalization function of Imaris Software was used to extract the Mander's and Pearson coe cients. Alternatively, measure of reciprocal puncta co-localisation was performed using cell segmentation (FIJI) followed by puncta segmentation according to their size in each channel (inferior or superior to 500 nm; Imaris). Signals from both channels were extracted and subjected to Pearson analysis to measure their correlation.
CrispR/Cas9 mutageneses pML104 plasmid expressing Cas9 enzyme and Guide RNA (from addgene) was linearized by PCR (Supplementary Table 3) and the 20-mer guide sequence targeting a speci c yeast genome site was inserted by Gibson cloning 54 . Mutagenesis was achieved by genomic cut and recombination performed by co-transforming yeast strain with the speci c plasmid and the codon optimized PCR product 55 . Short mutagenic PCR products (< 100 nucleotides) were obtained using overlapping forward and reverse primers (see Supplementary Table 2) while long mutagenic PCR products (> 100 nucleotides) were obtained using codon optimized gene sequences (GenScript; See Supplementary Table 3). Yeast were plated onto CSM-URA plates and then replicated on the same plates to decrease the number of false positive clones. CrispR e ciency was estimated by comparing the number of clones obtained with and without co-transformation with PCR product. 10 clones were then subjected to colony PCR (and subsequently sequenced to verify the mutagenesis. CrispR/Cas9 based Random PCR mutagenesis was performed using GeneMorphII Random Mutagenesis Kit (Agilent Technologies) aiming for 1-2 mutation(s) per 500 bp of PCR product. Transformants were plated onto 10-cm CSM-URA plates at a density of 100-200 clones per plate, and then replicated onto CSM-URA containing or not 2.5 nM Rapamycin. Rapamycin-sensitive clones were then sequenced using the same approach as previously described Western Blot analysis Yeast cultures were treated following standard TCA-Urea extraction procedures. Protein lysates were loaded on a 4-20 gradient SDS-PAGE gel and transferred to a nitrocellulose membrane using the iBlot ® system (ThermoFisher). The membrane was probed with primary antibodies overnight at 4 °C, washed and incubated with secondary antibodies for 45 min at RT. The membrane was developed using the Odyssey ® imaging system (LI-COR) and the results were quanti ed using ImageStudio™ Lite (LI-COR).
Primary antibodies used were rabbit polyclonal anti-Sch9, mouse monoclonal anti-P-Sch9 S758 (In house at a dilution of 1:5'000 and 1:2'500, respectively) and mouse monoclonal anti-HA (1:15'000, Sigma n°H9658). Secondary antibodies used were donkey anti-rabbit, IRDye 800 (Li-Cor Biosciencesand donkey anti-mouse, IRDye 680 (Li-Cor Biosciences). All secondary antibodies were used at a dilution of 1:10'000. Cells expressing KOG1 -TAP or kog1 HC -TAP were cultured in CSM and harvested either in exponential phase or PDS (OD 600 2.5 or OD 600 16, respectively). Cell pellets were frozen and further lysed by grinding Movies were collected at a magni cation of 37,000x, corresponding to a calibrated pixel size of 1.35 Å/pixel at the specimen level.
CryoEM data processing Motion correction and dose weighting of the recorded movies were performed using MotionCor2 57 , discarding the rst frame. Initial CTF estimation was performed on the aligned and dose-weighted summed frames using CTFFIND4 58 . TORC1 laments were picked manually using the e2helixboxer module in EMAN2 59 from a total of 3864 micrographs, resulting in 8625 picked laments. A total of 219,455 particles were extracted in RELION 2.0 60 using the --helix option, with an extract size of 600 pixels (810 Å), an outer diameter of 620 Å, and a helical rise of 26.75 Å. Following particle extraction, perparticle CTF correction was performed using Gctf 61 .
Initially, helical reconstruction was performed in RELION2.0 using binned particles (2.7 Å/pixel), a previously acquired low-resolution map of TOROID 27 low-pass ltered to 30 Å, and corresponding previously determined helical parameters as input, while imposing D1 symmetry and allowing for a We thus argued that the rather low resolution obtained after helical reconstruction in RELION and cryoSPARC could be the consequence of a varying pitch along the helices, resulting in an averaging of the structure while implying one set of helical parameters. To test this, we used the 9.1 Å helical reconstruction map (resampled to a voxel size of 1.35 Å) to create a mask containing the whole TOROID lament segment except for one central TORC1 assembly and its interacting regions with adjacent TORC1 complexes. We then performed signal subtraction in RELION2.0, using this mask and the structure and segment coordinates of the helical reconstruction, to generate a set of 219,455 subtracted particles containing one isolated TORC1 assembly per segment. Next, we employed the 'localrec' module in Scipion 43,44 to crop and re-center the signal subtracted particles in a smaller box of 300 pixels (405 Å), as well as assigning to each particle a re ned defocus based on the helical geometry. Single-particle analysis (SPA) of the subtracted particles using 3D re nement in RELION2.0, while imposing D1 symmetry, resulted in a map with a resolution of 4.5 Å (FSC = 0.143). We then imported the particles from the RELION2.0 re nement in cryoSPARC 3.01. After 2D classi cation and class selection, a set of 213808 selected particles was used for Non-Uniform re nement 45 in cryoSPARC 3.01, employing a dynamic mask, imposing D1 symmetry, using the option to keep particles from the same helix in the same half-set, and allowing high-order aberration estimation and correction, which resulted in a nal map with a resolution of 3.85 Å (FSC = 0.143). A nal post-processed and locally sharpened map was obtained using DeepEMhancer 64 . Local resolution estimation of the nal 3D reconstruction was calculated in cryoSPARC 3.01. A summary of cryo-EM data collection parameters and image processing procedures can be found in Supplementary Information (Supplementary Figure 3, Supplementary Table 1).

Model building and re nement
Initial homology models of Tor2 and Lst8 were generated using Phyre2 65 , while a model of Kog1 was generated using ITasser 66 . Homology models of Tor2, Lst8 and Kog1 were rst manually placed in the nal 3D reconstruction followed by rigid-body tting in Chimera 67 . The rigid-body tted models were subsequently subjected to a round of exible tting using Imod t 68 followed by automatic molecular dynamics exible tting using NAMDINATOR 69 . The exibly tted structure was then re ned using the model is equally predicted well, but displays a closed conformation (similar to active mTOR, Figure 4B) starkly contrasting the open conformation of Tor2 extracted from our TOROID structural model, thus resulting in a RMSD value of 8.484 Å (1894 aligned main-chain atoms). While the Kog1 Alphafold model shows a high overall agreement with Kog1 extracted from the TOROID structure (RMSD: 0.829 Å over 1016 aligned main-chain atoms), the 'Twix' region displays a tilt which markedly differs from the TOROID structure, and the 'Tack' region, although correctly predicted as a helix surrounded by large loop regions, is predicted to be in a completely incorrect location. Accordingly, the model con dence score of this particular region in the Kog1 Alphafold structural prediction is very low. Nonetheless, we were able to use the Kog1 Alphafold model to improve the TOROID Kog1 structure in regions were the EM density was very weak, notably the Kog1 N-terminus and the C-terminal WD domain, resulting in signi cantly improved protein geometry validation statistics. A summary of re nement and validation statistics can be found in Supplementary Table 1.

Data Availability
Cryo-EM TOROID maps obtained using either helical reconstruction or signal subtraction and ensuing single particle analysis have been submitted to the EM Data Bank (EMDB) with accession codes EMD submitted to the Protein Data Bank (PDB) with accession code 7PQH.      Helices a26 and a29 of Kog1 are required for interaction with the EGOC. a) Alignment of Kog1, Kog1 ∆twix and Kog1 HC sequences and corresponding structure models. Alpha helices are numbered according to the molecular model. b,e) WT, Δgtr1 Δgtr2, kog1 HC and kog1 ∆twix cells expressing GFP-Tor1 b) or Ego3-GFP e) in exponential phase or in PDS. c) Cells with the indicated genotypes spotted onto plates containing 2.5 nM Rapamycin. d,f) Bar graph representing the quanti cation of TOROID or EGOC puncta formation from b) and e) respectively (N=3 and n>300). Error bars correspond to standard deviation and ***<0.001. g) Western blot analysis of glucose-dependent TORC1 activity using the indicated strains. h) In vitro pull downs of TORC1 containing either Kog1-TAP or Kog1 HC -TAP using cobalt-Dynabeads pre-coated with puri ed (His) 6 -proteins (Coomassie). Inputs and Pull Downs were analyzed by western blot. i) Quanti cation of h) with N=3. Error bars correspond to standard deviation. *** indicates P-value lower than 0.001.

Figure 7
Random mutagenesis reveals that EGOC binding to Kog1 competes with TORC1 oligomerisation. a) Schematic representing the random mutagenesis screen of the KOG1 armadillo sequence to uncover constitutive TOROID mutants.