TORC1

TORC1 is an ~1.2 MDa complex composed of Lst8, Kog1, Tco89 and either of the two Tor proteins (i.e. Tor1 or Tor2). TORC1 is tethered to the limiting membrane of the vacuole by the EGO – Exit from rapamycin-induced GrOwth arrest – complex (EGOC). The EGOC acts as a signaling hub in that it receives signals triggered by the presence of cellular metabolites and communicates this information to TORC1. The « business end » of the EGOC is formed by an obligate dimer of the Rag-family GTPases Gtr1 and Gtr2. Under favourable growth conditions, Gtr1 is loaded with GTP and Gtr2 with GDP and TORC1 is active; when nutrients are lacking, the guanine-nucleotide loading of the two Gtrs is reversed and TORC1 is inactive. 

We made the surprising discovery that glucose starvation leads to TORC1 inhibition by triggering its assemble into a giant helix we named a TOROID [1] – a previously unrecognized mode of protein kinase activity regulation. In this conformation, access to the active site of the TOR kinase is occluded in a manner like that caused by rapamycin●FKBP12 binding. We further found that, in their active form, the RAG GTPases of yeast, Gtr1/2, extract TORC1 from the TOROID helix to restore kinase activity upon glucose addition [2]. Whether or not this mode of regulation is conserved in higher eukaryotes remains to be seen. The GTPase activators (GAPs) upstream of the RAG GTPases are known, but how their activities are regulated is not. To address this, by CryoEM we solved the structure of the yeast SEAC complex [3], and more recently the SEAC complex bound to its substrate the EGOC [4]. Collectively, these works help reveal the molecular mechanisms by which nutrient cues are signaled to TORC1.

TORC2

TORC2 is an ~1.4 MDa complex composed of Lst8, Avo1, Avo2, Avo3, Bit61/Bit2 and Tor2. 

Given its insensitivity to rapamycin, TORC2 has been trickier to study. We made the unexpected discovery that this complex is regulated primarily downstream of mechanical properties (e.g. tension) of the plasma membrane [1]. Our low-resolution cryoEM structure of TORC2 enabled us to engineer strains in which TORC2 but not TORC1 is inhibited by rapamycin [2] – a very powerful approach, used now by the larger community, to study TORC2. Using this tool, we demonstrated that TORC2 functions as a central hub in a homeostatic feedback loop needed to maintain the biophysical properties of the plasma membrane [3]. In parallel, a high throughput screening campaign yielded palmitoylcarnitine (PalmC) which intercalates the plasma membrane and consequently triggers TORC2 inhibition [3] and later revealed that stresses that lead to TORC2 inhibition do so through mobilization of sterols in the plasma membrane [4]. In contrast, stimuli that activate TORC2 seem to be sensed by dedicated plasma membrane structures known as eisosome [5], although how sensing is achieved was unclear. Our cryoEM structure of the eisosome protein coat together with its near-native membrane microdomain revealed that increased membrane tension triggers the release of specific lipids (PS, PI4,5P2 and ergosterol), in addition to proteins (Slm1/2), to regulate TORC2 [6]. Finally, we recently dramatically improved our cryoEM structure of TORC2 to ~2.2Å resolution [7]. This structure reveals how the PH domain of the Avo1 subunit wedges into the kinase pocket to inhibit kinase activity and allows us to propose the sequence of structural rearrangements needed for TORC2 activation.   

The lab is presently employing cryo-focused ion beam milling (cryo-FIB) and cryo-electron tomography (cryo-ET) to connect structural insights obtained in vitro with those observed in vivo, contributing to a comprehensive understanding of PM macrodomains and  their involvement in TORC2 signaling.

TOR in mammals

We are investigating the conservation of the findings in S. cerevisiae in mammalian cells using CRISPR-mediated genome editing and complementary biochemical techniques.

Additionally, a screening for small molecules that specifically inhibit mTORC2 has been performed, with the hope that such molecules may find clinical interest to treat diseases associated with dysregulated Akt signaling (e.g. cancers, metabolic syndrome) [1].