A conformational change in the N terminus of SLC38A9 signals mTORC1 activation

Abstract

mTORC1 is a central hub that integrates environmental cues, such as cellular stresses and nutrient availability to modulate metabolism and cellular responses. Recently, SLC38A9, a lysosomal amino acid transporter, emerged as a sensor for luminal arginine and as an activator of mTORC1. The amino acid-mediated activation of mTORC1 is regulated by the N-terminal domain of SLC38A9. Here, we determined the crystal structure of zebrafish SLC38A9 (drSLC38A9) and found the N-terminal fragment inserted deep within the transporter, bound in the substrate-binding pocket where normally arginine would bind. This represents a significant conformational change of the N-terminal domain (N-plug) when compared with our recent arginine-bound structure of drSLC38A9. We propose a ball-and-chain model for mTORC1 activation, where N-plug insertion and Rag GTPase binding with SLC38A9 is regulated by luminal arginine levels. This work provides important insights into nutrient sensing by SLC38A9 to activate the mTORC1 pathways in response to dietary amino acids.

A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling

“…Using groups of amino acids to activate mTORC1 in un-injected oocytes, we found an intriguing coincidence between the substrates of SNAT2 and their ability to activate mTORC1. The extended N-terminus of slc38 member SNAT9 has recently been shown to bind to the RagA/C heterodimer (Rebsamen et al, 2015;Wang et al, 2015;Fromm et al, 2020) and occupy the arginine binding site during mTORC1 inactivation (Lei et al, 2018;Lei et al, 2020). A similar mechanism could be envisioned for the N-terminus of SNAT2 (Gaccioli et al, 2006;Hundal and Taylor, 2009;Poncet and Taylor, 2013), although the N-terminal is predicted to be 50 residues shorter than that of SNAT9.…”

“…Multiple transporters have been advanced as direct or indirect activators of the mTORC1 pathway including SNAT2 (slc38a2) (Evans et al, 2007;Hyde et al, 2007;Evans et al, 2008), SNAT9 (slc38a9) (Rebsamen et al, 2015;Wang et al, 2015), LAT1-4F2hc (slc7a5-slc3a2) (Fuchs et al, 2007;Nicklin et al, 2009), ASCT2 (slc1a5) (Fuchs et al, 2007;Nicklin et al, 2009), PAT1 (slc36a1) (Heublein et al, 2010;Zoncu et al, 2011;Wu et al, 2016;Zhao et al, 2019) and PAT4 (slc36a4) (Heublein et al, 2010;Fan et al, 2016;Zheng et al, 2016). A number of studies have now firmly established that SNAT9 is a lysosomal arginine sensor for mTORC1 (Jung et al, 2015;Rebsamen et al, 2015;Wang et al, 2015;Rebsamen and Superti-Furga, 2016;Wyant et al, 2017) by direct interaction of the transport N-terminal with the Rag GTPase-Ragulator-FLCN:FNIP2 complex upon arginine sensing (Fromm et al, 2020;Lei et al, 2020). Another widely proposed mechanism is glutamine accumulation by ASCT2, followed by its exchange for leucine import via LAT1-4F2hc (Fuchs et al, 2007;Nicklin et al, 2009).…”

Plug-and-socket mechanisms in nutrient sensing by lysosomal amino acid transporters

“…Their study unveils a plug-and-socket mechanism where a flexible protruding loop of the complex plugs into the central cavity of the transporter, suggesting a mechanism for the interplay between amino acid transport and amino acid signaling. Interestingly, this mechanism bears some resemblance with the interaction between another lysosomal amino acid transporter and a signaling complex intervening in the mechanistic target of rapamycin complex 1 (mTORC1) pathway elucidated in very recent studies (6,7).…”

“…The large, cytosolic N-terminal tail of SNAT9 had been implicated in the interaction with the Ragulator-Rag complex (12,13); however, the arginine-sensing mechanism remained unclear. In a recent study (7), part of the N terminus was shown to form a β hairpin, which plugs into the central cavity of SNAT9 in cytosol-open conformation and interacts with the arginine-binding pocket at the bottom of the cavity. Arginine competes with the β hairpin, thus releasing it from the central cavity with two outcomes: The amino acid transport activity of SNAT9 is up-regulated because alternating-access movements are not hindered anymore, and the N-terminal tail of SNAT9 can engage in other interactions (Fig.…”

Principles of Alternating Access in LeuT-fold Transporters: Commonalities and Divergences

“…GTPases involved in downstream signaling [78,92]. In a potential case of convergent evolution, the C-terminal domain of the pH-dependent Glu/GABA exchanger GadC arrests transport by binding to the intracellular cavity at neutral pH in a nearly identical conformation [48].…”