Proton-Assisted Amino Acid Transporter PAT1 Complexes with Rag GTPases and Activates TORC1 on Late Endosomal and Lysosomal Membranes

Abstract

Mammalian Target of Rapamycin Complex 1 (mTORC1) is activated by growth factor-regulated phosphoinositide 3-kinase (PI3K)/Akt/Rheb signalling and extracellular amino acids (AAs) to promote growth and proliferation. These AAs induce translocation of mTOR to late endosomes and lysosomes (LELs), subsequent activation via mechanisms involving the presence of intralumenal AAs, and interaction between mTORC1 and a multiprotein assembly containing Rag GTPases and the heterotrimeric Ragulator complex. However, the mechanisms by which AAs control these different aspects of mTORC1 activation are not well understood. We have recently shown that intracellular Proton-assisted Amino acid Transporter 1 (PAT1)/SLC36A1 is an essential mediator of AA-dependent mTORC1 activation. Here we demonstrate in Human Embryonic Kidney (HEK-293) cells that PAT1 is primarily located on LELs, physically interacts with the Rag GTPases and is required for normal AA-dependent mTOR relocalisation. We also use the powerful in vivo genetic methodologies available in Drosophila to investigate the regulation of the PAT1/Rag/Ragulator complex. We show that GFP-tagged PATs reside at both the cell surface and LELs in vivo, mirroring PAT1 distribution in several normal mammalian cell types. Elevated PI3K/Akt/Rheb signalling increases intracellular levels of PATs and synergistically enhances PAT-induced growth via a mechanism requiring endocytosis. In light of the recent identification of the vacuolar H+-ATPase as another Rag-interacting component, we propose a model in which PATs function as part of an AA-sensing engine that drives mTORC1 activation from LEL compartments.

Cystinosin is a Component of the Vacuolar H+-ATPase-Ragulator-Rag Complex Controlling Mammalian Target of Rapamycin Complex 1 Signaling

“…For example, two members of the SCL38 family, proton-assisted amino acid transporter 1 (PAT1) and SLC38A9 have recently been found to directly interact with components of the Rag-Ragulator complex and to be involved in the regulation of mTORC1 activity. [22][23][24] Importantly, SLC38A9 was shown to interact with Rag-Ragulator in an amino acid-dependent manner, and to act as a positive regulator of mTORC1 activity, most likely independently of v-ATPase. The interaction between SLC38A9 and v-ATPase seems much less important than that of cystinosin and v-ATPase, suggesting that the mechanism of regulation of the mTORC1 pathway differs between these two amino acid transporters.…”

Preclinical Evaluation of 11C-Sarcosine as a Substrate of Proton-Coupled Amino Acid Transporters and First Human Application in Prostate Cancer

“…PAT4 is a high-affinity, low-capacity electroneutral transporter of neutral amino acids (26). Baseline PAT expression is ubiquitous (15) and resides at the cell surface and intracellular membranes (endosomes, lysosomes) (27). Elevated PAT expressions have been linked to cell proliferation (24).…”

“…Elevated PAT expressions have been linked to cell proliferation (24). Here, PATs function as part of an amino acid-sensing engine that drives activation of the mammalian target of rapamycin complex 1 (mTORC1) (27). Physiologically high PAT1/PAT2 expressions have been identified in the brain, pancreas, kidney, duodenum, and small intestine (24).…”

Autophagy in renal diseases

“…The Ragulator complex has been implicated in binding the Rag GTPase heterodimer to the lipid bilayer [33]. It was recently shown that the lysosomal membrane protein human member 9 of the solute carrier family 38 (SLC38A9), a physical and functional component of the amino acid sensing RagulatorRag GTPase complex, positively regulates the mTORC1 pathway [32,34].…”

“…It was recently shown that the lysosomal membrane protein human member 9 of the solute carrier family 38 (SLC38A9), a physical and functional component of the amino acid sensing RagulatorRag GTPase complex, positively regulates the mTORC1 pathway [32,34]. Additionally, the proton-assisted amino acid transporter 1 (PAT1) or SLC36A1 has been shown to be an essential modulator of amino acid-dependent mTORC1 activation [33]. Activation via this pathway leads to the translocation of mTORC1 to the lysosomal surface where it can be further activated by Rheb, stimulating the mTORC1 kinase activity [5].…”