Proton-assisted amino acid transporter PAT1 complexes with Rag GTPases and activates TORC1 on late endosomal and lysosomal membranes.

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.

Drosophila expresses a CD98 transporter with an evolutionarily conserved structure and amino acid-transport properties.

Mammalian CD98 heterodimeric amino acid transporters consist of a promiscuous single-pass transmembrane glycoprotein, CD98hc (CD98 heavy chain), and one of six multipass transmembrane proteins or 'light chains'. The heterodimeric complexes of CD98hc and the light chains LAT1 (L-type amino acid transporter 1) or LAT2 specifically promote sodium-independent System L exchange of neutral amino acids, including leucine. CD98hc is also implicated in other processes, including cell fusion, cell adhesion and activation of TOR (target of rapamycin) signalling. Surprisingly, recent reports suggested that insects lack a membrane-bound CD98hc, but in the present study we show that Drosophila CG2791 encodes a functional CD98hc orthologue with conservation in intracellular, transmembrane and extracellular domains. We demonstrate by RNA-interference knockdown in Drosophila Schneider cells that CG2791 and two Drosophila homologues of the mammalian CD98 light chains, Mnd (Minidiscs) and JhI-21, are required for normal levels of System L transport. Furthermore, we show that System L activity is increased by methoprene, an analogue of the developmentally regulated endocrine hormone juvenile hormone, an effect that is potentially mediated by elevated Mnd expression. Co-expression of CG2791 and JhI-21, but not CG2791 and Mnd, in Xenopus oocytes mediates System L transport. Finally, mapping of conserved sequences on to the recently determined crystal structure of the human CD98hc extracellular domain highlights two conserved exposed hydrophobic patches at either end of the domain that are potential protein-protein-interaction surfaces. Therefore our results not only show that there is functional conservation of CD98hc System L transporters in flies, but also provide new insights into the structure, functions and regulation of heterodimeric amino acid transporters.

Amino acid sensing and mTOR regulation: inside or out?

mTOR (mammalian target of rapamycin) plays a key role in determining how growth factor, nutrient and oxygen levels modulate intracellular events critical for the viability and growth of the cell. This is reflected in the impact of aberrant mTOR signalling on a number of major human diseases and has helped to drive research to understand how TOR (target of rapamycin) is itself regulated. While it is clear that amino acids can affect TOR signalling, how these molecules are sensed by TOR remains controversial, perhaps because cells use different mechanisms as environmental conditions change. Even the question of whether they have an effect inside the cell or at its surface remains unresolved. The present review summarizes current ideas and suggests ways in which some of the models proposed might be unified to produce an amino acid detection system that can adapt to environmental change.