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

PLoS One. 2012;7(5):e36616. doi: 10.1371/journal.pone.0036616. Epub 2012 May 4.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Transport Systems / metabolism*
  • Amino Acids / pharmacology
  • Animals
  • Drosophila melanogaster / cytology
  • Drosophila melanogaster / growth & development
  • Drosophila melanogaster / metabolism
  • Endocytosis / drug effects
  • Endosomes / drug effects
  • Endosomes / metabolism*
  • Female
  • GTP Phosphohydrolases / metabolism*
  • HEK293 Cells
  • Humans
  • Intracellular Membranes / drug effects
  • Intracellular Membranes / metabolism*
  • Lysosomes / drug effects
  • Lysosomes / metabolism*
  • Mechanistic Target of Rapamycin Complex 1
  • Monomeric GTP-Binding Proteins / metabolism
  • Multiprotein Complexes
  • Neuropeptides / metabolism
  • Phosphatidylinositol 3-Kinases / metabolism
  • Protein Binding / drug effects
  • Proteins / metabolism*
  • Proto-Oncogene Proteins c-akt / metabolism
  • Ras Homolog Enriched in Brain Protein
  • Signal Transduction / drug effects
  • Symporters / metabolism*
  • TOR Serine-Threonine Kinases

Substances

  • Amino Acid Transport Systems
  • Amino Acids
  • Multiprotein Complexes
  • Neuropeptides
  • Proteins
  • RHEB protein, human
  • Ras Homolog Enriched in Brain Protein
  • SLC36A1 protein, human
  • Symporters
  • Phosphatidylinositol 3-Kinases
  • TOR Serine-Threonine Kinases
  • Mechanistic Target of Rapamycin Complex 1
  • Proto-Oncogene Proteins c-akt
  • GTP Phosphohydrolases
  • Monomeric GTP-Binding Proteins