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

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

Margrét H Ögmundsdóttir  1 , Sabine Heublein, Shubana Kazi, Bruno Reynolds, Shivanthy M Visvalingam, Michael K Shaw, Deborah C I Goberdhan
Affiliations

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

Margrét H Ögmundsdóttir et al. PLoS One. 2012.

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. mTOR localises to LAMP2/PAT1-positive compartments upon AA stimulation.
(A, B) Flag-PAT1 (red; B) overexpressed in a stably transfected HEK-293 cell line has a similar intracellular localisation pattern to endogenous PAT1 (green; A). Cells under steady state conditions are shown. (C) Endogenous PAT1 strongly co-localises with LAMP2, which marks LEL compartments. Cells were stained after 50 min AA starvation followed by 10 min AA stimulation. Merge shows LAMP2 (red), PAT1 (green) and DAPI (blue). (D, E) Subcellular localisation of LAMP2, Flag-PAT1 and mTOR after 50 min AA starvation (D) and 50 min AA starvation followed by 10 min AA stimulation (E). Note that Flag-PAT1 and LAMP2 co-localise under both conditions, but mTOR is only recruited to a limited number of LELs upon AA stimulation. Merge shows Flag-PAT1 (red), mTOR (green) and DAPI (blue). Scale bars in B and E are 10 µm, A; scale bar in B also applies to A and C, scale bar in E also applies to D.
Figure 2
Figure 2. Flag-PAT1 and mTOR co-localise at the surface of the same intracellular compartments.
(A) Ultrathin sections from a stable HEK-293 cell line overexpressing Flag-PAT1 and either labelled with anti-Flag antibodies (5 nm gold) or (B–D) co-labelled with anti-Flag (5 nm gold) and anti-mTOR (10 nm gold) antibodies. Endogenous mTOR and Flag-PAT1 are found on the surface of the same membrane-bound compartments that often contain an electron-dense core. Arrowheads mark a subset of locations where immunolabelled mTOR is in close enough proximity to Flag-PAT1 to be part of a multimolecular complex. Scale bar is 200 nm in all panels.
Figure 3
Figure 3. PAT1 co-localises and can physically interact with Rag GTPases.
(A, B) Flag-PAT1 co-localises with a subset of the compartments containing endogenous RagC under both AA-starved (A) and AA-stimulated (B) conditions in a stable HEK-293 cell line overexpressing Flag-PAT1. (C) Under steady state conditions, immunoprecipitation of Flag-PAT1 leads to co-immunoprecipitation of endogenous RagC, but not tubulin. (D) Conversely, immunoprecipitation of Flag-RagD, but not Flag-Rap2A, both transiently expressed in HEK-293 cells, leads to co-immunoprecipitation of endogenous PAT1, but not tubulin, suggesting that the Rag GTPases complex with PAT1 in cells. Scale bar in A is 10 µm and applies to all panels in A and B.
Figure 4
Figure 4. PAT1 modulates the AA-dependent relocalisation of mTOR to LELs.
(A, B) Knockdown of PAT1 (PAT1 kd) in HEK-293 cells reduces the AA-stimulated accumulation of mTOR (green) to LAMP2-positive (red) LELs (B), when compared to cells treated with a scrambled siRNA (scr; A). (C, D) Importantly, PAT1 knockdown does not eliminate all PAT1 protein from cells (compare D with control cells in C), so residual mTOR relocalisation in B may result from the presence of low levels of PAT1. PAT1 antibody staining is shown in green, LAMP2 in red. Scale bar in A is 10 µm and applies to all panels.
Figure 5
Figure 5. Drosophila PAT-GFP fusion proteins have similar functional activities to untagged PATs in vivo.
Untagged transporters synthesised from GS insertions in path (pathGS13857; B, H, N) and CG1139 (CG1139GS10666; C, I, O), and tagged transporters synthesised from UAS-Path-GFP (D, J, P) and UAS-CG1139-GFP line 1 (E, K, Q) and line 2 (F, L, R) insertions were expressed in the differentiating cells of the fly eye with GMR-GAL4 in the presence or absence of other transgenes. (A–F) Overexpression of pathGS13857 (B) and CG1139GS10666 (C) produces a significant increase in ommatidial size relative to controls (A). A more subtle, but also significant, increase in growth was seen for PATH-GFP (D) and CG1139-GFP line 1 (E). CG1139 line 2 produced a bulging and disorganised eye phenotype (F). For size comparisons, n = 6; bottom of panels A–E, mean ± s.d. relative to control; * P<0.05, ** P<0.01, ***P<0.001. (G–L) The overgrowth induced by overexpressing UAS-Rheb with GMR-GAL4 (G) is synergistically enhanced by co-expression of tagged and untagged transporters (H–L), suggesting a role for Rheb signalling in controlling the growth-promoting activity of PATs. (M–R) The apoptotic, reduced eye phenotype produced by overexpression of foxoGS9928 with GMR-GAL4, which is most clearly seen at the ventro-posterior edge of the eye (arrow in M), is enhanced by co-expression of tagged and untagged transporters (N–R), consistent with these molecules acting through the TORC1 signalling cascade to inhibit Akt and enhance, FOXO activity. Scale bar is 100 µm and applies to all panels. Ommatidial size measurements were made when expression of transgenes did not disturb the ommatidial array.
Figure 6
Figure 6. Drosophila PATs are localised at the cell surface and on LEL compartments in vivo.
(A, B) Expression of PATH-GFP (green) in living Drosophila S2 cells stained with the acid-sensitive dye Lysotracker Red. Some PATH-GFP is found at the plasma membrane (white arrow), but most is at the surface of intracellular compartments. It does not generally co-localise with the most intensely stained Lysotracker Red-positive compartments (likely to be lysosomes, e.g. red arrows), but does co-localise with the less intensely stained Lysotracker Red-positive compartments (probably late endosomes and some lysosomes, e.g. yellow arrows), perhaps because GFP fluorescence or integrity is affected in highly acidic conditions. In addition, some PATH-GFP-containing compartments do not co-stain with Lysotracker Red (e.g. green arrows). (C, D) PATH-GFP (green) expressed under Lsp2-GAL4 control in the larval fat body also co-localises with only a subset of Lysotracker Red-positive compartments in living tissue (e.g. examples marked with arrows as in A and B), but is also expressed at high levels at the surface of cells. Note in D, PATH-GFP is specifically expressed at the surface of some larger Lysotracker Red-positive structures (yellow arrows) and other membrane structures (green arrows), consistent with its known membrane-association. (E) CG1139-GFP co-localises with many, but not all compartments stained with HRP-Lamp1, a late endosomal and lysosomal marker, in fixed larval fat bodies (e.g. yellow arrows), suggesting that some, but not all, intracellular CG1139 is in LELs. Nuclei are stained with DAPI (blue) in E″. Scale bar is 5 µm in A, B and D, and 20 µm in C and E.
Figure 7
Figure 7. The growth-promoting activity of PAT transporters is synergistically enhanced by hyperactivation of PI3K/Akt signalling.
(A–C) GAL4-UAS-induced overexpression of the fly PAT transporter genes, path (B) and CG1139 (C) in the differentiating eye with GMR-GAL4 promotes increased growth compared to normal animals (A). (D–F) When overexpressed in a PTEN mutant background (D), the effect of path (E) and CG1139 (F) on growth is synergistically enhanced, resulting in a highly overgrown, bulging eye phenotype. Ommatidial size measurements are given for eyes where the ommatidial array is regularly arranged (n = 6; bottom of panels A–C, mean ± s.d. relative to control (A); *P<0.001, increased relative to control). Fly genotypes are w; GMR-GAL4 (A), w; GMR-GAL4/pathGS13857 (B), w; GMR-GAL4/CG1139GS10666 (C), y w; PTEN1 FRT40A/P[w+]l(2)3.1 FRT 40A; GMR-GAL4 (D), y w; PTEN1 FRT40A/P[w+]l(2)3.1 FRT 40A; GMR-GAL4/pathGS13857 (E) and y w; PTEN1 FRT40A/P[w+]l(2)3.1 FRT 40A; GMR-GAL4/CG1139GS10666 (F).
Figure 8
Figure 8. PI3K/Akt/Rheb signalling promotes shuttling of the PATs to endosomal compartments.
(A, C) CG1139-GFP expressed in the larval fat body using Lsp2-GAL4 is localised to both the plasma membrane (white arrow) and also to intracellular LELs throughout the cytoplasm (yellow arrow; A). Overexpression of Rheb leads to an increase in the relative proportion of intracellular, including perinuclear, protein compared to cell surface CG1139-GFP (C). (B, D) When CG1139-GFP is co-expressed with a dominant negative version of Shibire, ShiK44A, which blocks endocytosis, in either the presence (D) or absence (B) of Rheb, the transporter is mostly located at the plasma membrane (white arrow), strongly suggesting that PATs are normally shuttled to LELs from the cell surface. (E, F) The ratio of the GFP signal intensity in a 2.25 µm perinuclear region and a 2.25 µm region at the plasma membrane (see E) was measured for genotypes in A–D (grey bars; error bars = s.d. in F). Average cell size for each genotype is also shown in F (blue bars; error bars = s.d.). n = 25; *P<0.001 (increased) and ■ P<0.001 (decreased) relative to non-Rheb/non-ShiK44A-expressing control; ■ ■ P<0.001, decreased relative to Rheb-overexpressing control. Rheb-induced changes in intracellular PATs and PAT-induced growth are entirely dependent on endocytosis. (G, H) In the larval eye imaginal disc, CG1139-GFP, expressed under GMR-GAL4 control (G), is mostly located at or near the plasma membrane, for example at the surfaces of flattened non-photoreceptor cells that surround each ommatidium (white arrows). Co-overexpression of Rheb (H) results in much larger ommatidia, with reduced staining around the ommatidial border (white arrow) and more CG1139-GFP cytoplasmic expression, including intense intracellular punctae of staining (yellow arrows), consistent with Rheb promoting endocytosis of PATs in this tissue. An outline of an individual ommatidium in G and H is marked with a dashed line. Scale bar in A is 20 µm and applies to panels A–D, scale bar in G is 5 µm and applies to panels G and H.
Figure 9
Figure 9. AA-dependent regulation of mTORC1in late endosomes and lysosomes via the PAT1/Rag/Ragulator/v-ATPase nutrisome complex.
We have shown that Proton-assisted Amino acid Transporters (PATs) localised on late endosomes and lysosomes (LELs) interact with Rag GTPases (Rags) and are required for mTORC1 activation. The Ragulator is a trimeric group of proteins involved in attaching the complex to the lipid bilayer. Recent studies have also demonstrated that the vacuolar H+-ATPase (v-ATPase) interacts with the activated Rag (Rag*)/Ragulator complex to control amino acid (AA)-dependent mTORC1 activation and that this is regulated by the rapid accumulation of extracellular AAs in LELs. This suggests a model where, in response to AAs (compare upper LEL to lower AA-stimulated LEL), these different molecules form a complex that we call the ‘nutrisome’. Cycling of protons through this nutrisomal engine induces conformational changes that activate mTORC1, leading to increased translation and cell growth. Importantly, signalling from the insulin receptor (InR) and subsequent activation of the PI3K/Akt/Rheb cascade promotes shuttling of PATs from the cell surface to LEL membranes, hence increasing PAT-dependent mTORC1 activation and cell growth. In addition, the accumulation of AAs in the LEL lumen presumably involves transport into intracellular endosomal compartments (depicted by compartment on left hand side) via currently unknown amino acid transporters (AATs) or potentially endocytosis. Cytoplasmic leucine (Leu), which may be brought into cells via the heterodimeric AAT, CD98 , has been shown to play a key role in activating mTORC1 in some cultured cells and may play a key role in this process. Influx of Leu or other AAs into the endolysosomal system may ultimately allow the AA substrates of PAT1 to accumulate in the LELs through AA exchange mechanisms, leading to PAT1-mediated activation of the nutrisome. Interactions leading to activation or inhibition of downstream components are depicted by solid arrows and solid bars respectively, movement of specific molecules by dashed arrows and processes involving membrane shuttling by dotted arrows.
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