mTORC1 controls key processes that regulate cell growth including mRNA translation

mTORC1 controls key processes that regulate cell growth including mRNA translation ribosome biogenesis and autophagy. binds directly to Ragulator as opposed to an indirect interaction that depends on other proteins or components of the cell. In these assays purified Ragulator bound c17orf59 (Figure 1D). Purified Rags failed to interact with FG-2216 purified c17orf59 even when Ragulator was present (Figure 1D). This confirms that c17orf59 likely interacts directly with members of Ragulator and that the Rags do not interact with c17orf59. c17orf59 localizes to the lysosome along with Ragulator Ragulator localizes to lysosomes and late endosomes by virtue of lipid modifications and targeting sequences on the N-terminus of p18 (Sancak et al. 2010 Nada et al. 2009 Consistent with its interaction with Ragulator HA-tagged c17orf59 co-localizes with the lysosomal marker LAMP2 (Figure 2A) indicating its presence at lysosomes. To determine the extent of co-localization between c17orf59 and Ragulator we re-expressed the cDNA for p18 in p18-null MEFs and examined the localization of c17orf59 and p18. Cells expressing HA-tagged c17orf59 display a highly significant co-localization with p18 (Figure 2B Supplemental Figure 1A and 1B). c17orf59 also co-localizes with another Ragulator subunit LAMTOR4 in a p18-dependent manner (Supplemental Figure 1C) further supporting the existence of a c17orf59-Ragulator interaction. The subcellular localization of c17orf59 was unaffected by the presence or absence of amino acids or insulin (Figure 2C and 2D). Figure 2 c17orf59 localizes to lysosomes with Ragulator c17orf59 loss does not FG-2216 alter mTORC1 activity in response to amino acids or insulin To examine the effects of loss of c17orf59 on mTORC1 activation we generated c17orf59-null HEK-293E and HeLa cells using the sgRNA/Cas9 system and reconstituted c17orf59 with expression of its cDNA driven by the c17orf59 promoter. The c17orf59-null cells showed no signaling defects in response to amino acid or serum starvation and re-stimulation as compared to non-targeting sgRNA or c17orf59-null cells reconstituted with c17orf59 (Figure 3A and 3B Supplemental Figure 2A and 2B) even when intermediate doses of either amino FG-2216 acids or insulin were added back to cells (Supplemental Figure 2C and 2D). In addition c17orf59 null cells had no alterations in mTORC1 signaling in response to cholesterol deprivation the only known stimulus that alters c17orf59 expression (Bartz et al. 2009 (Supplemental Figure 2E). Despite its interaction with Ragulator loss of c17orf59 does not cause alterations in mTORC1 activation by amino acids or insulin. This lack of signaling phenotype was consistent across Rabbit polyclonal to PDK4. multiple clones of c17orf59-null cells using multiple guides in both HEK-293E and HeLa cells as FG-2216 well as using shRNA-mediated knockdown of c17orf59 (data not shown) so we are confident that the results are not the product of re-wiring in the single cell clones that became the c17orf59-null cells. Based on these results we tested the effects of c17orf59 overexpression on Ragulator function and mTORC1 activity. Figure 3 Loss of c17orf59 does not alter mTORC1 signaling in response to amino acids or insulin c17orf59 disrupts the Rag-Ragulator interaction in cells and FG-2216 binding assays with purified proteins. Much like in cells when purified c17orf59 was pre-incubated with the GST-Ragulator there was a dose-dependent decrease in the amount of purified Rags that bound to immobilized Ragulator (Figure 4C). In a similar experiment using immobilized GST-tagged RagB with RagC increasing amounts of purified c17orf59 decreased Ragulator binding to the Rags (Figure 4D). Because Ragulator is required for the lysosomal localization of the Rag GTPases it is possible that the disruption of the Rag-Ragulator interaction due to c17orf59 overexpression results in a mis-localization of the Rag GTPases away from the lysosome. To test this we transiently expressed FLAG-tagged c17orf59 in HEK-293T cells marking cells that were transfected using GFP driven by an internal ribosome entry sequence (IRES) downstream of the c17orf59 cDNA. The amount of RagC that co-localizes with the lysosomal marker LAMP1 decreases in cells that overexpress c17orf59 but not FLAG-tagged GFP alone (Figure 4E GFP-positive cells). Importantly overexpression of c17orf59 does not alter the localization of Ragulator component LAMTOR4 (Supplemental Figure 3A) indicating that Ragulator is still intact and present at.