Figure 8.
Figure 8. SLC38A9 is required for amino acid regulation of the Ragulator–BORC interaction and lysosome positioning. (A) Control, nontargeting siRNA (C) or siRNA targeting SLC38A9 (SLC) was electroporated into myrlysin-KO HeLa cells stably rescued with myrlysin-GFP. Cells were incubated in medium with or without amino acids (AA) for 60 min and then subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblotting (IB) with antibodies to LAMTOR2 or GFP. The positions of molecular mass markers (in kilodaltons) are indicated on the left. Bar graphs show the ratios of LAMTOR2 to myrlysin-GFP quantified by densitometry (in arbitrary units). Values are the mean ± SD from three independent experiments. *, P < 0.05 (Student’s t test). (B) Cells were transfected and treated as in A and subjected to immunostaining with antibodies to GFP and LAMP1. Bar, 10 µm. The distribution of lysosomes relative to the MTOC was quantified using Imaris and normalized to the longest distance between the MTOC and the cell periphery. Values are the mean ± SD from 20 cells per condition. (C) Hypothetical model for the regulation of lysosome positioning by Ragulator and BORC. In amino acid–replete conditions, SLC38A9 tightly binds to Ragulator and the Rag GTPases, leading to RagA/B activation and mTORC1 recruitment to the lysosomal membrane. At the same time, SLC38A9 binding weakens the Ragulator–BORC interaction, allowing BORC and Arl8 to recruit kinesins and thus promote anterograde transport of lysosomes. In amino acid–depleted conditions, weakening of the interaction with SLC38A9 causes a conformational change in Ragulator that prevents activation of the Rags and recruitment of mTORC1 to lysosomes, while simultaneously enhancing an inhibitory effect on BORC. This enhancement does not interfere with Arl8 association with lysosomes, but does prevent the recruitment of kinesins to lysosomes, reducing their anterograde transport and leading to their clustering in the juxtanuclear area. The generic kinesin shown in the scheme represents either KIF1B or KIF5B in complex with the corresponding adaptors (Guardia et al., 2016).

SLC38A9 is required for amino acid regulation of the Ragulator–BORC interaction and lysosome positioning. (A) Control, nontargeting siRNA (C) or siRNA targeting SLC38A9 (SLC) was electroporated into myrlysin-KO HeLa cells stably rescued with myrlysin-GFP. Cells were incubated in medium with or without amino acids (AA) for 60 min and then subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblotting (IB) with antibodies to LAMTOR2 or GFP. The positions of molecular mass markers (in kilodaltons) are indicated on the left. Bar graphs show the ratios of LAMTOR2 to myrlysin-GFP quantified by densitometry (in arbitrary units). Values are the mean ± SD from three independent experiments. *, P < 0.05 (Student’s t test). (B) Cells were transfected and treated as in A and subjected to immunostaining with antibodies to GFP and LAMP1. Bar, 10 µm. The distribution of lysosomes relative to the MTOC was quantified using Imaris and normalized to the longest distance between the MTOC and the cell periphery. Values are the mean ± SD from 20 cells per condition. (C) Hypothetical model for the regulation of lysosome positioning by Ragulator and BORC. In amino acid–replete conditions, SLC38A9 tightly binds to Ragulator and the Rag GTPases, leading to RagA/B activation and mTORC1 recruitment to the lysosomal membrane. At the same time, SLC38A9 binding weakens the Ragulator–BORC interaction, allowing BORC and Arl8 to recruit kinesins and thus promote anterograde transport of lysosomes. In amino acid–depleted conditions, weakening of the interaction with SLC38A9 causes a conformational change in Ragulator that prevents activation of the Rags and recruitment of mTORC1 to lysosomes, while simultaneously enhancing an inhibitory effect on BORC. This enhancement does not interfere with Arl8 association with lysosomes, but does prevent the recruitment of kinesins to lysosomes, reducing their anterograde transport and leading to their clustering in the juxtanuclear area. The generic kinesin shown in the scheme represents either KIF1B or KIF5B in complex with the corresponding adaptors (Guardia et al., 2016).

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