Figure 5. The accumulation of Ub-Rheb renders lysosomal mTORC1 localization and its activation in Ragulator deficient cells.

5A. Levels of lysosomal ATXN3 are reduced in p18 deficient cells. Levels of endogenous ATXN3 and indicated proteins were monitored in the purified lysosomes. Levels of lysosomal ATXN3 were quantified as the ratio of ATXN3/Rab7. **p<0.01, mean±SEM, n=3

5B and 5C. Ub-Rheb is accumulated in p18 KO HEK293T and RagA/B KO MEF cells. Endogenous Rheb was IPed from p18 KO HEK293T cells (5B) and RagA/B KO MEF cells (5C).

5D and 5E. Ragulator or RagA/B deficient cells display constitutive and amino acid-independent mTORC1 activity. mTORC1 activity in p18 KO HEK293T cells (5D) and RagA/B KO MEF cells (5E) were shown.

5F. Blocking lysosome activity enhances mTORC1 activity in Ragulator deficient cells independent of amino acid availability. The indicated cells were starved for amino acids in the presence or absence of a lysosomal inhibitor, Chloroquine (CQ, 50 μM) or Concanamycin A (ConA, 5 μM) for 50 min, then replenished with amino acids for another 10 min.

5G. Blocking proteasome activity also enhances mTORC1 activity in Ragulator deficient cells independent of amino acid availability. Similar experiments were performed as Fig. 5F using a proteasome inhibitor (MG-132; 20 μM).

5H. Blocking lysosome or proteasome activity enhances mTORC1 activity in RagA/B deficient cells independent of amino acid availability. Wild-type (RagA/B FF) or RagA/B KO MEF cells were treated as Fig. 5G.

5I Blocking both lysosome and proteasome function additively enhances mTORC1 activity in Ragulator or RagA/B deficient cells. The indicated cells were starved for amino acids for 15 min, then treated with Chloroquine (CQ; 50 μM), MG-132 (MG; 20 μM), or Chloroquine and MG-132 (CQ+MG) for another 45 min.

5J. Blocking both proteasome and lysosome function further accumulates Ub-Rheb in Ragulator deficient cells. p18 KO HEK293T cells were treated as Fig. 5I.