Figure 6. Localization of the TSC complex, but not mTORC1, to the lysosome is dependent on farnesylated Rheb.

(A) HeLa cells with siRNA-mediated knockdown of Rheb1 and 2, or control siRNAs (siC), were serum starved then stimulated with insulin (100 nM, 15 min). Note: the siC and siRheb1/2 samples are from the same blot and exposure.

(B) Cells treated as in (A) were co-labeled for TSC2 (red) and LAMP2 (green). Representative cells are shown and percent colocalization is graphed as a mean±SEM (right). *p< 1X10⁻¹² for comparison with unstimulated siC cells.

(C) Cells treated as in (A) were co-labeled for mTOR and LAMP2 (images in Figure S6A) and quantified as in (B).

(D) HeLa cells serum starved (16 hr) with or without FTI-277 (FTI; 10 μM) were left unstimulated or were stimulated with insulin (15 min).

(E) Cells were treated as in (D) prior to labeling and quantification of colocalization as in (B) (graph to right). *p< 1X10⁻¹⁰ for comparison with untreated, serum-starved cells.

(F) Cells treated as in (D) were co-labeled for TSC2 and Rheb1 (images in Figure S6B). Colocalization was quantified as in (B). *p< 1X10⁻⁹ for comparison with untreated cells.

(G) Cells treated as in (D) were co-labeled for mTOR and LAMP2 (images in Figure S6C). Colocalization was quantified as in (B).

(H) Cells treated as in (D) were co-labeled for mTOR and Rheb1 (images in Figure S6D). Colocalization was quantified as in (B). *p< 1X10⁻⁸ for comparison with untreated cells.

(I) Endogenous TSC complex components were pulled down from lysates of serum starved HeLa cells with recombinant purified GST-Rheb preloaded with no nucleotide, GTPγS, or GDPβS compared to control pull downs with GST or GST-Rab5A preloaded with GDPβS. GST-fused bait proteins were detected with Ponceau S stain.

(J) GST-Rheb pulldowns were performed as in (I), but cells were stimulated with insulin (15 min). See supporting data in Figure S6.