Figure 4.

Membrane recruitment of SPG15 is independent of mTORC1. (A) Diagram showing the relationships between some of the players in the mTORC1 pathway for nutrient sensing on the lysosome surface. The Ragulator complex recruits the Rag heterodimer, which in turn recruits mTORC1, consisting of RAPTOR, mTOR, and three other subunits; however, Rag binding to mTORC1 only occurs when RagA/B is GTP bound and RagC/D is GDP bound. This is the nucleotide loading state in fed cells, while starved cells typically have GDP-loaded RagA/B and GTP-loaded RagC/D. (B) IF double labeling of SPG15-GFP (amplified with anti-GFP) and mTOR in cells that had been treated with siRNA to knock down either RagC, the Ragulator LAMTOR1 subunit, or RAPTOR, compared with cells transfected with a nontargeting siRNA (control). Knocking down RagC or Ragulator affected the recruitment of both SPG15-GFP and mTOR, while knocking down RAPTOR affected mTOR recruitment only. Scale bar: 20 µm. (C) Western blot showing the efficiency of the RAPTOR and Ragulator knockdowns. Shown here is a control versus RAPTOR knockdown, but it is representative of the loading for all knockdowns. kd, knockdown.