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. Author manuscript; available in PMC: 2021 Nov 9.
Published in final edited form as: Dev Cell. 2020 Sep 10;55(3):289–297.e4. doi: 10.1016/j.devcel.2020.08.008

Figure 3. Endolysomal LC3 lipidation induces selective lysosome turnover.

Figure 3.

(A) Treatment with monensin, nigericin, or ammonium chloride for two hours induces colocalization of GFP-LC3 (green) with LAMP1 (red) on enlarged endolysosomes. Insets at bottom are indicated by white hatched boxes. Scale bar = 10μm. (B) Treatment with monensin, nigericin, or ammonium chloride for eight hours induces turnover of GFP-TRPML1 in an ATG5-regulated, ATG13-independent manner. Actin loading controls are also shown. (C) Graph shows quantification of data from part B from three independent experiments; error bars show SEM. (D) Treatment with ammonium chloride for 24 hours induces selective turnover of GFP-TRPML1 and GFP-SNAT7. Graph shows relative turnover of the indicated GFP-tagged lysosomal transmembrane proteins in control or ammonium-treated cells, normalized to turnover in response to LLOMe. Data are from three independent experiments for each reporter; error bars show SEM. *p<.05; **p<.01; ***p<.001. Note that CTNS-GFP, LAMP1-GFP, and PQLC2-GFP turnover to a significantly lesser extent in response to ammonium as compared to LLOMe. Representative western blots are shown below the graph. For each reporter, free GFP is indicated with an arrow and full-length protein is indicated with an asterisk. (E) GFP-TRPML1 turnover in cells starved for glucose for 24 hours requires the presence glutamine. Right schematic: glutamine catabolism generates ammonium.