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. 2022 Sep 15;19(4):1239–1257. doi: 10.1080/15548627.2022.2123098

Figure 5.

Figure 5.

RAB37 induces LC3 lipidation through interaction with LC3. (A) MIN6 cells were transfected with Vector or different amounts (μg) of pLp’s-RAB37-Flag plasmid DNA. The expression levels of Flag and LC3 were determined by immunoblotting analysis using specific antibodies. ACTB/β-actin was used as the internal control. (B) the recombinant or heat-treated RAB37 protein (100°C for 15 min, 4 μM) was incubated with GTP (1 mM), GDP (1 mM), or GTPγS (analog of GTP, 1 mM) at 30°C for 2 h. The solution was mixed with BIOMOL GREEN™, and then incubated at 30°C for 30 min. The produced phosphate was detected using a spectrophotometric multi-well plate reader. (C) LC3 lipidation reaction. The reaction mixture containing 2 μM ATG7, 2 μM ATG3, 10 μM LC3B, 2 mM lipid (400-nm liposomes containing 10 mol% bl-PI, 30 mol% DOPE, 58 mol% POPC, 1 mol% Rhod, and 1 mol% NBD), 1 mm dithiothreitol, 1 mM ATP with or without 4 μM RAB37 protein, and 1 mM GTP was incubated at 37°C for 90 min followed by SDS PAGE analysis of the mixture solution. NT: The reaction solution without RAB37 protein. (D) the PE-NTA lipidation reaction mixture (400 nm liposomes containing POPE:POPC:Rhod:DOGS-NTA = molar ratio 20:73:1:6) plus 4 μM RAB37 protein (RAB37) and various concentrations of RAB37-12xhis-bound liposomes (20, 100, 300 μM RAB37-12xhis with 1.5 mM liposome plus 1 mm GTP) were incubated at 37°C for 90 min. (C and D) the reaction mixtures were analyzed in 12% Bis-Tris PAGE, and total protein levels were determined by Coomassie Brilliant Blue staining. (E) MIN6 cells were treated with LG or HG for 1 h and total protein extraction was immunoprecipitated by anti-LC3 antibody. (F) Human embryonic kidney 293T cells were transfected with plasmid DNA (24 μg) of pCMV-HA-RAB37-WT, pCMV-HA-RAB37-mLIR1, pCMV-HA-RAB37-mLIR2, or pCMV-HA-RAB37-mLIR3, and the total protein extraction was immunoprecipitated by anti-LC3 antibody. The expression levels of HA in the precipitates and whole-cell lysates were determined using anti-HA antibody by immunoblotting. Schematic diagrams of RAB37 mutants in the LIR motif are shown.