FIGURE 8.

The LIR motif in ULK1 is needed for amino acid starvation-induced co-localization of ULK1 with endogenous LC3B, GABARAP L1, and WIPI2 on autophagosomes. A, co-localization of wild type GFP-ULK1 with endogenous GABARAP L1 and LC3B in punctuated structures in HEK293 Flp-In T-Rex cells. Expression of GFP-ULK1 (wild type or mutated) was induced for 24 h with tetracycline. Cells were then incubated for 1 h in starvation medium without amino acids, fixed, and stained with anti-LC3B or anti-GABARAP L1 antibodies as indicated. Representative images are shown, the circles indicate dots with co-localization. Bars, 2 μm. B, quantitative analysis of intracellular punctuated structures in HEK293 Flp-In T-Rex cells expressing GFP-ULK1 (wild type or LIR-mutated). Expression of GFP-ULK1 was induced as in A, and cells were then incubated for 1 h in full medium or starvation medium. Cells were fixed and stained with anti-WIPI2, anti-LC3B, or anti-GABARAP L1 antibodies before being analyzed by confocal microscopy. Each bar indicating the number of GFP-ULK1 positive dots per cell (green color) is based on an analysis of 490–570 cells, whereas 100–180 cells were analyzed for co-localization with endogenous WIPI2, LC3B, and GABARAP L1. Under starvation conditions, the average co-localization of WIPI2, LC3B, and GABARAP L1 with wild type ULK1 dots was 47, 45, or 43%, respectively, and for the LIR mutant ULK1 the corresponding numbers were 25, 15, and 22%.