FIG. 5.

CTDs of ULK1 and ULK2 inhibit autophagy. (A) Control (pLUC) and expression constructs for Myc-tagged ULK1 and ULK2 CTDs were transfected into 293/GFP-LC3 cells. Inhibition of starvation-induced GFP-LC3 lipidation was measured as described in the legend for Fig. 2. **, P < 0.006 in pairwise comparisons with pLUC-transfected, starved cells. (B) 293/GFP-LC3 cells transfected as described for panel A were labeled overnight with [¹⁴C]valine. Cells were then either left untreated or starved in EBSS for 2 h, and autophagic degradation of long-lived proteins (prot deg) was analyzed. Cell samples transfected in parallel were used as a control to detect overexpressed constructs. Each bar represents the average from three independent samples ± the standard deviation. (C) HEK293A cells were transfected for 24 h with control pLUC plasmid, a Myc-tagged ULK1(1-351) deletion mutant as an internal control, or Myc-tagged ULK1 CTD. Cells were then left untreated or starved in EBSS-leupeptin for 2 h and then lysed for SDS-PAGE analysis of LC3 lipidation. The ratio of LC3-II/LC3-I measured for each sample is indicated at the bottom. (D) Myc-tagged ULK1 CTD constructs with C-terminal truncations were transfected into 293/GFP-LC3 cells, and GFP-LC3 lipidation was measured as described in the legend for Fig. 2. *, P < 0.05; NS, P > 0.4 (in pairwise comparisons with full-length-CTD-transfected, starved cells). A schematic of the truncations is shown in Fig. 2. βTub, β-tubulin.