OATL1 directly interacted with mammalian Atg8 homologues. (A) Schematic representation of the OATL1 protein. The solid bar indicates a truncated mutant of OATL1 (OATL1-N) that includes a putative LRS. The sequence alignment of the LRS in OATL1, p62, and NBR1 is shown. Residues in the sequences that are conserved and similar are shown against a shaded background. The asterisks indicate the positions of amino acid residues that were substituted in the OATL1 mutants in this study (see E). TBC, the Tre-2/Bub2/Cdc16 domain. (B and C) Yeast cells (pJ69-4A) possessing the indicated genes on pGAD-C1 (prey) or pGBD-C1 (bait) vectors were patched on synthetic complete medium lacking adenine, histidine, leucine, and uracil (SC-AHLW; selection medium). The baits and preys in B are reversed in C. (D) Purified LC3 (LC), GABARAP (GB), or GATE-16 (GT) proteins were mixed with purified GST or GST–OATL1-N proteins and then incubated with glutathione–Sepharose beads. Proteins bound to the beads were analyzed by 14% SDS-PAGE followed by Coomassie Brilliant Blue R-250 staining. (E) Purified GABARAP was mixed with GST, GST–OATL1-N (WT), GST–OATL1-N-W136A (WA), or GST–OATL1-N-E134A/D135A (ED/AA) and then incubated with glutathione–Sepharose beads. Proteins bound to the beads were analyzed by 14% SDS-PAGE followed by Coomassie Brilliant Blue R-250 staining. All proteins used in D and E were produced in bacteria and affinity purified by standard protocols.