CKA binds to Atg8 through a region containing a conserved LIR motif. (A) Diagram of the CKA protein structure. Protein domains are indicated by boxes and labeled above the diagram. BD, binding domain. Three putative LIR motifs within CKA that are conserved in the mammalian striatin family orthologues are shown and denoted by a black line under the diagram with their amino acid coordinates. Bacterially expressed and purified protein fragments of CKA used for GST pull-downs are indicated under the diagram, and their binding ability to Atg8a is indicated to the right. (B) CKA directly bound to Atg8a through a region containing the LIR2 motif. All indicated protein fragments of CKA pulled down with GST-Atg8a except the N-terminal fragment that contains only the first LIR domain. (C) GFP-CKA and endogenous CKA from cultured Drosophila cells pulled down readily with GST-Atg8a, but not with GST alone. Mutating the LIR2 motif within CKA diminished the binding of GFP-CKA^mutLIR2 to GST-Atg8a compared with GFP-CKA. Binding of endogenous CKA was similar between the GST-Atg8a pull-downs. The amount of GFP-CKA protein pulled down by GST-Atg8a normalized to endogenous CKA and GST-Atg8a protein levels for four separate experiments, and means and SEM are shown. IB, immunoblot. Paired, one-tailed t test results: *, P < 0.05. Representative blots are shown. For His pull-downs, CKA full-length, 1–300, and 1–400, n ≥ 3 repeats; 1–466 and 166–END, n = 2 repeats.