(A) Amino acids 386–409 in SpHfl1 are required for SpAtg8 binding. (B) Schematic summarizing the results shown in Figure 3—figure supplement 1A and B, and Figure 3—figure supplement 2A. ND, not determined. (C) Amino acids 386–409 in SpHfl1 are sufficient for specific in-vitro interactions with SpAtg8 and ScAtg8. (D) Sequences corresponding to the Atg8-interacting region of SpHfl1 in SpHfl1-related proteins from nine representative fungal species. SpHfl1-related proteins were identified using NCBI BLASTP and aligned using MAFFT. The alignment was manually adjusted and visualized using Jalview. The accession numbers of the sequences (from top to bottom) are: NP_012977.3, XP_002554458.1, XP_011275000.1, XP_013933679.1, CDK27935.1, XP_002494035.1, NP_593211.1, XP_012053683.1, and EPZ35735.1. (E, I) Stereo views of the 2Fo-Fc electron density maps for the SpAtg8-SpHfl1(386-409) complex (E) and the ScAtg8-ScHfl1(368-389) complex (I) calculated using COOT. (F) Structural comparison between SpAtg8 and ScAtg8. Crystal structure of ScAtg8 (PDB 2ZPN) was superimposed on that of SpAtg8 by minimizing the rms differences of main-chain atoms. (G) Intermolecular β-sheet is not formed between Atg8 and Hfl1. Crystal structures of the ScAtg8-ScAtg19(412-415) complex (top; PDB 2ZPN), the SpAtg8-SpHfl1(386-409) complex (middle), and the ScAtg8-ScHfl1(368-389) complex (bottom) are shown with stick models. Broken lines indicate hydrogen bonds responsible for forming the intra- and inter-molecular β-sheets. Numbers indicate distances in Å. For clarity, only main chains are shown except for the two hydrophobic residues that bind to the W-site and the L-site. (H) Amino acids 368–389 in ScHfl1 are important for strong in-vitro interactions with ScAtg8. (J) Head-to-tail interactions of seven copies of the ScHfl1(368-389)-ScAtg8 fusion protein observed in the asymmetric unit. (K) Superimposition of seven copies of the ScAtg8-ScHfl1 complex shown in a main-chain trace. Two copies, colored dark and light gray, show a slightly different conformation. (L) Crystal-packing interactions observed between two copies of ScHfl1. Dark and light gray copies correspond to those in (K).