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. Author manuscript; available in PMC: 2020 Apr 18.
Published in final edited form as: Cell. 2019 Apr 4;177(3):766–781.e24. doi: 10.1016/j.cell.2019.02.009

Figure 4. The UIM-UDS Interface Mediates ATG8 Binding in Yeast and Humans.

Figure 4.

(A) A collection of yeast UIM proteins bind Atg8 by Y2H. Growth of cells on medium lacking leucine and tryptophan is shown in Figure S4 (also for panels (B), (C) and (E)).

(B) Y2H assays reveal that UIM regions in yeast Ent1 and Ent2 bind Atg8 at the UDS.

(C) Ubx5 interacts with the UDS of Atg8 via a UIM, from Y2H analysis of Ubx5 truncations (upper panel) or mutations (lower panel) paired with WT, ΔLDS or ΔUDS forms of Atg8.

(D) In vitro binding assays confirm the UIM-UDS interaction interface between Ubx5 and Atg8. Purified proteins were mixed and pulled down with Ni-NTA agarose beads. Input and bound proteins were visualized as in Figure 1F.

(E) Several human UIM-containing proteins interact with the ATG8 homologs MAP1LC3a and GABARAP by Y2H.

(F) Screening of a yeast cDNA library by Y2H using WT, ΔLDS or ΔUDS forms of yeast Atg8 identified multiple LDS- and UDS-binding proteins. A Venn diagram of the interaction categories is included; asterisks indicate proteins previously identified as Atg8 interactors.

(G) Dot blot binding assays confirm the candidate interactors identified in panel (F). The assay was performed as in Figure 2J.