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. 2012 Oct 5;287(47):39275–39290. doi: 10.1074/jbc.M112.378109

FIGURE 2.

FIGURE 2.

Identification of a GABARAP interacting LIR motif in ULK1. A, schematic representation of the domain organization of human ULK1. The ULK1 fragments used to map the GABARAP-interacting region are indicated. (+) and (−) indicate the presence or absence of GABARAP binding, respectively. B, GST pulldown assay showing a direct interaction between GABARAP and a deletion fragment of ULK1 encompassing amino acids 351–400. All proteins were produced in E. coli, and MBP or MBP-GABARAP was eluted from the beads and tested for interaction with immobilized GST or GST-ULK1(351–400). Precipitated GST or GST-ULK1(351–400) and co-precipitated MBP or MBP-GABARAP were analyzed by Western blotting using the indicated antibodies. C, GST pulldown assay demonstrating loss of binding of the F357A and F357A/V360A point mutations of ULK1(351–400) to in vitro translated GFP-GABARAP. D, full-length ULK1 with the F357A and F357A/V360A point mutations that lost binding affinity for GABARAP. The GST pulldown assay was carried out using in vitro translated full-length ULK1 constructs and bacterially expressed and immobilized GST or GST-GABARAP. E, the F357A/V360A point mutations prevent co-precipitation of ULK1 with GFP-GABARAP from HEK293 cell extracts. Cells were co-transfected with the indicated constructs, and GFP or GFP-GABARAP were immunoprecipitated (IP) using GFP antibodies. The inputs of wild type or mutated 3xFLAG-ULK1 (lower panel), precipitated GFP or GFP-GABARAP (middle panel), and co-precipitated FLAG-ULK1 (upper panel) were analyzed by Western blotting (WB) using the indicated antibodies. F, yeast Atg1 interacts with yeast Atg8. In vitro translated yAtg1 was tested in a GST pulldown assay for interaction with GST or GST-yAtg8. Autoradiograph (AR) and Coomassie staining of immobilized GST or GST-yAtg8 (CBB) are shown. G, Drosophila Atg1B interacts with Drosophila Atg8A via a LIR motif. In vitro translated DmAtg1B, wild-type, and LIR mutant, were tested in a GST pulldown assay for interaction with GST or GST-DmAtg8A. Autoradiograph and Coomassie staining of immobilized GST or GST-DmAtg8A (CBB) are shown.