Fig. 8: Several transmembrane cargo receptors can bind WIPI proteins.
a, AF3 screen for interaction between all known cargo receptors (soluble and transmembrane) and WIPI2. Predicted interactions are plotted for their ipTM score. b, Microscopy-based bead assay of GST-tagged NIX, CCPG1, FAM134C, TEX264 and FKBP8 or GST alone as a negative control and incubated with mCherry-tagged WIPI2d. c,d, As in b, but with a GFP-tagged C-terminal region of FIP200 (CTR). The laser power was either very low to visualize CCPG1-FIP200 interaction (c) or with higher laser power to visualize FAM134C, TEX264, FKBP8 and FIP200 interaction (d). In c, we used the Fire LUT to better visualize the difference in binding strength between the different receptors. e, As in b, but with mCherry-tagged WIPI2d and/or GFP-tagged C-terminal region of FIP200 (CTR). f, Co-immunoprecipitation of TEX264-GFP or FAM134C-GFP after 6-h starvation treatment with EBSS and immunoblotting for the interaction with WIPI2. g, Turnover analysis of different ER-phagy receptors upon starvation treatment for 12 h with EBSS in WT, WIPI2 KO or FIP200 KO HeLa cells. Data are presented as mean ± s.d. (n = 3 biologically independent experiments). h, Schematic overview of the different selective autophagy pathways. Soluble cargo receptors are recruited to ubiquitinylated organelles and recruit the ULK1 complex through FIP200 to initiate autophagosome biogenesis. Transmembrane cargo receptors can initiate autophagosome biogenesis either by recruiting FIP200 or by recruiting WIPI proteins. The latter then recruits the ULK1 complex through interactions with ATG13. Depending on the cargo receptor, autophagosome biogenesis can be initiated through FIP200- and/or WIPI-driven mechanisms. Results are representative of three independent replicates (b–g). Scale bars, 100 µm. Unprocessed blots are available in the source data. Schematic generated with BioRender.
