Fig. 3: Membrane-associated IDRs trigger ER-phagy.
a, CLSM images of delivery of CNX-positive ER portions (red) to LAMP1-positive EL (cyan) upon the expression of GT17 in MEF treated with 50 nm BafA1. GT17 consists of a luminal GFP (G in the schematics on the left) tethered at the ER membrane with a transmembrane domain of 17 residues (T17)68. Schematics of GT17-FAM134BIDR, GT17-SEC62IDR and GT17-TEX264IDR chimeras and the control GT17 construct without the IDR are shown. Scale bars, 1 μm. b, Same as a, where cells express GT17-FAM134BIDR (that is, the GT17 moiety displaying the IDR module of FAM134B at the cytoplasmic face of the ER membrane (left)). c, Same as b for GT17-SEC62IDR. d, Same as b for GT17-TEX264IDR. e–g, Same as b–d for cells expressing GT17-FAM134BIDRLIR (e), GT17-SEC62IDRLIR (f) and GT17-TEX264IDRLIR (g) mutants. Please also refer to Extended Data Fig. 2a–g for micrographs of entire cells. h, LysoQuant quantification of LAMP1-positive EL accumulating CNX-positive ER portions in a–g (n = 15, 21, 16, 19, 17, 16 and 19 cells), N = 3 biological replicates. A one-way ANOVA with Turkey’s multiple comparisons test was performed, F = 64.15. Adjusted P value: ****P < 0.0001. The mean bar is shown. i, GFP native in gel fluorescence and HaloTag assay showing enhanced delivery of ER-phagy reporter HT17 to hydrolytically active EL upon expression of GT17-SEC62IDR (lane 4), GT17-FAM134BIDR (lane 6) and GT17-TEX264IDR (lane 8), as demonstrated by the increase in Halo fragment (high contrast). N = 1 biological replicate. Bottom: the Coomassie loading control. j, IEM showing the localization of gold-labelled GT17-SEC62IDR in the lumen of ER and ER portions in the cytoplasm (red arrowheads) or within EL (blue arrowheads). The dashed line shows the EL limiting membrane. Scale bar, 500 nm. N = 1 biological replicate. k, Same as j for GT17-SEC62IDRLIR.
