Fig. 5: FKBPL-CKAP4 interaction and FKBPL oligomerization facilitate stress-induced ER-phagy.
From: Cytosolic FKBPL and ER-resident CKAP4 co-regulates ER-phagy and protein secretion
a, c, e HeLa/ssRFP-EGFP-KDEL WT, FKBPL KO (a), CKAP4 KO (c), and FKBPL-CKAP4 DKO (e) cells were incubated in full medium containing 1 μg/ml doxycycline for 14 h. After briefly washed with PBS, cells were left untreated or treated with 1 μM A23187, 0.5 mM dithiothreitol (DTT), 2 μg/ml tunicamycin (TM), 100 nM rapamycin (Rapa), or EBSS for another 6 h. Cell extracts were analyzed by immunoblotting. b, d, f Signal intensities of the cleaved relative to full-length ssRFP-EGFP-KDEL were quantified. g Schematic representation of biotinylation assays using V5-APEX-FKBPL or V5-split-TurboID-FKBPL. h, i HEK293T cells were transfected with V5-APEX-FKBPL for 24 h and left untreated or treated with 1 μM A23187, 0.5 mM DTT, 2 μg/ml TM, 100 nM rapamycin (Rapa), or EBSS for 1 h (h) or 2 h (i). Cells were treated with 500 μM biotin-phenol for another 30 min at 37 °C. Then, cells were incubated with 1 mM H2O2 for 1 min and quickly quenched with 5 mM Trolox, 10 mM sodium azide, and 10 mM sodium ascorbate. j, k HEK293T cells were co-transfected with V5-TurboID(N)-FKBPL and V5-TurboID(C)-FKBPL (represented as V5-split-TurboID-FKBPL) for 24 h and left untreated or treated with 1 μM A23187, 0.5 mM DTT, 2 μg/ml TM, 100 nM Rapa, or EBSS for 1 h (j) or 2 h (k). Cells were treated with 100 mM biotin for another 1 h. Biotinylated proteins pulled down by Streptavidin-Agarose and whole cell lysates (WCL) were analyzed by immunoblotting. l Schematic representation of FKBPL-CKAP4-dependent ER-phagy induction under stress conditions. The illustration created with BioRender.com is released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en. Data represents the mean ± SEM of three independent experiments in (b, d, and f). Differences were statistically analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test in (b, d, and f). Arbitrary units, arb. units.
