The ΔN, ΔIDR2, I56E, and L63R mutants of NCOA4 showed defects in ferritin microautophagy, and TAX1BP1 is required for both macroautophagy and microautophagy of ferritin–NCOA4 condensates. (A) NCOA4 KO HeLa cells expressing mGFP-FTH1 and FLAG-NCOA4 (WT or mutants) were treated with 2 μg/ml doxycycline for 30 h to induce mRuby3-RAB5Q79L expression and then observed by fluorescence microscopy. Scale bars, 10 μm (main) and 1 μm (inset). (B) WT, FIP200 KO, and TAX1BP1 KO HeLa cells expressing Halo-NCOA4 were treated with 20 nM HaloTag SaraFluor 650T ligand for 24 h in DMEM. The cells were further treated with LysoTracker Red for 15 min in DMEM and then observed by fluorescence microscopy. Scale bars, 10 μm (main) and 3 μm (inset). (C) Fluorescence intensity of Halo-NCOA4 per LysoTracker Red-positive structure in B was quantified (n = 2,235–3,626 LysoTracker Red-positive structures from a subset of observations reproduced in at least two independent experiments). Differences were statistically analyzed by Welch’s t test with Holm–Sidak method for multiple comparison (two-tailed test). Data distribution was assumed to be normal, which was not formally tested.
