Figure 3. TP53INP1 acts as the receptor... | Rockefeller University Press

Figure 3.

$TP53INP1 acts as the receptor for autophagic sequestration of SOD1. (A) In a GFP-Trap assay, Flag-LC3C, and to a lesser extent Flag-LC3A and Flag-LC3B, are immunoprecipitated by GFP-TP53INP1. (B) In a GFP-Trap assay, Flag-LC3C is immunoprecipitated by GFP-TP53INP1, but not GFP-TP53INP1(D19A/W31A/V34A, ΔLIR). (C) The schematic shows the generation of TP53INP1 truncations. (D) In a GFP-Trap assay, mCherry-SOD1 is immunoprecipitated by WT GFP-TP53INP1, GFP-TP53INP1(ΔN), and GFP-TP53INP1(ΔM), but not GFP-TP53INP1(ΔC). (E) In an in vitro pull-down assay, TrxA-TP53INP1 is immunoprecipitated by MBP-SOD1. (F and G) Confocal images of 293T cells coexpressing mCherry-SOD1 and GFP-TP53INP1 under starvation (Strv) conditions (F). Cells were treated with digitonin before fixation to remove cytosolic signals. (G) shows relative fluorescence intensity plots along the dotted line in the inset image in F. Bars: 5 μm; insets, 2 μm. (H and I) Confocal images of 293T cells coexpressing mCherry-SOD1 and GFP-TP53INP1 under starvation plus Baf (Strv + Baf) conditions (H). Cells were treated with digitonin before fixation to remove cytosolic signals. (I) shows relative fluorescence intensity plots along the dotted line in the inset in H. Bars: 5 μm; insets, 2 μm. (J and K) Confocal images of siTP53INP1 293T cells expressing mCherry-GFP-SOD1 under control (Ctrl) or starvation (Strv) conditions (J). Quantification of mCherry-only puncta is presented as mean ± SEM (Ctrl, n = 20; Strv, n = 20) (K). Bars: 5 μm; insets, 2 μm. (L) Immunoblotting shows reduced levels of endogenous SOD1 in the lysosomal fraction in siTP53INP1 cells compared with NC cells. (M) Immunoblotting shows that the endogenous LC3-II level is reduced by siTP53INP1, and the reduction is rescued by the overexpression of WT RNAi-resistant SNAP-TP53INP1r and SNAP-TP53INP1(ΔC)r, but not SNAP-TP53INP1(ΔLIR)r. Quantifications of LC3-II levels (normalized by ACTB levels) are shown. (N and O) Confocal images show that siTP53INP1 reduces the increased number of mCherry-only puncta induced by starvation (Strv). This reduction is rescued by the overexpression of WT RNAi-resistant SNAP-TP53INP1r, but not SNAP-TP53INP1(ΔLIR)r or SNAP-TP53INP1(ΔC)r (N). Quantification of mCherry-only puncta is presented as mean ± SEM (NC + SNAP Ctrl, n = 20; NC + SNAP Strv, n = 20; siTP53INP1 + SNAP-TP53INP1r Strv, n = 20; siTP53INP1 + SNAP-TP53INP1(ΔLIR)r Strv, n = 20; siTP53INP1 + SNAP-TP53INP1(ΔC)r Strv, n = 20) (O). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. CTSL, cathepsin L. Source data are available for this figure: SourceData F3$

TP53INP1 acts as the receptor for autophagic sequestration of SOD1. (A) In a GFP-Trap assay, Flag-LC3C, and to a lesser extent Flag-LC3A and Flag-LC3B, are immunoprecipitated by GFP-TP53INP1. (B) In a GFP-Trap assay, Flag-LC3C is immunoprecipitated by GFP-TP53INP1, but not GFP-TP53INP1(D19A/W31A/V34A, ΔLIR). (C) The schematic shows the generation of TP53INP1 truncations. (D) In a GFP-Trap assay, mCherry-SOD1 is immunoprecipitated by WT GFP-TP53INP1, GFP-TP53INP1(ΔN), and GFP-TP53INP1(ΔM), but not GFP-TP53INP1(ΔC). (E) In an in vitro pull-down assay, TrxA-TP53INP1 is immunoprecipitated by MBP-SOD1. (F and G) Confocal images of 293T cells coexpressing mCherry-SOD1 and GFP-TP53INP1 under starvation (Strv) conditions (F). Cells were treated with digitonin before fixation to remove cytosolic signals. (G) shows relative fluorescence intensity plots along the dotted line in the inset image in F. Bars: 5 μm; insets, 2 μm. (H and I) Confocal images of 293T cells coexpressing mCherry-SOD1 and GFP-TP53INP1 under starvation plus Baf (Strv + Baf) conditions (H). Cells were treated with digitonin before fixation to remove cytosolic signals. (I) shows relative fluorescence intensity plots along the dotted line in the inset in H. Bars: 5 μm; insets, 2 μm. (J and K) Confocal images of siTP53INP1 293T cells expressing mCherry-GFP-SOD1 under control (Ctrl) or starvation (Strv) conditions (J). Quantification of mCherry-only puncta is presented as mean ± SEM (Ctrl, n = 20; Strv, n = 20) (K). Bars: 5 μm; insets, 2 μm. (L) Immunoblotting shows reduced levels of endogenous SOD1 in the lysosomal fraction in siTP53INP1 cells compared with NC cells. (M) Immunoblotting shows that the endogenous LC3-II level is reduced by siTP53INP1, and the reduction is rescued by the overexpression of WT RNAi-resistant SNAP-TP53INP1^r and SNAP-TP53INP1(ΔC)^r, but not SNAP-TP53INP1(ΔLIR)^r. Quantifications of LC3-II levels (normalized by ACTB levels) are shown. (N and O) Confocal images show that siTP53INP1 reduces the increased number of mCherry-only puncta induced by starvation (Strv). This reduction is rescued by the overexpression of WT RNAi-resistant SNAP-TP53INP1^r, but not SNAP-TP53INP1(ΔLIR)^r or SNAP-TP53INP1(ΔC)^r (N). Quantification of mCherry-only puncta is presented as mean ± SEM (NC + SNAP Ctrl, n = 20; NC + SNAP Strv, n = 20; siTP53INP1 + SNAP-TP53INP1^r Strv, n = 20; siTP53INP1 + SNAP-TP53INP1(ΔLIR)^r Strv, n = 20; siTP53INP1 + SNAP-TP53INP1(ΔC)^r Strv, n = 20) (O). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. CTSL, cathepsin L. Source data are available for this figure: SourceData F3