Figure 5.
SIRT7 deacetylates FAM134B at K160 to reduce ER-phagy. (A) K160 acetylation of endogenous FAM134B increased by being treated with deacetylase inhibitor NAM (5 mM) for 12 h. IP was performed with an antibody to acetylated lysine. K160 acetylation and FAM134B were detected by Western blot (WB). (B) The interaction of FAM134B with deacetylase SIRT. Myc-tagged SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7 were expressed individually in HEK293T cells, which simultaneously expressed FAM134B-Flag. IP was performed with anti-Myc magnetic beads, which was followed by Western blot for FAM134B-Flag. (C and D) K160 acetylation accelerated by SIRT7. Myc-tagged Ctrl, SIRT2, SIRT3, or SIRT7 was expressed individually in HEK293T. The indicated proteins were detected by Western blot. Quantification of K160 acetylation was shown in D (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (E) FAM134B K160 deacetylation by SIRT7 in vitro. FAM134B-Flag purified from HEK293T with anti-Flag beads were incubated with HA-SIRT7 in deacetylation buffer at 37°C for 2 h. FAM134B acetylation was analyzed by Western blot. The experiments were performed twice. (F) CBP and SIRT7 were translocated from nucleus to ER under Tg treatment. HEK293T cells treated with Tg (1 μM) for 0, 0.5, 1 h were performed with ER isolation assay. ER components were labeled with SEC61B. Nucleus was labeled with Lamin-B1. Mitochondria were labeled with COXII. The indicated proteins were detected by Western blot. The experiments were performed twice. (G and H) Endogenous SIRT7 was translocated from nucleus to ER labeled by CLIMP63 upon Tg treatment (1 μM, 1 h). Scale bars, 10 µm. The colocalization was analyzed by Pearson’s correlation coefficient in H (n = 20 cells per group). Data are shown as means ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (I and J) SIRT7 KD enhanced FAM134B-mediated ER membrane fragmentation. FAM134B KO U2OS cells were engineered to inducibly express EGFP-FAM134B (WT) at endogenous levels. Cells were treated with Tg (1 μM, 1 h) or DMSO. Lysosomal degradation of EGFP-FAM134B was blocked by 10 nM BafA1 for 2 h. EGFP-positive puncta were quantified in J (n = 25 cells per group). Scale bars, 10 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. **P < 0.01, ***P < 0.001. (K and L) SIRT7 KD accelerated FAM134B-mediated ER-phagy. SIRT7 WT or KD U2OS cells transiently expressing 0.2 µg mCherry-EGFP-FAM134B (WT). MCherry-positive but EGFP-negative puncta were quantified for in L (n = 30 cells per group). Scale bars, 10 µm. The scale bars in the magnification. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (M and N) ER membrane protein degradation analysis in SIRT7 WT or KD cells. Cells were treated with Tg (1 μM) for 0, 0.5, 1, 3, 6 h. The indicated proteins were detected by Western blot. Quantification of K160 acetylation was shown in N (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. **P < 0.01. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F5.

SIRT7 deacetylates FAM134B at K160 to reduce ER-phagy. (A) K160 acetylation of endogenous FAM134B increased by being treated with deacetylase inhibitor NAM (5 mM) for 12 h. IP was performed with an antibody to acetylated lysine. K160 acetylation and FAM134B were detected by Western blot (WB). (B) The interaction of FAM134B with deacetylase SIRT. Myc-tagged SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7 were expressed individually in HEK293T cells, which simultaneously expressed FAM134B-Flag. IP was performed with anti-Myc magnetic beads, which was followed by Western blot for FAM134B-Flag. (C and D) K160 acetylation accelerated by SIRT7. Myc-tagged Ctrl, SIRT2, SIRT3, or SIRT7 was expressed individually in HEK293T. The indicated proteins were detected by Western blot. Quantification of K160 acetylation was shown in D (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (E) FAM134B K160 deacetylation by SIRT7 in vitro. FAM134B-Flag purified from HEK293T with anti-Flag beads were incubated with HA-SIRT7 in deacetylation buffer at 37°C for 2 h. FAM134B acetylation was analyzed by Western blot. The experiments were performed twice. (F) CBP and SIRT7 were translocated from nucleus to ER under Tg treatment. HEK293T cells treated with Tg (1 μM) for 0, 0.5, 1 h were performed with ER isolation assay. ER components were labeled with SEC61B. Nucleus was labeled with Lamin-B1. Mitochondria were labeled with COXII. The indicated proteins were detected by Western blot. The experiments were performed twice. (G and H) Endogenous SIRT7 was translocated from nucleus to ER labeled by CLIMP63 upon Tg treatment (1 μM, 1 h). Scale bars, 10 µm. The colocalization was analyzed by Pearson’s correlation coefficient in H (n = 20 cells per group). Data are shown as means ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (I and J) SIRT7 KD enhanced FAM134B-mediated ER membrane fragmentation. FAM134B KO U2OS cells were engineered to inducibly express EGFP-FAM134B (WT) at endogenous levels. Cells were treated with Tg (1 μM, 1 h) or DMSO. Lysosomal degradation of EGFP-FAM134B was blocked by 10 nM BafA1 for 2 h. EGFP-positive puncta were quantified in J (n = 25 cells per group). Scale bars, 10 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. **P < 0.01, ***P < 0.001. (K and L) SIRT7 KD accelerated FAM134B-mediated ER-phagy. SIRT7 WT or KD U2OS cells transiently expressing 0.2 µg mCherry-EGFP-FAM134B (WT). MCherry-positive but EGFP-negative puncta were quantified for in L (n = 30 cells per group). Scale bars, 10 µm. The scale bars in the magnification. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (M and N) ER membrane protein degradation analysis in SIRT7 WT or KD cells. Cells were treated with Tg (1 μM) for 0, 0.5, 1, 3, 6 h. The indicated proteins were detected by Western blot. Quantification of K160 acetylation was shown in N (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. **P < 0.01. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F5.

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