Figure S2.
FAM134B acetylation dramatically enhances FAM134B oligomerization to induce ER fragmentation and ER-phagy. (A) Purified recombinant protein FAM134B for in vitro liposome fragmentation assay (Fig. 2 C). (B) Induction of expression of mCherry-EGFP-FAM134B or EGFP-FAM134B to endogenous levels. Construction of mCherry-EGFP-FAM134B or EGFP-FAM134B inducible cell line in FAM134B KO U2OS cells. Cells were induced with 0.5 µg Doxycycline for 24 h. Western blot was performed using anti-FAM134B antibody. (C and D) FAM134B K160Q enhanced ER membrane fragmentation. U2OS cells transiently expressed 0.2 µg EGFP-FAM134B (WT), EGFP-FAM134B (K160R), or EGFP-FAM134B (K160Q). Lysosomal degradation of EGFP-FAM134B was blocked by 10 nM BafA1 for 2 h. EGFP-positive puncta were quantified in D (n = 30 cells per group). Scale bars, 10 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (E and F) FAM134B K160Q enhanced ER-phagy. U2OS cells transiently expressed 0.2 µg mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (K160R), or mCherry-EGFP-FAM134B (K160Q). MCherry-positive but EGFP-negative puncta were quantified for in F (n = 30 cells per group). Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (G) Analysis of the oligomer size of recombinant FAM134B protein (WT and K160Q) using Superose6 SEC. (H) Analysis of the secondary structure of FAM134B protein by circular dichroism assay. FAM134B-WT and FAM134B-K160Q protein at peaks collected by SEC were analyzed by circular dichroism (JASCO, J-1500-150ST). (I) Analysis of the oligomer size of FAM134B (WT and K160Q) using native PAGE. FAM134B-Flag purified from HEK293T was eluted with 3× Flag peptide, then loaded into a linear 3–12% gradient native PAGE gel. (J) ER stress markers including ATF6, p-IRE1, and p-PERK were responsive to Tg treatment but not overexpression of FAM134B. HEK293T cells were treated with 1 μM of Tg for 1 h or transfected with FAM134B-Flag. The indicated proteins were detected by Western blot. (K) FAM134B was colocalized with LC3 and BAP31. U2OS cells transfected with EGFP-FAM134B were stained for endogenous LC3 and BAP31, which was as a marker for ER-derived membrane structure. Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. (L and M) More ER membrane fragments were induced by K160Q, which were colocalized with LC3. The number of LC3 puncta not involved in ER-phagy (not colocalized with FAM134B) were the same in each group. LC3-positive puncta were quantified in M (n = 27 cells per group). Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. **P < 0.01, ns means no significance. (N) ER-phagy substrates instead of macroautophagy substrates were accumulated in FAM134B KO U2OS cells. The indicated proteins were detected by Western blot. (O) Overexpression of FAM134B failed to activate macroautophagy. HEK293T cells treated with Torin1 (250 nM) for 4 h or transfected with FAM134B-Flag were analyzed by Western blot for NDP52 and p62 degradation. (P) Lysosomal cleavage of GFP was analyzed by Western blot in U2OS cells transfected with 1.5 μg GFP-SEC61B and 0.5 μg FAM134B-Flag. Cells were treated with 10 nM BafA1 for 4 h or DMSO. WCL: whole-cell lysate. (Q) ULK1 acetylation increased in response to serum starvation but not overexpression of FAM134B. HEK293T cells were serum starved for 12 h or transfected with HA-FAM134B, which simultaneously expressed ULK1-Flag. IP was performed with an antibody to acetylated lysine. ULK1-Flag was detected by Western blot (WB). The experiments were performed twice. (R) VPS34 acetylation decreased in response to Torin1 treatment but not overexpression of FAM134B. HEK293T cells were treated with Torin1 (250 nM) for 2 h or transfected with HA-FAM134B, which simultaneously expressed VPS34-Flag. IP was performed with an antibody to acetylated lysine. VPS34-Flag was detected by Western blot. The experiments were performed twice. Molecular weight measurements are in kD. Source data are available for this figure: SourceData FS2.

FAM134B acetylation dramatically enhances FAM134B oligomerization to induce ER fragmentation and ER-phagy. (A) Purified recombinant protein FAM134B for in vitro liposome fragmentation assay (Fig. 2 C). (B) Induction of expression of mCherry-EGFP-FAM134B or EGFP-FAM134B to endogenous levels. Construction of mCherry-EGFP-FAM134B or EGFP-FAM134B inducible cell line in FAM134B KO U2OS cells. Cells were induced with 0.5 µg Doxycycline for 24 h. Western blot was performed using anti-FAM134B antibody. (C and D) FAM134B K160Q enhanced ER membrane fragmentation. U2OS cells transiently expressed 0.2 µg EGFP-FAM134B (WT), EGFP-FAM134B (K160R), or EGFP-FAM134B (K160Q). Lysosomal degradation of EGFP-FAM134B was blocked by 10 nM BafA1 for 2 h. EGFP-positive puncta were quantified in D (n = 30 cells per group). Scale bars, 10 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (E and F) FAM134B K160Q enhanced ER-phagy. U2OS cells transiently expressed 0.2 µg mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (K160R), or mCherry-EGFP-FAM134B (K160Q). MCherry-positive but EGFP-negative puncta were quantified for in F (n = 30 cells per group). Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (G) Analysis of the oligomer size of recombinant FAM134B protein (WT and K160Q) using Superose6 SEC. (H) Analysis of the secondary structure of FAM134B protein by circular dichroism assay. FAM134B-WT and FAM134B-K160Q protein at peaks collected by SEC were analyzed by circular dichroism (JASCO, J-1500-150ST). (I) Analysis of the oligomer size of FAM134B (WT and K160Q) using native PAGE. FAM134B-Flag purified from HEK293T was eluted with 3× Flag peptide, then loaded into a linear 3–12% gradient native PAGE gel. (J) ER stress markers including ATF6, p-IRE1, and p-PERK were responsive to Tg treatment but not overexpression of FAM134B. HEK293T cells were treated with 1 μM of Tg for 1 h or transfected with FAM134B-Flag. The indicated proteins were detected by Western blot. (K) FAM134B was colocalized with LC3 and BAP31. U2OS cells transfected with EGFP-FAM134B were stained for endogenous LC3 and BAP31, which was as a marker for ER-derived membrane structure. Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. (L and M) More ER membrane fragments were induced by K160Q, which were colocalized with LC3. The number of LC3 puncta not involved in ER-phagy (not colocalized with FAM134B) were the same in each group. LC3-positive puncta were quantified in M (n = 27 cells per group). Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. **P < 0.01, ns means no significance. (N) ER-phagy substrates instead of macroautophagy substrates were accumulated in FAM134B KO U2OS cells. The indicated proteins were detected by Western blot. (O) Overexpression of FAM134B failed to activate macroautophagy. HEK293T cells treated with Torin1 (250 nM) for 4 h or transfected with FAM134B-Flag were analyzed by Western blot for NDP52 and p62 degradation. (P) Lysosomal cleavage of GFP was analyzed by Western blot in U2OS cells transfected with 1.5 μg GFP-SEC61B and 0.5 μg FAM134B-Flag. Cells were treated with 10 nM BafA1 for 4 h or DMSO. WCL: whole-cell lysate. (Q) ULK1 acetylation increased in response to serum starvation but not overexpression of FAM134B. HEK293T cells were serum starved for 12 h or transfected with HA-FAM134B, which simultaneously expressed ULK1-Flag. IP was performed with an antibody to acetylated lysine. ULK1-Flag was detected by Western blot (WB). The experiments were performed twice. (R) VPS34 acetylation decreased in response to Torin1 treatment but not overexpression of FAM134B. HEK293T cells were treated with Torin1 (250 nM) for 2 h or transfected with HA-FAM134B, which simultaneously expressed VPS34-Flag. IP was performed with an antibody to acetylated lysine. VPS34-Flag was detected by Western blot. The experiments were performed twice. Molecular weight measurements are in kD. Source data are available for this figure: SourceData FS2.

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