Figure 2.
FAM134B acetylation dramatically enhances FAM134B oligomerization to induce ER fragmentation and ER-phagy. (A) Topology of FAM134B. Schematic of the full-length FAM134B; K160 resides in the RTND of FAM134B. CAMK2B-mediated FAM134B S151 phosphorylation, which further enhanced FAM134B8 oligomerization, ER fragmentation, and ER-phagy. G216R, a type II HSAN patient-derived FAM134B variant, exhibits hyperactive in FAM134B oligomerization, ER fragmentation, and ER-phagy. TM: transmembrane. (B) Comparison of self-interaction of FAM134B mutants using co-IP. Mimicking dephosphorylation by SA and deacetylation by K160R reduced FAM134B self-interaction. Mimicking permanent phosphorylation by SD enhanced FAM134B self-interaction. K160Q which mimicking permanent acetylation and G216R dramatically enhanced FAM134B self-interaction and oligomerization. HEK293T cells were transfected with mutants of HA-FAM134B and FAM134B-Flag. IP was performed with anti-Flag beads. The indicated proteins were detected by Western blot (WB). SA: mutanting Serine into Alanine. SD: mutanting Serine into Aspartic acid. (C–E) Acetylation of FAM134B enhanced liposome fragmentation in vitro. After injecting recombinant protein (25 μg) into the chamber (500 μl), the morphological changes of liposomes were monitored by live imaging for 20 min. The images at different time points as indicated were presented. The time required for liposome fragmentation was quantified in E (n = 3 experimental replicates). Scale bars, 10 μm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (F and G) FAM134B K160Q enhanced ER membrane fragmentation. FAM134B KO U2OS cells were engineered to inducibly express EGFP-FAM134B (WT), EGFP-FAM134B (K160R), or EGFP-FAM134B (K160Q) at endogenous levels. Lysosomal degradation of EGFP-FAM134B was blocked by 10 nM BafA1 for 2 h. EGFP-positive puncta were quantified in G (n = 30 cells per group). Scale bars, 10 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (H and I) FAM134B K160Q enhanced ER-phagy. FAM134B KO U2OS cells were engineered to inducibly express mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (K160R), or mCherry-EGFP-FAM134B (K160Q) at endogenous levels. MCherry-positive but EGFP-negative puncta were quantified in I (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. (J) Lysosomal cleavage of GFP was analyzed by Western blot for U2OS cells transfected with 1.5 μg GFP-SEC61B and 0.5 μg FAM134B-Flag mutants. WCL: whole-cell lysate. (K) Analysis of FAM134B-mediated ER membrane fragmentation by CLEM. U2OS cells grown on glass gridded coverslips were transfected with EGFP-FAM134B and imaged by Zeiss Airyscan to collect light microscopy images. Samples were then prepared for imaging by FIB-SEM. Light and electron microscope images were superimposed. Scale bars, 10 μm. (L) Analysis of FAM134B-mediated ER membrane fragmentation by IEM. U2OS cells were transfected with EGFP-FAM134B. White arrowheads indicated that the ER sheet labeled by immuno-gold was wrapped by autophagosomes. Scale bar, 200 nm. (M and N) FAM134B was colocalized with LAMP2 and LC3. U2OS cells transfected with EGFP-FAM134B were stained for endogenous LAMP2 and LC3. Lysosomal degradation of EGFP-FAM134B was blocked by 10 nM BafA1 for 2 h. EGFP-positive puncta were quantified in N (n = 20 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, ***P < 0.001. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F2.

FAM134B acetylation dramatically enhances FAM134B oligomerization to induce ER fragmentation and ER-phagy. (A) Topology of FAM134B. Schematic of the full-length FAM134B; K160 resides in the RTND of FAM134B. CAMK2B-mediated FAM134B S151 phosphorylation, which further enhanced FAM134B8 oligomerization, ER fragmentation, and ER-phagy. G216R, a type II HSAN patient-derived FAM134B variant, exhibits hyperactive in FAM134B oligomerization, ER fragmentation, and ER-phagy. TM: transmembrane. (B) Comparison of self-interaction of FAM134B mutants using co-IP. Mimicking dephosphorylation by SA and deacetylation by K160R reduced FAM134B self-interaction. Mimicking permanent phosphorylation by SD enhanced FAM134B self-interaction. K160Q which mimicking permanent acetylation and G216R dramatically enhanced FAM134B self-interaction and oligomerization. HEK293T cells were transfected with mutants of HA-FAM134B and FAM134B-Flag. IP was performed with anti-Flag beads. The indicated proteins were detected by Western blot (WB). SA: mutanting Serine into Alanine. SD: mutanting Serine into Aspartic acid. (C–E) Acetylation of FAM134B enhanced liposome fragmentation in vitro. After injecting recombinant protein (25 μg) into the chamber (500 μl), the morphological changes of liposomes were monitored by live imaging for 20 min. The images at different time points as indicated were presented. The time required for liposome fragmentation was quantified in E (n = 3 experimental replicates). Scale bars, 10 μm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (F and G) FAM134B K160Q enhanced ER membrane fragmentation. FAM134B KO U2OS cells were engineered to inducibly express EGFP-FAM134B (WT), EGFP-FAM134B (K160R), or EGFP-FAM134B (K160Q) at endogenous levels. Lysosomal degradation of EGFP-FAM134B was blocked by 10 nM BafA1 for 2 h. EGFP-positive puncta were quantified in G (n = 30 cells per group). Scale bars, 10 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (H and I) FAM134B K160Q enhanced ER-phagy. FAM134B KO U2OS cells were engineered to inducibly express mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (K160R), or mCherry-EGFP-FAM134B (K160Q) at endogenous levels. MCherry-positive but EGFP-negative puncta were quantified in I (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. (J) Lysosomal cleavage of GFP was analyzed by Western blot for U2OS cells transfected with 1.5 μg GFP-SEC61B and 0.5 μg FAM134B-Flag mutants. WCL: whole-cell lysate. (K) Analysis of FAM134B-mediated ER membrane fragmentation by CLEM. U2OS cells grown on glass gridded coverslips were transfected with EGFP-FAM134B and imaged by Zeiss Airyscan to collect light microscopy images. Samples were then prepared for imaging by FIB-SEM. Light and electron microscope images were superimposed. Scale bars, 10 μm. (L) Analysis of FAM134B-mediated ER membrane fragmentation by IEM. U2OS cells were transfected with EGFP-FAM134B. White arrowheads indicated that the ER sheet labeled by immuno-gold was wrapped by autophagosomes. Scale bar, 200 nm. (M and N) FAM134B was colocalized with LAMP2 and LC3. U2OS cells transfected with EGFP-FAM134B were stained for endogenous LAMP2 and LC3. Lysosomal degradation of EGFP-FAM134B was blocked by 10 nM BafA1 for 2 h. EGFP-positive puncta were quantified in N (n = 20 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, ***P < 0.001. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F2.

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