FAM134B oligomerization drives endoplasmic reticulum membrane scission for ER‐phagy

A

Mass spectrometry analysis uncovered that FAM134B was phosphorylated at serine 151.

B

Comparison of the self‐interaction of FAM134B mono‐phosphorylation mutants using co‐IP.

C

Comparison of the self‐interaction of FAM134 tri‐phosphorylation mutants using co‐IP.

D

Comparison of membrane scission activity using in vitro liposome fragmentation assay. Scale bars, 10 μm. ***P < 0.001, one‐way ANOVA; error bars indicate SEM (n ≧ 3).

E

Lysosomal cleavage of GFP was analyzed by Western blot for the cells co‐expressing GFP‐SEC61B and FAM134B (WT)‐Flag, FAM134B (SA)‐Flag, or FAM134B (SD)‐Flag.

F, G

Measurement of the intracellular ER‐scission activity. FAM134B knockout (KO) U2OS cells were engineered to express EGFP‐FAM134B (WT), EGFP‐FAM134B (SA), or EGFP‐FAM134B (SD) at endogenous levels, and lysosomal degradation of EGFP‐FAM134B was blocked by Bafilomycin A1 (Baf A1). GFP‐positive puncta were quantified for each cell in (G). For control, 25 cells were counted (n = 25); for EGFP‐FAM134B (WT), n = 27; for EGFP‐FAM134B (SA), n = 29; for EGFP‐FAM134B (SD), n = 25. Scale bars, 10 μm. ***P < 0.001, one‐way ANOVA; error bars indicate SEM.

H, I

Measurement of the ER‐phagy activity. FAM134B knockout (KO) U2OS cells were engineered to express mCherry‐EGFP‐FAM134B (WT), mCherry‐EGFP‐FAM134B (S151A), or mCherry‐EGFP‐FAM134B (S151D) at endogenous levels. Lysosomal mCherry‐positive but GFP‐negative puncta were quantified for each cell in (I). For mCherry‐EGFP‐FAM134B (WT), 25 cells were counted (n = 25); for mCherry‐EGFP‐FAM134B (SA), n = 23; for mCherry‐EGFP‐FAM134B (SD), n = 21. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. ***P < 0.001, one‐way ANOVA; error bars indicate SEM.

Figure 2. Phosphorylation enhances FAM134B oligomerization and its activity in membrane scission and ER‐phagy.