Figure 4. A HSAN II patient‐derived variant, FAM134B^{G 216R}, is a gain‐of‐function mutant in oligomerization, ER scission, and ER‐phagy.

• A
  Comparison of the self‐interaction of FAM134B WT with mutants as indicated using co‐IP. SA‐G216R was constructed by simultaneously mutating S149, S151, and S153 to alanine (SA) on the basis of FAM134B‐G216R.
• B
  In vitro liposome fragmentation assay. Scale bars, 10 μm. ***P < 0.001, one‐way ANOVA; error bars indicate SEM (n ≧ 3).
• C, D
  Measurement of the intracellular ER‐scission activity of FAM134B WT, G216R, S151A‐G216R, and SA‐G216R. GFP‐tagged FAM134B proteins were expressed in U2OS cells at same levels. The autolysosomal degradation of ER fragments labeled by GFP‐FAM134B was blocked by Baf A1 or DMSO. GFP‐positive puncta were quantified for each cell in (D). For all of the groups, at least 30 cells were included for quantification; Scale bars, 10 μm. ***P < 0.001, one‐way ANOVA; error bars indicate SEM.
• E, F
  Measurement of the ER‐phagy activity of FAM134B^G216R. U2OS cells transiently expressing mCherry‐EGFP‐FAM134B (WT), mCherry‐EGFP‐FAM134B (G216R), mCherry‐EGFP‐FAM134B (S151A‐G216R), or mCherry‐EGFP‐FAM134B (SA‐G216R) at same levels. Lysosomal mCherry‐positive but GFP‐negative puncta were quantified for each cell in (F). For WT, 50 cells were counted (n = 50); for G216R, n = 55; for S151A‐G216R, n = 51; for SA‐G216R, n = 52. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. *P < 0.05, ***P < 0.001, one‐way ANOVA; error bars indicate SEM.

Source data are available online for this figure.