FIGURE 4.

Gly-60 directly affects the intramembrane cleavage of Bri2 by SPPL2b and not shedding of Bri2. A, Bri2 ΔE variants lacking the BRICHOS domain affect Bri2 ICD production similar to the respective full-length constructs. Co-expression of SPPL2b wt and the indicated Bri2ΔE mutants revealed a significantly reduced Bri2 ICD generation only in cells expressing Bri2ΔE G60A or Bri2ΔE GallA. Bri ΔE and its corresponding ICDs were detected using the anti-FLAG antibody. The corresponding Bri2 C-peptides were isolated from conditioned media after 4 h using the polyclonal V5 antibody. B, quantification of the experiment shown in A. Data represent the means ± S.D. of at least twelve independent experiments. C, Bri2 ΔE variants all share a similar subcellular localization. Immunohistochemistry of HEK293 TR cells transfected with the indicated Bri2 ΔE variants reveals similar localization of all protein variants at the plasma membrane (staining without Triton X-100 and within the secretory pathway (staining with Triton X-100). Scale bar, 10 μm.