Glycine residues within the TMD of BRi2 greatly facilitate its intramembrane cleavage by SPPL2b. A, model depicting the proteolytic processing of Bri2 by furin, shedding, and subsequent intramembrane proteolysis. B, amino acid sequence of the Bri2 TMD and the flanking regions. The potential helix-destabilizing residues are enlarged and the corresponding alanine mutations are indicated below. C, Bri2 GallA is less efficiently processed by SPPL2b than Bri2 wt. Co-expression of Bri2 wt or Bri2 GallA with SPPL2b wt or the catalytically inactive SPPL2b D421A mutant shows reduced ICD generation for Bri2 GallA. Bri2 and Bri2 ICD were detected using the anti-FLAG antibody. The corresponding BRICHOS domain was isolated from conditioned media using the polyclonal V5 antibody. D, quantitative analysis of the experiment shown in C. Data represent the means ± S.D. of twelve independent experiments. SPPL2b-dependent ICD generation is significantly reduced to 42.4 ± 17.3% for Bri2 GallA (p < 0.000004). E, proteasomal degradation of Bri2 GallA is not increased. Cells co-expressing SPPL2b wt and either Bri2 wt or Bri2 GallA were treated with 10 μm MG132 or the respective carrier for 24h. Bri2 and Bri2 ICD were detected using the anti-FLAG antibody. The corresponding BRICHOS domain was isolated from conditioned media using the polyclonal V5 antibody. F, quantitative analysis of the experiment shown in E. Data represent the means ± S.D. of six independent experiments. G, cellular localization of Bri2 GallA is similar to Bri2 wt. Immunohistochemistry of HEK293 TR cells transfected with Bri2 wt or Bri2 GallA reveals similar localization of both proteins at the plasma membrane (staining without Triton X-100) and within the secretory pathway (staining with Triton X-100). Bri2 wt and Bri2 GallA were detected using an anti-V5 antibody. Scale bar, 10 μm.

The GXXXG dimerization motif is not a major determinant for the intramembrane cleavage of Bri2. A, GXXXG to AXXXG mutation does not change Bri2 ICD generation. Co-expression of Bri2 wt or Bri2 G67A with SPPL2b wt or the inactive SPPL2b D421A revealed similar ICD production from both Bri2 variants. All proteolytic products were detected as described in Fig. 1C. B, quantitative analysis of the experiment shown in A. Data represent the means ± S.D. of eight independent experiments. C, GXXXG to IXXXG mutation slightly reduced SPPL2b dependent Bri2 ICD generation. Co-expression of Bri2 wt or Bri2 G67I with SPPL2b wt or the inactive SPPL2b D421A revealed a slightly reduced ICD production from Bri2 G67I. All proteolytic products were detected as described in Fig. 1C. D, quantitative analysis of the experiment shown in C. Data represent the means ± S.D. of eighteen independent experiments. E, mutations in GXXXG motif did not affect subcellular localization of Bri2. Immunohistochemistry of HEK293 TR cells transfected with Bri2 wt, Bri2 G67A, or Bri2 G67I reveals similar localization of all Bri2 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.

Gly-60 in the N-terminal part of the Bri2 TMD enables efficient cleavage by SPPL2b. A, G60A mutation in the TMD of Bri2 strongly reduces intramembrane cleavage. Co-expression of SPPL2b wt and the indicated Bri2 mutants revealed a strongly reduced Bri2 ICD generation only in cells expressing the G60A or the GallA mutant. All proteolytic products were detected as described in Fig. 1C. B, quantification of the experiment shown in A. Data represent the means ± S.D. of at least twelve independent experiments. C, glycine mutations in the Bri2 TMD do not affect subcellular localization of Bri2. Immunohistochemistry of HEK293 TR cells transfected with the indicated Bri2 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.

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.

Gly-60 significantly disturbs the α-helical structure of the Bri2 TMD. CD spectroscopy of synthetic Bri2 peptides covering the TMD and its flanking regions. The indicated peptides were incorporated in lipid vesicles and CD spectra were recorded at 20 °C. As indicated by the single minimum of the ellipticity between 220 nm and 230 nm the Bri2 wt peptide is mainly in a β-strand like conformation. Similarly, peptides with the G71A or the G72A mutation are predominantly in a β-strand like conformation. Peptides mimicking the G60A or the Bri2 GallA mutation revealed spectra with a smaller ellipticity at 210 nm to 215 nm suggesting a α-helical conformation.