Volume 8 issue 6 June 2007
The cellular trafficking of the secretory proprotein convertase PCSK9 and its dependence on
the LDLR
Nasha Nassoury, Daniel A. Blasiole, Angie Tebon Oler, Suzanne Benjannet, Josée Hamelin, Vivianne Poupon, Peter S. McPherson, Alan D. Attie, Annik Prat, and Nabil G. Seidah

Figure S1: Secretability of PCSK9 and its fragment PCSK9-L455X and CHRD constructs. HEK293 cells were either transfected with cDNAs coding for PCSK9, or its derivatives PCSK9_455X (L455X) or the CHRD alone or together with the LDLR. Forty eight hours post transfection; the cells were pulse-labeled for 3h with ³⁵S-Met/Cys and media immunoprecipitated with the mAb-V5. The migration positions of proPCSK9, PCSK9 and CHRD are emphasized.

Figure 1 (jpg)

Figure S2: Specificity of the LDLR and proPCSK9 complex formation. HEK293 cells were either transfected with an empty pIRES2-EGFP vector or co-transfected with cDNAs coding for LDLR and either an empty vector, PCSK9, SKI-1 mutated at its active site His (H249A) or wild type SKI-1 (31). Forty eight hours post transfection; the cells were pulse-labeled for 3h with ³⁵S-Met/Cys and then cell lysates and media immunoprecipitated with the LDLR-C7 antibody. The migration positions of proPCSK9, proSKI-1 and SKI-1 are emphasized. Note the effect of PCSK9 on the increased ratio LDLR in the ER (LDLR_ER) versus the Golgi (LDL_RG).

Figure 2 (jpg)

Figure S3: Immunocytochemical localization of natural PCSK9 mutants in the ER. Immunofluorescence analysis was performed on HuH7 cells transiently transfected with recombinant cDNAs of G106R, L253F, Q554E, C679X and the H226A mutant. The cells were analyzed using both V5 (red) and anti-calnexin (blue) antibodies. Bar = 20 µm.

Figure 3 (jpg)

Figure S4: Targeting of PCSK9 to the ER does not change the sorting of the LDLR to the cell surface. Immunofluorescence analysis was performed on CHO cells transiently transfected with cDNAs of either PCSK9, or its mutants H226A, C679X and R682X. Bar = 20 µm.

Figure 4 (jpg)