Fig 2: Intracellular localization of human ABCC6 variants expressed in vivo in mouse liver.The human and mouse ABCC6/Abcc6 were detected on frozen sections by immunofluorescence using the M6II-31 monoclonal antibody (green) and the S-20 polyclonal antibody (red), respectively, Panel A: A low magnification image shows the overall distribution of the human WT ABCC6 in a cross-section of single liver lobe. B: The basolateral plasma membrane localization of the WT ABCC6 expressed in mouse liver hepatocytes was confirmed by immunofluorescent imaging. The Z-stack cross-section images are also shown and arrows point to the basolateral membrane. C: Localization of the human WT and mutant ABCC6. Individual channels and the merged images of the endogenous Abcc6 (red) and the human ABCC6 variants (green) are shown. The scale bar represents 50 µm.

Fig 3: Biochemical characterization of the human WT and mutant ABCC6. A: Expression of ABCC6 variants in Sf9 insect cells as detected by western blotting. The WT ABCC6 and R1339C mutant were detected with the HB6 antibody, whereas the expression of ?ABCC6, V1298F, G1321S, R1138Q and R1314W was revealed with the M6II-7 antibody. B: Upper panel: specific MgATP-binding as revealed by photo-crosslinking of Sf9 membrane preparations with 8-N3-[a-32P] ATP under non-catalytic conditions (at 0°C).. b-gal: control membrane isolated from cells overexpressing b-galactoside. Lower panel: detection of the trapped ADP-vanadate complex (occluded nucleotide intermediate) as revealed by photo-crosslinking of the membrane preparation derived from Sf9 cells with 8-N3-[a-32P] ATP under catalytic conditions (at 37°C). b-gal: control membrane isolated from cells overexpressing b-galactoside. The arrow indicates the position of the labelled proteins C: Relative transport activities of ABCC6 and PXE-causing missense variants. Dark grey columns represent LTC4-transport activities whereas light grey columns indicate GS-NEM transport activities.

Fig 4: Membrane topology and homology model of the human ABCC6. A: The membrane topology was based on prediction previously described [53]. The locations of the various domains are indicated as horizontal arrows above the topology model. Various lines indicate the locations of the epitopes for the polyclonal antibody HB6 and K-14 and monoclonal antibodies M6II-7, M6II-31 utilized in the present study and arrows point to the position of the PXE missense mutations. B: The location of mutants in the three-dimensional homology model of the human ABCC6 protein [23] are shown. The entire model is shown with the two ABC halves of the molecule distinguished by grey and pink colors. The left panel is a schematic illustrating the domain swap characteristic of the ABC-protein superfamily. Protein segments of the ABC-domains forming the transmission interface are space-filled and colored in blue. Protein segments of the intracellular loops forming the transmission interface are colored in red.

Fig 5: Co-localization of the endoplasmic reticulum (ER) marker Calnexin and selected ABCC6 mutants.The liver cells expressing the human ABCC6 variants (WT, R1339C, R1138Q and R1314W) were visualized by immunofluorescence on liver frozen sections using the M6II-31 monoclonal antibody (green) and a rabbit polyclonal antibody to the mouse ER marker Calnexin (red). Minimal co-localization (yellow) was seen between Calnexin and the WT and R1138Q mutant, while some spotty co-localization was visible for the R1339C mutant, primarily around the nuclei indicating partial ER retention or slower trafficking of this variant. The R1314W mutant showed the highest level of co-localization, which suggested that a large fraction of this protein is unable to leave the ER and translocate to the plasma membrane. Scale is shown on the upper left image (50 µm).