Intracellular localization of chimeric VWFpp coexpressed in trans with human propeptide-deleted VWF (Δpro).

AtT-20 cells were transiently transfected with human/canine chimeric VWFpp and human propeptide-deleted VWF (Δpro) to explore what region of VWFpp must contain human sequence to regain association and storage of human Δpro. The expressed chimeric VWFpp is depicted at left, with the portion containing canine sequence in orange and the portion containing human sequence in blue. Transfected cells were fixed, permeabilized, dual-stained as described in “Methods,” and examined by confocal microscopy. Panels A, D, G, and J show cells stained for VWFpp (green). Panels B, E, H, and K show cells stained for VWF (red). The merges of VWFpp and VWF staining are shown in panels C, F, I, and L. Colocalization of VWFpp and VWF is shown in yellow. Expression of C/H-119-VWFpp with human Δpro (A-C) resulted in granular storage of both the chimeric VWFpp (A) and human VWF (B), and the 2 proteins were colocalized (C). The converse chimeric VWFpp, H/C-119-VWFpp, did not sort human VWF to storage (D-F). The chimera C/H-386-VWFpp sorted human VWF to storage granules where they colocalized (G-I). The converse construct, H/C-386-VWFpp, did not traffic human VWF to granules (K-L), although the VWFpp was stored (J). These results demonstrate that the chimeric VWFpp proteins retain the necessary signal(s) for sorting to storage granules and can direct human VWF to storage. Bar, 10 μm.