The obstruction of diffusion by hypertonicity requires the presence of two N-linked oligosaccharides. (A and B) FRAP of the YFP-KDEL receptor (A), DiOC6 (A), and YE variants (B). The translational diffusion was measured as for Fig. 1. YE(gly)1 or YE(gly)2 indicates a YE variant containing one or two glycans, respectively. The averaged recoveries of the indicated molecules are shown. (C) Deletion of VSVG N-glycosylation abolished the diffusional restriction caused by hypertonicity. YFP-fused VSVG variants (see Results) were expressed, and their sensitivity to hyperosmolarity was examined at 38°C using FRAP. Glycosylation of each molecule was examined by immunoblotting (top). The asterisk indicates a nonspecific band. Colors are as in Fig. 1.
