Figure 2

VR1 is glycosylated at N604. (A) Cartoon (not to scale) of proposed subunit topology for a single VR1 subunit. Shown are the epitopes for the N-terminal and FLAG antibodies used in Western blot experiments, and the consensus sequence for glycosylation, located just distal to the fifth transmembrane domain at position 604. The red circle depicts the approximate localization of this consensus sequence. The line parting from the circle points to the sequence of this N-glycosylation site and the yellow box shows the asparagine which was substituted for a serine in order to produce the glycosylation mutant (N604S). (B) Western blot of oocytes expressing VR1 probed with the N-terminal antibody. Uninjected oocytes and oocytes expressing VR1 were prepared as described in Experimental Procedures. The monomer was observed as a doublet at 80 kDa and at 84 kDa in VR1-injected oocytes and a third band of 200 kDa was also observed in these oocytes. No bands were observed in the uninjected oocytes. (C) Western blot of oocytes expressing VR1 probed with the FLAG antibody. Bands are as in (B). (D) Western blot of oocytes expressing VR1 or N604S. Uninjected oocytes and oocytes expressing VR1 or N604S were prepared as described in Experimental Procedures. The monomer doublet in the VR1 is not present in the N604S mutant; only the 80 kDa band is observed. No bands were observed in the uninjected oocytes. This blot was probed with the FLAG antibody.