Fig. 3. Parietal cell cultures double stained for expression of H,K-ATPase (HK) and ezrin (or CFP-ezrin). Resting cells infected with control virus displayed native ezrin primarily on apical membrane vacuoles and basolateral membrane to some extent, and HK staining was distributed throughout the cytoplasm, presumably on tubulovesicles. After stimulation ezrin and HK were localized to some extent on expanded apical secretory vacuoles. Cells infected with rAD-CFP-ezrin WT, CFP-ezrin T567A mutant and CFP-ezrin T567D mutant were stained for HK and CFP (using a GFP antibody). In resting cells expressing CFP-ezrin WT and T567A mutant where it colocalized with a subset of CFP staining. This was highly unusual because the resting cells displayed none of the morphological characteristics of the secretory phenotype (which promptly occurred after the cells were stimulated with histamine plus IBMX). The targeting of CFP-ezrin T567D mutant was similar to that described in Fig. 2, where the CFP signal was detected primarily at the basolateral membrane. Not only was the normal secretory response absent in the T567D-expressing cells, the HK was completely `repolaraized' to the basolateral membrane, there colocalizing with CFP-ezrin T567D.