FIG. 4.

Inactivating point mutations do not disrupt heteromultimer formation. (A) Cells expressing wild-type and mutant forms of VacA are vacuolated. HeLa cells were infected with vT7 and were then transfected with plasmids encoding the indicated protein or protein fusions as described in Materials and Methods. After 24 h, cells were analyzed for neutral red uptake and total protein, as described in Materials and Methods. Data were collected in triplicate for three independent experiments and are expressed relative to cells expressing VacA alone. (B) Wild-type and mutant forms of VacA associate within cells. HeLa cells were infected with vT7 and were then cotransfected with plasmids expressing the indicated fluorescent protein fusions as described in Materials and Methods. After 18 to 24 h, the fluorescence emission of the transfected cells was analyzed by fluorescence microscopy, and FRET images were collected and analyzed as described for Fig. 1. The data were manipulated as described in the text. Values represent the average of at least 12 independent cells and 3 independent experiments.