Acidic pH–induced coflotation of WT and mutant RSPs with liposomes. RSPs were incubated with liposomes at pH 5.4 (solid lines) and 8.0 (dotted lines), back-neutralized, and then subjected to centrifugation in sucrose step gradients. The gradients were fractionated, and the amount of E protein in each fraction was determined by a quantitative four-layer ELISA. The top fractions containing RSPs coflotated with the liposomes are indicated by a bracket. (A–C) Representative examples of the analysis of the step gradients obtained with WT (A) and the fusion-negative mutants H323A (B) and H248N-H287A (C). The inset in C shows the fluorescence spectrum of the coflotated fractions of the mutant (gray) relative to those of the WT (black) from experiments using pyrene-labeled RSPs. AU, arbitrary units. (D) Extent of acidic pH–induced coflotation of E with liposomes obtained with mutant RSPs relative to the WT (set at 100%). The data are the means from at least two independent experiments; the error bars represent the standard errors of the means.