Figure 3.

Sequential activation of actin assembly sites by ACF1 and ACF2. (A) An extract was fractionated over a Q-Sepharose column to yield the flow through and the 0.5 M KCl eluate, which were subsequently desalted and concentrated. The urea-treated, permeabilized cells were incubated with the flow through (a), the eluate (b), or a 1:1 mixture of the two fractions (c) before Rd-actin polymerization. Rhodamine fluorescence images of representative groups of cells are shown. (B) The complementing factors in the flow through and in the eluate are designated ACF1 and ACF2, respectively. The histograms show the percentages of the urea-treated cells that preferentially incorporated Rd-actin into the bud after the cells were incubated with the buffer, a mixture of ACF1 and ACF2, ACF1 first and then ACF2, or ACF2 first and then ACF1. In the latter two treatments, the cells were washed with the buffer after the incubation with the first factor. The percentages shown are averages of the results from two experiments, and the error bars are standard deviations. Bar, 10 μm.