Figure 6. Coimmunoprecipitation of Cvt19 and prAPI.

(A) Spheroplasts isolated from ape1Δ (THY101) cells containing either Δ 9–11 API or P22L API on a plasmid were pulse labeled for 20 min, chased for 30 min, and lysed in native immunoprecipitation buffer. The resulting extract was cleared by centrifugation at top speed in a microcentrifuge for 5 min before immunoprecipitating with antisera as indicated by “First Ab.” A second, nonnative immunoprecipitation reaction was then performed using the antisera denoted as “Second Ab.”

(B) As in (A) except that wild-type (SEY6210) cells were used. The positions of Cvt19, prAPI, and mAPI are indicated. The lower molecular weight band in the Cvt19 immunoprecipitations is a Cvt19 degradation product.

(C) In vitro binding of Cvt19 and prAPI. Bacterial lysate from cells containing either the control vector alone (Con), or expressing Cvt19 (19) were incubated with in vitro-translated prAPI. API was then recovered by native immunoprecipitation with Cvt19 antiserum. The prAPI recovered is plotted as a percent of the total added to the binding reaction. Error bars represent the standard deviation from three separate experiments.