FIG. 7.

Association of Kar2p with the immunotoxin precursor. Cells of P. pastoris strain PAOX-2c were pulse-chase-labeled as described in the legend to Fig. 6 and subsequently lysed in 2% CHAPS in the presence of 30 U/ml apyrase to preserve Kar2p binding. The cell lysates were immunoprecipitated with the anti-Kar2p antibody. The precipitates were then subjected to SDS-PAGE and phosphorimaging (A) or to immunoprecipitation by anti-DT antibody before SDS-PAGE analysis (B). Anti-DT immunoprecipitation was performed after the anti-Kar2p precipitates were boiled for 5 min in the presence of 1% SDS and then diluted 1:6 in IP buffer (see Materials and Methods). The relative amounts of proimmunotoxin (Pro-IT) coprecipitated by Kar2p from PAOX-1c and PAOX-2c are shown in panel C.