Figure 5.

(A) prAPI assembles into higher-order, pelletable Cvt complexes in apg5^ts. Spheroplasts were shifted to 38°C for 5 min, pulse labeled for 5 min, and subjected to a cold chase for 20 min. The labeled spheroplasts were lysed by resuspension in PS200 with or without 5 mM MgCl₂ and then separated into a soluble fraction and a membrane-containing pellet fraction, as described in MATERIALS AND METHODS. The percentage of prAPI recovered in the pellet fraction is represented. (B) prAPI is membrane associated in the apg5^ts mutant. apg5^ts spheroplasts were pulse labeled for 5 min, chased for 60 min, and subjected to fractionation into total (T), supernatant (S), and pellet (P) fractions, as described in MATERIALS AND METHODS. The P fraction was resuspended in 60% sucrose in the absence or presence of 1% Triton X-100 (TX-100) and overlaid with 55% sucrose followed by 35% sucrose. After centrifugation at 100,000 × g for 60 min, float (F), nonfloat (NF), and pellet (P2) fractions were collected and API was recovered by immunoprecipitation. Samples were analyzed by SDS-PAGE, and API was detected with a STORM PhosphorImager.