Figure 5.

A minor population of Laa1p associates with CCV. LAA1-13Myc cells expressing AUX1/SWA2 from its endogenous promoter (GPY2983, WT) or a galactose-inducible promoter (GPY3011, GAL-AUX1) were grown for at least 24 h at 30°C in YPD, a repressive medium for GAL-AUX1. Extracts were fractionated by differential centrifugation, and medium-speed supernatants were applied to a Sephacryl S-1000 column. Proteins in each fraction were trichloroacetic acid (TCA)-precipitated and analyzed by SDS-PAGE and immunoblotting with antibodies against clathrin heavy chain (Chc1p), myc, and the β subunits of AP-1 (Apl2p) and AP-3 (Apl6p). CCV-containing fractions are indicated with a solid line, and soluble triskelion fractions are indicated with a dotted line. Each lane contains 25% of TCA precipitate (fractions) or 0.5% of total material loaded on column (load).