Membrane protein retention in the yeast Golgi apparatus: dipeptidyl aminopeptidase A is retained by a cytoplasmic signal containing aromatic residues

Yeast strains were grown at 30°C to midlogarithmic phase in selective synthetic media lacking methionine and cysteine. 0.50Ds of cells (~5 x 10 ~) were then pelleted and resuspended in fresh media. The cells were pulsed by the addition of NEN aSS-express label and chased by addition of 50 t~g/ml methionine and 50 #g/ml cysteine. To end the chase, the cultures were adjusted to 10 mM NaN3 on ice. The cells were then spheroplasted , pelleted, and lysed by incubating in 1% SDS, 8 M urea, 0.5 mM PMSF, 1 tzg/mi leupeptin, and 1/~g/ml pepstatin at 100°C for 5 min. The volume was then adjusted on ice to 1 ml with IP buffer (10 mM Tris, pH 8.0, 0.1% Triton X-100, 2 mM EDTA) and IgGsorb was added to 0.5%. After preadsorbing on ice for 15 min, the IgGsorb was pelleted, and either anti-ALP polycloual antibody (Raymond et al., 1992) or anti-DPAP A polycional antibody (Roberts et al., 1992) was added to the supernatant followed by a 1-h incubation on ice. Immune complexes were precipitated by adding IgGsorb to 0.5% and incubating I h on ice. The precipitates were washed twice with 0.1 M Tris, pH 7.6, containing 2.0 M urea, 0.2 M NaCI, and 1% Triton X-100, and then twice with 1% 2-mercaptoethanol. The samples were then analyzed by SDS-PAGE and fluorography as described previously . Gels were quantified using Radioanalytic Imaging System (AMBIS Inc., San Diego, CA).