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

For simultaneous detection of A-ALP, ALP, or DPAP A with the 60-kD V-ATPase subunit, the cells were incubated with the following solutions followed by extensive washing with 5 mg/ml BSA in PBS after each step: (a) 1:10 dilution each of rabbit anti-ALP or anti-DPAP A antibody and the anti-60-kD V-ATPase subunit mouse mAb, (b) 1:1,000 dilution of biotinconjugated goat anti-rabbit IgG (H + L), and (c) 1:500 dilution each of FITC-conjugated streptavidin and rhodamine-conjugated goat anti-mouse IgG (H + L). The procedure for costaining of A-ALP, ALP, or RS-ALP with Kex2p was essentially as described by Franzusoff et al. (1991) and involved the following incubations: (a) I:10 dilution of rabbit anti-ALP, (b) 10 #g/ml Fab fragment of goat-anti-rabbit IgG (H + L), (c) 1:50 dilution of rabbit anti-Kex2p, (d) 1.5/~g/ml mouse anti-rabbit IgG (H + L), and (e) 1.5 ~g/ml each of rhodamine-conjugated rabbit anti-goat F(ab')2 and FITC-conjugated rabbit anti-mouse F(alY)2.