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

A series of deletions were made in the cytoplasmic domain of DPAP A (in the context of the A-ALP fusion protein) to identify the region responsible for its retention in the Golgi (Fig. 1 C) . The A-ALP deletion mutants were expressed in a phoSA strain and localized by indirect immunofluorescence microscopy. In accordance with the vacuolar default model for membrane protein sorting in yeast (Roberts et al., 1992) , the deletion mutants that failed to be retained in the Golgi were delivered to the vacuolar membrane rather than to the cell surface. Table I shows quantitation of the immunolocalization data focusing on the percentage of cells that exhibit vacuolar staining. As indicated in Fig. 4 , wildtype A-ALP exhibited a Golgi staining pattern, with very few cells (<1%) exhibiting vacuolar membrane staining. Removal of residues 2-51 or even 2-80 within the ll8-amino acid cytoplasmic domain had little effect on localization of A-ALP. However, deletion of 20 additional residues (A2-100) resulted in dramatic mislocalization of A 2-100-A-ALP to the vacuole, as the anti-ALP antibody decorated the vacuolar membrane in 100% of the stained cells examined. The residues immediately adjacent to the transmembrane domain were not required for retention since the A109-116-A-ALP protein exhibited essentially wild-type localization. Removal of residues 68-106, as well as the smaller deletion A85-106, prevented retention in the Golgi apparatus. The observation that the 22-amino acid deletion (A85-106) resulted in as severe a mislocalization phenotype (99% of cells exhibiting vacuolar staining) as removal of almost the entire cytoplasmic tail (A2-100, 100%) suggested that the most important elements of the retention signal were within this region. To define region 85-106 further, three smaller deletions (A85-92, A92-99, and A99-106) were analyzed. The A85-92-A-ALP protein was mislocalized to the vacuole (100% of cells * Each construct was analyzed in SF838-1D-8A2 cells (pho8A, pep4-3) contaning CEN-based plasmids encoding either wild-type or mutant A-ALP proteins (see Materials and Methods for detailed description of plasmids). The cells were fixed, spheroplasted, and costained with rabbit anti-ALP and mouse anti-60-kD V-ATPase subunit, and analyzed by indirect immunofluorescence microscopy as described in Materials and Methods. Staining for all constructs was apparent in at least 90% of the cells examined, and of these stained cells at least 100 cells were quantified. ¢ The percentages refer to the percent of cells that exhibit significant vacuolar staining for each protein. Vacuolar staining was scored by comparing the staining pattern of each construct in a given cell with the pattern obtained using an antibody against the 60-kD V-ATPase subunit, a marker for the vacuolar membrane (see Materials and Methods). Depending on the protein being analyzed, other staining patterns such as Golgi staining were sometimes observed rather than (or in addition to) vacuolar staining.