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

The constructs were analyzed in a strain containing a null allele of STE/3, the structural gene for DPAP A (Julius et al., 1983) . Immunoprecipitations of DPAP A, A85-106-AAA, A-X-A, and A85-106-A-X-A expressed from 2 #m plasmids were carried out using a polyclonal antibody against the luminal domain of DPAP A (Roberts et ai., 1992). Fig. 2 from DPAP A whereas the A85-106-AAA and A85-106-A-X-A mutants have a slightly greater mobility, consistent with a 22-amino acid deletion. Non-glycosylated DPAP A is reduced in size ~,5-kD compared to the glycosylated protein (Roberts et al., 1992) , therefore these results indicate that the mutant enzymes are translocated into the ER and receive carbohydrate modifications in the ER and Golgi similarly to DPAP A. To determine the cellular locations of the constructs, indirect immunofluorescence microscopy was performed using an anti-DPAP A antibody (Roberts et al., 1992) . Fig. 3 B shows that A-X-A, like DPAP A (Roberts et al., 1992 ; data not shown), is localized to discrete punctate patches in the cytoplasm distinct from the ER and vacuole and typical of the yeast Golgi apparatus . Comparison of this staining pattern with that of the 60-kD vacuolar proton-translocating ATPase (V-ATPase) subunit ( Fig. 3 C) , a marker for the yeast vacuolar membrane (Yamashiro et al., 1990) , demonstrates that the location of A-X-A is nonvacuolar. A-X-A exhibited a Golgi staining pattern in 100% of the cells examined, and was found on the vacuolar membrane in <1% of cells ( domain of DPAP A does not appear to be involved in its retention in the Golgi other than acting as a membrane anchor. In addition, the A-X-A protein complemented a stel3A strain for processing of the u-factor mating pheromone demonstrating that it was correctly folded and in the correct Golgi compartment (data not shown). In contrast to the Golgi localization of A-X-A, ARS-106-A-X-A exhibited a vacuolar membrane staining pattern (Fig. 3 , compare panels E and F) indistinguishable from that of A85-106-AAA (Roberts et al,, 1992 ). A85-106-A-X-A decorated the vacuolar membrane in 100% of the cells examined and in a small percentage of cells (11%) was also localized to the Golgi apparatus. These data show that mislocalization of DPAP A to the vacuole as caused by the A85-106 mutation occurs norreally when the transmembrane domain is replaced with a synthetic hydrophobic sequence, demonstrating that transport to the vacuole does not require a specific signal within this domain.