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

Western blot analysis of extracts ofpho8A cells carrying a CEN-based plasmid encoding the A-ALP fusion protein demonstrated that A-ALP was synthesized as a 95-kD polypeptide, which behaved as an integral membrane protein under high pH carbonate extraction conditions (data not shown). Immunolocalization experiments showed that A-ALP exhibits a cytoplasmic punctate staining pattern (Fig. 4 A) that is distinct from the vacuolar staining pattern obtained using a monoclonal antibody against the 60-kD V-ATPase subunit and distinct from wild-type ALP (see below). The staining pattern of A-ALP was indistinguishable from the staining pattern observed for the Golgi membrane proteins DPAP A (Roberts et al., 1992) , Kex2p , and Kexlp (Cooper and Bussey, 1992) . As a direct test of whether A-ALP is localized to the same Golgi structures as Kex2p, double-labeling experiments were carded out to simultaneously detect the two antigens. Fig. 4 A shows representative cells expressing both A-ALP and Kex2p, where Kex2p was overexpressed (*15-fold) by placing the KEX2 gene under the control of the GAL/promoter to aid in its visualization. This level of KEX2 overexpression does not qualitatively affect its localization . Comparison of the Kex2p staining pattern with that of A-ALP revealed (Fig. 4 A) striking overlap of the staining patterns for the two antigens. Quantitation of the immunofluorescence data (for details see Materials and Methods) indicated that 97 % of the cells that stain for both antigens exhibited extensive colocalization. Thus, the data indicate that the vast majority of A-ALP colocalizes with Kex2p, a marker for the yeast Golgi apparatus (Redding et ai., 1991) .