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

Mutational analysis clearly identified an eight amino stretch containing a critical Phe-X-Phe-X-Asp sequence as necessary for retention; however, it was important to determine whether this sequence motif could function independently to retain a non-Golgi membrane protein. A sufficiency test was conducted by incorporating the 10-amino acid sequence surrounding F85 and F87 that had been analyzed by alanine scanning mutagenesis (residues 81-90 of DPAP A; Table III) into the cytoplasmic domain of ALP, removing residues 11-17 of ALP in the process (Fig. 8 A) . This new hybrid protein (RS-ALP) as well as wild-type ALP were analyzed in a pho8A strain by indirect immunofluorescence microscopy. Fig. 8 B shows that the antibody against ALP clearly labels the vacuolar membrane of cells expressing wild-type ALP, as expected (compare to the 60-kD V-ATPase subunit staining pattern). In contrast, cells expressing RS-ALP exhibit a punctate, predominantly nonvacuolar staining pattern reminiscent of the Golgi membrane proteins DPAP A and Kex2p. All of the cells analyzed clearly exhibited a Golgi-type of staining pattern, whereas 27 % also exhibited a detectable level of vacuolar staining in addition to Golgi staining (see Materials and Methods for details).