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

In mammalian cells, there is much evidence that aromatic residues in the cytoplasmic domains of membrane proteins can act as sorting signals for clustering into coated pits (for review see Trowbridge, 1991) . Therefore, it was intriguing that there were two phenylalanine residues at positions 85 and 87 within the eight amino stretch found to be essential for Golgi retention of A-ALP. To test whether the phenylalanine residues at positions 85 and 87, and the intervening nonaromatic glutamine residue at position 86, were important for retention, extensive site saturation mutagenesis was performed at each of these three positions, The mutant A-ALP hybrids were analyzed by monitoring the kinetics of PEP4-dependent processing, enzymatic activity, as well as indirect immunofluorescence microscopy. Cells expressing either wild-type A-ALP or the mutant protein with F85 substituted by alanine (F85A-A-ALP) were pulse-labeled with 35S for 30 min and chased for the indicated times (Fig. 6) . After a 60-min chase, no processing was detected for wildtype A-ALP whereas processing of the F85A-A-ALP protein was evident at the beginning of the chase period and was ~50% complete by 60-min of chase. Processing of the F85A-A-ALP hybrid protein, which was completely PEP4- . Each strain was 3sS-labcled for 15 rain and chased for 0, 20, 60, and 180 rain, spheroplasted, lysed,and subjected to immunoprecipitation using an anti-ALP antibody. The percent processing at each time point was quantified by scanning SDS-PAGE gels with an AMBIS Redioanalytic Imaging System and the half-time was determined by linear regression analysis. For proteins having <20% processing after a 180-min chase or proteins having no detectable processing at the 180-rain time point, the half-time of processing was expressed as ">180" or by ~no processing," respectively. teins containing amino acid replacements at positions 85, 86, or 87 (Table II) . No processing of wild-type A-ALP was detected after 3 h of chase while every amino acid replacement at positions 85 and 87 caused processing to occur with half times that ranged from 55 to 165 min. Replacing both phenylalanines with alanines (A85,A87-A-ALP; t~a of 70 min) did not decrease the half time of processing as compared with replacing F85 or F87 alone (t~ of 70 and 55, respectively), nor did the double replacement increase the already severe mislocalization defect of the F85A-A-ALP (Fig. 5 ) and F87A-A-ALP (Table I; 96% of cells show vacuolar staining) mutants as judged by immunofluorescence microscopy. Whereas there appears to be a specific requirement for a phenylalanine at position 85, position 87 is somewhat less strict since a tyrosine or valine substitution exhibited a less severe phenotype than the other residues tested. In contrast to replacement of F85 and F87, replacements for Q86 had little if any affect on Golgi retention as judged by the extremely slow rates of processing. While these data suggest that the most critical element of the retention signal consists ofa Phe-X-Phe motif, it is possible that amino acids surrounding this sequence are also important for retention. To test this possibility, alanine scanning mutagenesis was carded out at positions 81-84 and 88-90 (Table HI) Table II. and F87A mutations, did result in a half-time of processing of 105 min indicating that D89 is also important for Golgi retention of A-ALP.