THE NEWLY IDENTIFIED YEAST GRD GENES ARE REQUIRED FOR RETENTION OF LATE-GOLGI MEMBRANE-PROTEINS

Processing of A-ALP, a late-Golgi membrane protein constructed by fusi ng the cytosolic domain of dipeptidyl aminopeptidase A to the transmem brane and lumenal domains of alkaline phosphatase (ALP), serves as a c onvenient assay for loss of retention of late-Golgi membrane proteins in Saccharomyces cerevisiae. In this study, a large group of novel grd (for Golgi retention defective) yeast mutants, representing 18 comple mentation groups, were identified on the basis of their mislocalizatio n of A-ALP to the vacuole, where it was proteolytically processed and thus became enzymatically activated. All of the grd mutants exhibited significant mislocalization of A-ALP, as measured by determining the k inetics of A-ALP processing and by analyzing its localization by indir ect immunofluorescence microscopy. The mutants were evaluated in a var iety of other phenotypic tests relevant to yeast Golgi function, inclu ding processing of the alpha-factor mating pheromone, sorting of the v acuolar hydrolase carboxypeptidase Y, and retention of an early-Golgi membrane protein. Mutants from three grd complementation groups also f ailed to retain an early-Golgi membrane protein, suggesting that these mutations may have more global effects on Golgi retention and functio n, However, the majority of the grd mutants appeared to be defective s pecifically for the retention of several late-Golgi membrane proteins. A subset of the grd mutants appeared defective only in retention of A -ALP and not other late-Golgi membrane proteins. The grd mutants defin e a new set of genes required for Golgi membrane protein retention in S. cerevisiae.