The Newly Identified Yeast GRD Genes Are Required for Retention of Late-Golgi Membrane Proteins

Abstract

Processing of A-ALP, a late-Golgi membrane protein constructed by fusing the cytosolic domain of dipeptidyl aminopeptidase A to the transmembrane and lumenal domains of alkaline phosphatase (ALP), serves as a convenient 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 complementation groups, were identified on the basis of their mislocalization 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 kinetics of A-ALP processing and by analyzing its localization by indirect immunofluorescence microscopy. The mutants were evaluated in a variety of other phenotypic tests relevant to yeast Golgi function, including processing of the a-factor mating pheromone, sorting of the vacuolar hydrolase carboxypeptidase Y, and retention of an early-Golgi membrane protein. Mutants from three grd complementation groups also failed to retain an early-Golgi membrane protein, suggesting that these mutations may have more global effects on Golgi retention and function. However, the majority of the grd mutants appeared to be defective specifically 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 define a new set of genes required for Golgi membrane protein retention in S. cerevisiae.