Genome-wide analysis of membrane transport using yeast knockout arrays.

ABSTRACT

The transport of membrane-bound proteins through post-Golgi compartments depends on the coordinated function of multiple genes that direct the recognition and routing of protein cargoes to their final cellular destination. As many of these sorting components are nonessential for viability, genome-wide screening of the yeast gene-deletion mutant collection provides a useful strategy for their identification. The potential of this approach is limited only by the availability of transport assays suitable for the high-throughput screening of yeast colony arrays. Two large-scale phenotypic screens to identify novel transport genes are described here. The fluorescence-based Calcofluor white assay identifies mutants with altered plasma membrane localization of the chitin synthase Chs3, which recycles between the cell surface, endosomes, and the late Golgi. The carboxypeptidase Y (CPY) assay allows mutants of a distinct Golgi-to-vacuole transport pathway to be identified, due to the missorting and secretion of the vacuolar hydrolase CPY from the cell.