Plate-based assay of AP-3 traffic in budding yeast

AP-3 (adaptor complex 3) mediates traffic between late Golgi or endolysosomal organelles, and late endolysosomal organelles. In mammals, mutations in AP-3 cause Hermansky-Pudlak Syndrome type 2, cyclic neutropenias, and a form of epileptic encephalopathy. In budding yeast, AP-3 carries cargo directly from the trans-Golgi to the lysosomal vacuole. Despite the pathway9s importance, yeast-based selections and screens for AP-3 mutants have not been available. We now report GNSI, a synthetic, genetically-encoded reporter that allows rapid plate-based assessment of AP-3 functional deficiency, using either colorimetric or growth phenotype readouts. This system identifies defects in both the formation and consumption of AP-3 carrier vesicles and will be adaptable to high-throughput screening in both plate array and liquid batch culture formats. A plasmid encoding GNSI has been deposited in the Addgene repository.