| Abstract: | AP‐3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. 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 pathway's importance and its discovery two decades ago, rapid screens and selections 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 chromogenic or growth phenotype readouts. This system identifies defects in both the formation and consumption of AP‐3 carrier vesicles and is adaptable to high‐throughput screening or selection in both plate array and liquid batch culture formats. Episomal and integrating plasmids encoding GNSI have been submitted to the Addgene repository. |
