Department of Biochemistry, University of Washington, Seattle, Washington.
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado.
Traffic. 2021 Jan;22(1-2):38-44. doi: 10.1111/tra.12772. Epub 2020 Dec 7.
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.
AP-3(衔接复合物 3)介导晚期高尔基体或早期内体到晚期内体隔室的运输。在哺乳动物中,AP-3 的突变会导致 Hermansky-Pudlak 综合征 2 型、周期性中性粒细胞减少症和一种癫痫性脑病。在出芽酵母中,AP-3 将货物直接从跨高尔基器运输到溶酶体空泡。尽管该途径非常重要,并且在二十年前就已经发现,但目前还没有用于 AP-3 突变体的快速筛选和选择方法。我们现在报告了 GNSI,这是一种合成的、基因编码的报告分子,可使用显色或生长表型读数,快速进行基于平板的 AP-3 功能缺陷评估。该系统可识别 AP-3 载体囊泡形成和消耗的缺陷,并且可适应平板阵列和液体批量培养格式的高通量筛选或选择。编码 GNSI 的附加体质粒和整合质粒已提交给 Addgene 库。