Children's Medical Research Institute, The University of Sydney, Westmead, Australia.
Children's Medical Research Institute, The University of Sydney, Westmead, Australia; Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Westmead, Australia.
Neurochem Int. 2019 Oct;129:104474. doi: 10.1016/j.neuint.2019.104474. Epub 2019 May 23.
Clathrin-mediated endocytosis at the nerve terminal is dependent on assembly protein 180 (AP180) and adapter protein complex 2 (AP2). Both membrane adapter proteins bind to each other and to clathrin, to drive assembly of the clathrin coat over nascent synaptic vesicles. Using knowledge of in vivo phosphorylation sites, AP180 was mutated to determine the effect on binding. N-terminally truncated AP180 exhibited phospho-mimetic (Ser/Thr to Glu)-dependent interaction with AP2, but not clathrin. C-terminally truncated and full length phospho-mutant AP180 bound less AP2 than wild type. However, there was no difference in AP2 binding for the phospho-mimetic or phospho-deficient (Ser/Thr to Ala) AP180 mutants. Thus, the phospho-mutant approach did not provide clarity for the role of phosphorylation in AP180-AP2 binding. Clathrin exhibited a phospho-mimetic-dependent interaction with full-length AP180. Furthermore, phospho-mimetic AP180 was deficient at assembling clathrin cages. These latter discoveries support a model where AP180 phosphorylation inhibits clathrin binding and assembly.
网格蛋白介导入胞作用依赖于衔接蛋白复合物 2（AP2）和装配蛋白 180（AP180）。这两种膜衔接蛋白彼此结合，与网格蛋白一起，驱动网格蛋白衣被在新生突触小泡上的组装。利用体内磷酸化位点的知识，对 AP180 进行突变以确定对结合的影响。N 端截断的 AP180 表现出与 AP2 的磷酸模拟（Ser/Thr 突变为 Glu）依赖性相互作用，但不与网格蛋白相互作用。C 端截断和全长磷酸突变体 AP180 与野生型相比，与 AP2 的结合减少。然而，磷酸模拟或磷酸缺陷（Ser/Thr 突变为 Ala）AP180 突变体的 AP2 结合没有差异。因此，磷酸突变体方法并没有为磷酸化在 AP180-AP2 结合中的作用提供明确的解释。网格蛋白与全长 AP180 表现出磷酸模拟依赖性相互作用。此外，磷酸模拟的 AP180 在组装网格蛋白笼方面存在缺陷。这些新发现支持了一种模型，即 AP180 磷酸化抑制网格蛋白的结合和组装。
