Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.
EMBO Rep. 2023 Oct 9;24(10):e53408. doi: 10.15252/embr.202153408. Epub 2023 Aug 2.
Monoamine transporters retrieve serotonin (SERT), dopamine (DAT), and norepinephrine (NET) from the synaptic cleft. Transporter internalization contributes to the regulation of their surface expression. Clathrin-mediated endocytosis of plasma membrane proteins requires adaptor protein-2 (AP2), which recruits cargo to the nascent clathrin cage. However, the intracellular portions of monoamine transporters are devoid of a conventional AP2-binding site. Here, we identify a MAD2 (mitotic arrest deficient-2) interaction motif in the C-terminus of SERT, which binds the closed conformation of MAD2 and allows for the recruitment of two additional mitotic spindle assembly checkpoint (SAC) proteins, BubR1 and p31 , and of AP2. We visualize MAD2, BubR1, and p31 in dorsal raphe neurons, and depletion of MAD2 in primary serotonergic rat neurons decreases SERT endocytosis in the soma. Our findings do not only provide mechanistic insights into transporter internalization but also allow for rationalizing why SAC proteins are present in post-mitotic neurons.
单胺转运体从突触间隙中回收 5-羟色胺（SERT）、多巴胺（DAT）和去甲肾上腺素（NET）。转运体内化有助于调节其表面表达。网格蛋白介导的质膜蛋白内吞作用需要衔接蛋白-2（AP2），它将货物募集到新生的网格蛋白笼中。然而，单胺转运体的细胞内部分缺乏传统的 AP2 结合位点。在这里，我们在 SERT 的 C 末端鉴定出一个 MAD2（有丝分裂阻滞缺陷-2）相互作用基序，该基序与 MAD2 的封闭构象结合，并允许募集另外两个有丝分裂纺锤体组装检查点（SAC）蛋白，BubR1 和 p31，以及 AP2。我们在背缝核神经元中可视化 MAD2、BubR1 和 p31，并在原代血清素能大鼠神经元中耗尽 MAD2，导致 SERT 在内体中的内化减少。我们的发现不仅为转运体内化提供了机制上的见解，而且还解释了为什么 SAC 蛋白存在于有丝分裂后神经元中。
