The cell cycle protein MAD2 facilitates endocytosis of the serotonin transporter in the neuronal soma

Abstract 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 comet , and of AP2. We visualize MAD2, BubR1, and p31 comet 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.