Trafficking of the vesicular acetylcholine transporter in SN56 cells: a dynamin‐sensitive step and interaction with the AP‐2 adaptor complex

The pathways by which synaptic vesicle proteins reach their destination are not completely defined. Here we investigated the traffic of a green fluorescent protein (GFP)‐tagged version of the vesicular acetylcholine transporter (VAChT) in cholinergic SN56 cells, a model system for neuronal processing of this cargo. GFP‐VAChT accumulates in small vesicular compartments in varicosities, but perturbation of endocytosis with a dominant negative mutant of dynamin I‐K44A impaired GFP‐VAChT trafficking to these processes. The protein in this condition accumulated in the cell body plasma membrane and in large vesicular patches therein. A VAChT endocytic mutant (L485A/L486A) was also located at the plasma membrane, however, the protein was not sorted to dynamin I‐K44A generated vesicles. A fusion protein containing the VAChT C‐terminal tail precipitated the AP‐2 adaptor protein complex from rat brain, suggesting that VAChT directly interacts with the endocytic complex. In addition, yeast two hybrid experiments indicated that the C‐terminal tail of VAChT interacts with the µ subunit of AP‐2 in a di‐leucine (L485A/L486A) dependent fashion. These observations suggest that the di‐leucine motif regulates sorting of VAChT from the soma plasma membrane through a clathrin dependent mechanism prior to the targeting of the transporter to varicosities.