Trafficking of the vesicular acetylcholine transporter in SN56 cells: a dynamin-sensitive step and interaction with the AP-2 adaptor complex |
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Authors: | Barbosa José Ferreira Lucimar T Martins-Silva C Santos Magda S Torres Gonzalo E Caron Marc G Gomez Marcus V Ferguson Stephen S G Prado Marco A M Prado Vania F |
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Institution: | Laboratório de Neurofarmacologia, Departamento de Farmacologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. |
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Abstract: | 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 micro 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. |
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Keywords: | cholinergic mechanisms endocytosis exocytosis synaptic vesicle |
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