title: The vesicular acetylcholine transporter interacts with clathrin-associated adaptor complexes AP-1 and AP-2.
abstract: In neuronal cells the neurotransmitter acetylcholine is transferred from the cytoplasm into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). The cytoplasmic tail of VAChT has been shown to contain signals that direct its sorting and trafficking. The role of clathrin-associated protein complexes in VAChT sorting to synaptic vesicles has been examined. A fusion protein between the VAChT cytoplasmic tail and glutathione S-transferase was used to identify VAChT-clathrin-associated protein adaptor protein 1, adaptor protein 2 and adaptor protein 180 complexes from a rat brain extract. In vivo coimmunoprecipitation confirmed adaptin alpha and adaptin gamma complexes, but adaptor protein 180 complexes were not detected by this technique. Deletion and site directed mutagenesis show that the VAChT cytoplasmic tail contains multiple trafficking signals. These include a non-classical tyrosine motif that serves as the signal for adaptin alpha and a dileucine motif that serves as the signal for adaptin gamma. A classical tyrosine motif is also involved in VAChT trafficking, but does not interact with any known adaptor proteins. There appear to be two endocytosis motifs, one involving the adaptor protein 1 binding site and the other involving the adaptor protein 2 binding site. These results suggest a complex trafficking pathway for VAChT.
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, 800 Rose Street, Lexington, KY 40536, USA.
authors.forename: Myung-Hee
authors.initials: MH
authors.lastname: Kim
authors.forename: Louis B
authors.initials: LB
authors.lastname: Hersh
mesh_terms: D033961:Adaptor Protein Complex 1 / Q000737:chemistry*; D033962:Adaptor Protein Complex 2 / Q000737:chemistry*; D033965:Adaptor Protein Complex alpha Subunits / Q000737:chemistry; D033982:Adaptor Protein Complex gamma Subunits / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002966:Clathrin / Q000737:chemistry*; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D026901:Membrane Transport Proteins; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016716:PC12 Cells; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013395:Sucrose / Q000737:chemistry / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins
chemical_list: D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D033982:Adaptor Protein Complex gamma Subunits; D002352:Carrier Proteins; D002966:Clathrin; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; D014443:Tyrosine; D013395:Sucrose; D005982:Glutathione Transferase