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 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.
D033942:Adaptor Proteins, Vesicular Transport; D020816:Amino Acid Motifs / Q000502:physiology; D019943:Amino Acid Substitution; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D034284:Dynamin I; D034281:Dynamins; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C493460:Slc18a3 protein, mouse; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D034284:Dynamin I; D020558:GTP Phosphohydrolases; D034281:Dynamins