Trafficking of green fluorescent protein tagged-vesicular acetylcholine transporter to varicosities in a cholinergic cell line

Synaptic vesicle proteins are suggested to travel from the trans-Golgi netw ork to active zones via tubulovesicular organelles, but the participation o f different populations of endosomes in trafficking remains a matter of deb ate. Therefore, we generated a green fluorescent protein (GFP)-tagged versi on of the vesicular acetylcholine transporter (VAChT) and studied the local ization of VAChT in organelles in the cell body and varicosities of living cholinergic cells. GFP-VAChT is distributed to both early and recycling end osomes in the cell body and is also observed to accumulate in endocytic org anelles within varicosities of SN56 cells. GFP-VAChT positive organelles in varicosities are localized close to plasma membrane and are labeled with F M4-64 and GFP-Rab5, markers of endocytic vesicles and early endosomes, resp ectively. A GFP-VAChT mutant lacking a dileucine endocytosis motif (leucine residues 485 and 486 changed to alanine residues) accumulated at the plasm a membrane in SN56 cells. This endocytosis-defective GFP-VAChT mutant is lo calized primarily at the somal plasma membrane and exhibits reduced neuriti c targeting. Furthermore, the VAChT mutant did not accumulate in varicositi es, as did VAChT. Our data suggest that clathrin-mediated internalization o f VAChT to endosomes at the cell body might be involved in proper sorting a nd trafficking of VAChT to varicosities. We conclude that genesis of compet ent cholinergic secretory vesicles depends on multiple interactions of VACh T with endocytic proteins.