A phosphorylation site regulates sorting of the vesicular acetylcholine transporter to dense core vesicles

Vesicular transport proteins package classical neurotransmitters for regula ted exocytotic release, and localize to at least two distinct types of secr etory vesicles. In PC12 cells, the vesicular acetylcholine transporter (VAC hT) localizes preferentially to synaptic-like microvesicles (SLMVs), wherea s the closely related vesicular monoamine transporters (VMATs) localize pre ferentially to large dense core vesicles (LDCVs). VAChT and the VMATs conta in COOH-terminal, cytoplasmic dileucine motifs required for internalization from the plasma membrane. We now show that VAChT undergoes regulated phosp horylation by protein kinase C on a serine (Ser-480) five residues upstream of the dileucine motif. Replacement of Ser-480 by glutamate, to mimic the phosphorylation event, increases the localization of VAChT to LDCVs. Conver sely, the VMATs contain two glutamates upstream of their dileucine-like mot if, and replacement of these residues by alanine conversely reduces sorting to LDCVs. The results provide some of the first information about sequence s involved in sorting to LDCVs. Since the location of the transporters dete rmines which vesicles store classical neurotransmitters, a change in VAChT trafficking due to phosphorylation may also influence the mode of transmitt er release.