Phosphorylation of the rat vesicular acetylcholine transporter

Metabolic labeling of a mutant PC12 cell line, A123.7, expressing recombina nt rat vesicular acetylcholine transporter (VAChT) with radiolabeled inorga nic phosphate was used to demonstrate phosphorylation of the transporter on a serine residue. Mutational analysis was used to demonstrate that serine 480, which is located on the COOH-terminal cytoplasmic tail, is the sole ph osphorylation site. Phosphorylation of serine 480 was attributable to the a ction of protein kinase C. Using a permanently dephosphorylated form of rat VAChT, S480A rVAChT, it was shown that this mutant displays the same kinet ics for the transport of acetylcholine and the binding of the inhibitor ves amicol as does the wild type transporter. However, sucrose gradient density centrifugation showed that, unlike wild type VAChT, the S480A mutant did n ot localize to synaptic vesicles. These results suggest that phosphorylatio n of serine 480 of VAChT is involved in the trafficking of this transporter .