The cytoplasmic tail of the vesicular acetylcholine transporter contains a synaptic vesicle targeting signal.

The human homologue of the vesicular acetylcholine transporter (hVAChT) and the neuronal isoform of the vesicular monoamine transporter (hVMAT2) are differentially targeted to two populations of regulated secretory organelles when expressed in PC12 cells. Western blot analysis of subcellular fractions from sucrose equilibrium density gradients and glycerol velocity gradients of homogenates from stably transfected cells revealed hVAChT immunoreactivity in fractions that contain synaptophysin, a marker of synaptic vesicles, while hVMAT2 immunoreactivity was confined to heavy fractions containing chromogranin B, a marker of large dense core vesicles. In cells treated with nerve growth factor, hVAChT immunoreactivity alone co-localized with synaptophysin and was abundantly expressed on synaptic vesicle clusters. Chimeras between hVMAT2 and hVAChT were utilized to identify the domain of hVAChT required for its expression on synaptic vesicles and which would shift the expression of hVMAT2 from large dense core vesicles to synaptic vesicles. Biochemical, immunocytochemical, and electron microscopic analyses revealed that a chimera in which the cytoplasmic tail of hVMAT2 was replaced with hVAChT sequences was now preferentially targeted to synaptic vesicles. In addition, hVAChT expression on synaptic vesicles was nearly abolished when the hVMAT2 cytoplasmic tail was present. Thus, structural information resides within the terminal cytoplasmic domain of VAChT, which specifically targets it to synaptic vesicles.