ENDOCYTOSIS OF VAMP IS FACILITATED BY A SYNAPTIC VESICLE TARGETING SIGNAL

After synaptic vesicles fuse with the plasma membrane and release thei r contents, vesicle membrane proteins recycle by endocytosis and are t argeted to newly formed synaptic vesicles. The membrane traffic of an epitope-tagged form of VAMP-2 (VAMP-TAg) was observed in transfected c ells to identify sequence requirements for recycling of a synaptic ves icle membrane protein. In the neuroendocrine PC12 cell line VAMP-TAg i s found not only in synaptic vesicles, but also in endosomes and on th e plasma membrane. Endocytosis of VAMP-TAg is a rapid and saturable pr ocess. At high expression levels VAMP-TAg accumulates at the cell surf ace. Rapid endocytosis of VAMP-TAg also occurs in transfected CHO cell s and is therefore independent of other synaptic proteins. The majorit y of the measured endocytosis is not directly into synaptic vesicles s ince mutations in VAMP-TAg that enhance synaptic vesicle targeting did not affect endocytosis. Nonetheless, mutations that inhibited synapti c vesicle targeting, in particular replacement of methionine-46 by ala nine, inhibited endocytosis by 85% in PC12 cells and by 35% in CHO cel ls. These results demonstrate that the synaptic vesicle targeting sign al is also used for endocytosis and can be recognized in cells lacking synaptic vesicles.