Synthesis of the Mammalian Synaptic Vesicle Protein Synaptophysin in Insect Cells: A Model for Vesicle Biogenesis

Abstract

The N-glycosylated integral membrane protein synaptophysin is one of the major polypeptide components of small presynaptic transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine cells of mammals. Functional properties, including a possible participation in channel formation, have been investigated by integration of purified synaptophysin into planar lipid bilayers. To overcome some of the inherent limitations of such anin vitroapproach we have overexpressed the rat synaptophysin cDNA in nonneuronal, non-neuroendocrine insect cells with the help of recombinant baculovirus. The complete polypeptide was produced in infected ovarian Sf9 cells at levels exceeding those observed in rat brain. The partially N-glycosylated molecules could be extracted from membranes with non-ionic detergents, most effectively withn-octyl-β-d-glucopyranoside, and could be enriched on chromatofocusing columns. By immunoelectron microscopy synaptophysin was shown to be integrated in the correct orientation into the endoplasmic reticulum, various pleomorphic vesicles and the plasma membrane. Using cell fractionation, including density gradient centrifugation and immunoisolation, we characterized distinct synaptophysin-rich vesicles. These vesicles may help to understand molecular principles of vesicle biogenesis in general and the function of synaptophysin in particular.