Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue.

Abstract

Two different monoclonal antibodies, characterized initially as binding synaptic terminal regions of rat brain, bind a 65,000-dalton protein, which is exposed on the outer surface of brain synaptic vesicles . Immunocytochemical experiments at the electron microscope level demonstrate that these antibodies bind the vesicles in many different types of nerve terminals. The antibodies have been used successfully to purify synaptic vesicles from crude brain homogenates by immunoprecipitation onto the surface of polyacrylamide beads. The profiles of the structures precipitated by these beads are almost exclusively vesicular, confirming the vesicle-specificity of the antibodies . In SDS gels, the antibodies bind a single protein of 65,000 daltons. The two antibodies are not identical, but compete for binding sites on this protein .Immune competition experiments also demonstrate that the antigenic components on the 65,000-dalton protein are widely distributed in neuronal and neural secretory tissues. Detectable antigen is not found in uninnervated tissue-blood cells and extrajunctional muscle . Low levels are found in nonneural secretory tissues ; it is not certain whether this reflects the presence of low amounts of the antigen on all the exocytotic vesicles in these tissues or whether the antigen is found only in neuronal fibers within these tissues . The molecular weight and at least two antigenic determinants of the 65,000-dalton protein are highly conserved throughout vertebrate phylogeny . The two antibodies recognize a 65,000-dalton protein present in shark, amphibia, birds, and mammals. The highly conserved nature of the determinants on this protein and their specific localization on secretory vesicles of many different types suggest that this protein may be essential for the normal function of neuronal secretory vesicles .

Effects of synaptotagmin reveal two distinct mechanisms of agonist‐stimulated internalization of the M4 muscarinic acetylcholine receptor

“…As shown in Figure 1d-g, the distribution of the two signals was identical, and both wild-type and mutant synaptotagmin were expressed almost exclusively at the plasma membrane. This intracellular sorting contrasts sharply with that normally seen in neurons and neuroendocrine cells, where synaptotagmin is delivered to synaptic vesicles and secretory granules, respectively (Matthew et al, 1981), but is in accord with the observed targeting of synaptotagmin to the plasma membrane in Chinese hamster ovary (CHO) cells (Jarousse & Kelly, 2001). We observed similar targeting using wild-type…”

The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis

“…This protein is localized to synaptic vesicles and large dense core secretory vesicles (Matthew et al 1981). It spans the vesicle membrane once and has a short luminal domain and a large cytoplasmic region largely comprised of two C2 domains, designated C2A and C2B.…”

Phosphatidylinositol 3-Kinase C2α Is Essential for ATP-dependent Priming of Neurosecretory Granule Exocytosis

“…We used affinity purified polyclonal antibody against PI3K-C2␣ ( Arcaro et al, 2000), anti-hGH (Sigma), anti-Syt I (Matthew et al, 1981), anti-P18 (Wendler et al, 2001), anti-EEA1 (a kind gift from H.T. McMahon) and anti-clathrin heavy chain (Transduction Laboratories).…”