Expression and localization of synaptotagmin I in rat parotid gland.

Synaptotagmins are a gene family of membrane proteins with distinct expression patterns. Synaptotagmin I is an abundant protein of the synaptic vesicle membrane and was implicated as the Ca2+ sensor in fast responding synapses. Yet, its precise role along the synaptic vesicle life cycle is not fully understood. In this report we show that synaptotagmin I is not exclusively confined to neuronal and neuroendocrine systems, rather, it is also expressed in the exocrine system of the parotid gland. The gene for synaptotagmin I was isolated and sequenced from rat parotid cDNA. The identity of synaptotagmin I protein was further confirmed by several independent antibodies. The protein is exclusively found in the membranous fraction of purified granules, similarly to VAMP-2, another major integral membrane protein of synaptic vesicles. Synaptotagmin I represents 0.4% of the total membrane protein mass of the granule. Using immunoelectron microscopy the two proteins were also localized primarily to the granules' membranes. These findings suggest that synaptotagmin I which regulates Ca(2+)-dependent neurotransmitter release also plays a role which is common to all secretory organelles-neuronal, endocrine and exocrine. A role for synaptotagmin I in integrating signals with protein secretion in the parotid gland is suggested.

The effect of calcium levels on synaptic proteins. A study on VAT-1 from Torpedo.

In this study we compare major synaptic proteins from Torpedo electric organ to their homologues from mammalian brain. Most of these proteins are members of small gene families. We demonstrate a high degree of evolutionary conservation of most synaptic proteins. However, in the electric organ each gene family is represented only by a single member. We focus on VAT-1, a major protein of the vesicle membrane in Torpedo. VAT-1 is located on the synaptic vesicle membrane and is highly concentrated on the plasma membrane following the application of alpha-latrotoxin. Taking advantage of the relative simplicity of Torpedo synapses, we performed an in vitro study on the properties of VAT-1 affected by changes in Ca2+ levels. VAT-1 is a low affinity Ca2+ binding protein whose ability to bind Ca2+ resides mainly, but not entirely, on the carboxy-terminal domain of the protein. In the presence of Ca2+, the protein is organized in a high molecular mass complex, which is destabilized by depleting Ca2+. This effect occurs only by chelating Ca2+ ions, but not with other divalent ions. VAT-1 is not complexed to any of the proteins which were implicated in the docking/fusion complex such as VAMP, synaptophysin or syntaxin, regardless of Ca2+ levels. Dependence of the stability of protein complexes on Ca2+ levels is also demonstrated on Torpedo n-Sec1. The possible physiological implications of such Ca2+ dependence are discussed.

VAT-1 from Torpedo synaptic vesicles is a calcium binding protein: a study in bacterial expression systems.

1. Calcium binding properties were examined in VAT-1, an abundant 41-kDa membrane protein expressed in the cholinergic cynaptic vesicles of Torpedo. 2. An overlay assay, using 45Ca2+ as a tracer, demonstrated the ability of a recombinant VAT-1 produced from the IPTG-inducible pKK223-3 expression vector to bind calcium. 3. A high yield of recombinant VAT-1 was obtained from the glutathione S-transferase (GST) expression system. The fusion product enabled VAT-1 purification via affinity chromatography. Subsequent cleavage by thrombin resulted in its separation from the GST carrier protein. 4. A direct Ca(2+)-binding study was performed with purified VAT-1 by a quick-spin column technique, in the presence of 45Ca2+. Quantitative analysis revealed a 1:1 molar stoichiometry for binding of Ca2+ to VAT-1, with a dissociation constant of 130 microM. 5. A GST-linked truncated protein consisting of 13 kDa from the VAT-1 carboxy-terminal domain was found to retain the capacity to bind Ca2+. 6. A data search for homologies between VAT-1 and known Ca(2+)-binding proteins revealed considerable similarity to members of the annexin family in a 140-amino acid region from the carboxy terminal of VAT-1, which overlaps two tandem Ca(2+)-binding domains of the annexin proteins.

The protein VAT-1 from Torpedo electric organ exhibits an ATPase activity.

VAT-1 is an abundant protein in Torpedo electric organ which copurifies with a major ATPase activity from synaptic vesicles. VAT-1 was expressed in E. coli and the product was purified and analyzed. The protein binds specifically to an ATP column and displays an ATPase activity as measured by the kinetics of [32P]phosphate release. The activity is dependent on divalent ions, with both Mg2+ and Ca2+ supporting the reaction. The apparent Km for ATP is 18 microM. This ATPase activity is not affected by known inhibitors of the vesicular V- and P-type ATPases such as vanadate and N-ethylmaleimide. We suggest that VAT-1 activity may affect ATP-dependent reactions in Torpedo nerve terminals, such as phosphorylation and dephosphorylation of proteins.

VAT-1 from Torpedo is a membranous homologue of zeta crystallin.

VAT-1 is a major protein from Torpedo synaptic vesicles. A protein data-base search revealed a striking homology to zeta crystallin from guinea pig lens. The overall amino-acid identity is 27%, and 58% similarity is reached by including conserved substitutions. The highest similarity (60% to 85%) between the two proteins is observed in five discrete domains, which are also conserved in zinc-dependent dehydrogenases, particularly in the alcohol dehydrogenase family. The cofactor-binding domain of oxidoreductases is conserved in VAT-1 and in zeta crystallin. VAT-1 preferably binds NADPH in the presence of zinc. In contrast with its homologous proteins, VAT-1 is an integral membrane protein of synaptic vesicles.