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Syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa), associated with the neuronal plasmalemma, and synaptobrevin, a membrane protein of synaptic vesicles, are essential components of the exocytotic apparatus of synaptic vesicles. All three can be proteolytically cleaved by tetanus and/or botulinum neurotoxins. As a consequence of their cleavage, exocytosis of neurotransmitters is blocked. In adrenal chromaffin cells botulinum neurotoxin A only incompletely inhibits exocytosis. This incomplete inhibition of exocytosis is associated with only partial cleavage of SNAP-25 by the toxin, indicating that distinct pools of SNAP-25 may exist in chromaffin cells which differ in their sensitivities to botulinum neurotoxin A. In line with this result we localized SNAP-25 by immunogold electron microscopy not only to the plasmalemma but also to the chromaffin vesicle membrane. Moreover, in addition to SNAP-25 monomers, stable SNAP-25/syntaxin heterodimers were found in chromaffin cells. Subfractionation studies revealed the presence of SNAP-25/syntaxin heterodimers in an enriched fraction of chromaffin vesicles. This complex proved to be stable in SDS, and SNAP-25 within heterodimers was resistant to proteolytic attack by botulinum neurotoxin A. We suggest that these preexisting heterodimers may serve as receptors of soluble NSF attachment proteins (SNAP receptors) during chromaffin vesicle exocytosis.

SNAP-25 is located on the plasma membrane and essential for exocytosis of neurotransmitters. It was suggested that SNAP-25 and syntaxin 1 via the interaction with VAMP-2 located on synaptic vesicles mediate the docking of the vesicles with the plasma membrane. In the present study, by means of biochemical and morphological analyses, we showed that SNAP-25 is present on chromaffin granules as well as on the plasma membrane. Reconstitution and immunoprecipitation analyses revealed that SNAP-25 on chromaffin granules has essentially the same properties as does SNAP-25 on the plasma membrane.

Syntaxin 1 (HPC-1), a component of the receptor for SNAPs (soluble N-ethylmaleimide-sensitive factor attachment proteins), has been implicated in the docking and fusion of synaptic vesicles with the plasma membrane. It was reported that syntaxin 1 in rat brain and chromaffin cells (PC12) is exclusively located on the plasma membrane (Bennett, M. K., Calakos, N., and Scheller, R. H. (1992) Science 257, 255-259; Söllner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993) Cell 75, 409-418). By means of biochemical and morphological analyses, we now show that syntaxin 1 is associated with chromaffin granules in the adrenal medulla. This finding raises the possibility that syntaxin 1 in chromaffin cells is a component of vesicle-SNAP receptor as well as one of target-SNAP receptor on the plasma membrane.

The subcellular distribution of three proteins of synaptic vesicles (synaptin/synaptophysin, p65 and SV2) was determined in bovine adrenal medulla and sympathetic nerve axons. In adrenals most p65 and SV2 is confined to chromaffin granules. Part of synaptin/synaptophysin is apparently also present in these organelles, but a considerable portion is found in a light vesicle which does not contain significant concentrations of typical markers of chromaffin granules (cytochrome b-561, dopamine beta-hydroxylase or the amine carrier). An analogous finding was obtained for sympathetic axons. The large dense core vesicles contain most p65 and also SV2 but only a smaller portion of synaptin/synaptophysin. A lighter vesicle containing this latter antigen and some SV2 has also been found. These results establish that in adrenal medulla and sympathetic axons three typical antigens of synaptic vesicles are not restricted to light vesicles. Apparently, a varying part of these antigens is found in chromaffin granules and large dense core vesicles. On the other hand, the light vesicles do not contain significant concentrations of functional antigens of chromaffin granules. Thus, the biogenesis of small presynaptic vesicles which contain all three antigens as well as functional components like the amine carrier is likely to involve considerable membrane sorting.