CYSTEINE STRING PROTEINS ASSOCIATED WITH SECRETORY GRANULES OF THE RAT NEUROHYPOPHYSIS

The properties and subcellular distribution of cysteine string protein s (csps) were analyzed in peptidergic nerve terminals of the rat neuro hypophysis. Polyclonal antibodies raised against recombinant rat brain csp recognized a 36 kDa protein in isolated neurosecretosomes from th e post-pituitary. After chemical deacylation, a single 27 kDa form was detected that displayed identical properties to csps in a whole-brain synaptosomal fraction. Immunoisolation demonstrated that synaptophysi n and csps were located in the same vesicles. Density gradient centrif ugation of postsynaptosomal supernatants of neurohypophysial homogenat es revealed that csps and VAMP were present in two distinct vesicle po pulations. Synaptophysin was only detected in the slowly migrating pop ulation corresponding to small synaptic vesicles, whereas arginine vas opressin was present in the more rapidly sedimenting population indica ting that it contains large dense core vesicles (LDCVs). Immobilized a ntibodies against csp, synaptotagmin, or VAMP captured vesicular argin ine vasopressin confirming the association of these proteins with LDCV s. Co-immunoprecipitation assays with proteins solubilized from neuroh ypophysial or whole-brain nerve terminals failed to reveal complexes c ontaining csp and [I-125]omega GVIA receptors. These results indicate that csps in the CNS are associated with both small synaptic vesicles and LDCVs. However, they do not provide support for the hypothesis tha t protein complexes implicated in exocytosis, which interact with pres ynaptic N-type calcium channels, contain csps.