A Similar Calmodulin‐Binding Protein Expressed in Chromaffin, Synaptic, and Neurohypophyseal Secretory Vesicles

Abstract

The presence of calmodulin-binding proteins in three neurosecretory vesicles (bovine adrenal chromaffin granules, bovine posterior pituitary secretory granules, and rat brain synaptic vesicles) was investigated. When detergent-solubilized membrane proteins from each type of secretory organelle were applied to calmodulin-affinity columns in the presence of calcium, several calmodulin-binding proteins were retained and these were eluted by EGTA from the columns. In all three membranes, a 65-kilodalton (63 kilodaltons in rat brain synaptic vesicles) and a 53-kilodalton protein were found consistently in the EGTA eluate. 125I-Calmodulin overlay tests on nitrocellulose sheets containing transferred chromaffin and posterior pituitary secretory granule membrane proteins showed a similarity in the protein bands labeled with radioactive calmodulin. In the presence of 10(-4) M calcium, eight major protein bands (240, 180, 145, 125, 65, 60, 53, and 49 kilodaltons) were labeled with 125I-calmodulin. The presence of 10 microM trifluoperazine (a calmodulin antagonist) significantly reduced this labeling, while no labeling was seen in the presence of 1 mM EGTA. Two monoclonal antibodies (mAb 30, mAb 48), previously shown to react with a cholinergic synaptic vesicle membrane protein of approximate molecular mass of 65 kilodaltons, were tested on total membrane proteins from the three different secretory vesicles and on calmodulin-binding proteins isolated from these membranes using calmodulin-affinity chromatography. Both monoclonal antibodies reacted with a 65-kilodalton protein present in membranes from chromaffin and posterior pituitary secretory granules and with a 63-kilodalton protein present in rat brain synaptic vesicle membranes. When the immunoblotting was repeated on secretory vesicle membrane calmodulin-binding proteins isolated by calmodulin-affinity chromatography, an identical staining pattern was obtained. These results clearly indicate that an immunologically identical calmodulin-binding protein is expressed in at least three different neurosecretory vesicle types, thus suggesting a common role for this protein in secretory vesicle function.

Cellular localization of synaptotagmin I, II, and III mRNAs in the central nervous system and pituitary and adrenal glands of the rat

“…Structural, biochemical, and physiological data are consistent with a crucial role of synaptotagmin in neurotransmitter release (reviewed by DeBello et al, 1993). It is one of the major membrane components of synaptic vesicles (Matthew et al, 1981;Fournier and Trifaro, 1988) and is exclusively expressed in neurons and neuroendocrine cells. Synaptotagmin binds Ca*+ at concentrations in the lo-100 PM range in a phospholipid-dependent manner (Brose et al, 1992) and has been postulated to be the Caz+ sensor which induces exocytosis.…”

“…Although synaptotagmin's role in the exocytosis of secretory vesicles is not clearly established in neuroendocrine cells (see Shoji-Kasai et al, 1992, andElferink et al, 1993), several laboratories have demonstrated synaptotagmin to be present on chromaffin granules (Matthew et al, 1981;Fournier and Trifaro, 1988;Perin et al, 1990). Furthermore, Walch-Solimena et al (1993 showed that synaptotagmin I is present on large dense core vesicle membranes of the posterior pituitary.…”

Distinct effects of alpha-SNAP, 14-3-3 proteins, and calmodulin on priming and triggering of regulated exocytosis.

“…Its site of action is unclear but an action mediated via calmodulindependent protein kinases is unlikely due to its lack of requirement for MgATP. One possible target is the vesicle protein synaptotagmin, which is a component of the SNARE complex (S611ner et al, 1993a), which may be a Ca 2+ receptor in exocytosis (Geppert et al, 1994) and is a known calmodulin-binding protein (Fournier and Trifaro, 1988;Tugal et al, 1991). The ability of synaptotagmin to bind calmodulin is controversial and calmodulin may have an alternative mechanism of action.…”

Calmodulin Regulates Endosome Fusion

“…Another possible target is the vesicle protein synaptotagmin, which is a Ca 2ϩ -binding protein and also a CaM-binding protein (48,49). Synaptotagmin is likely to be a Ca 2ϩ sensor in the final fusion step of exocytosis (23,24).…”