| CAPS (Ca2+-dependent Activator Protein for Secretion) is a protein discovered for its ability to promote Ca2+-triggered dense-core vesicle exocytosis in permeabilized neuroendocrine cells. To directly test whether CAPS is required for the process of regulated exocytosis in intact cells, C. elegans neurons that lacked the sole CAPS ortholog UNC-31, were monitored for vesicle exocytosis. Neuropeptide secretion from dense-core vesicles was strongly impaired in unc-31 mutants, both in cultured neurons and in living worms. In contrast, cultured neurons from unc-31 mutants exhibited normal stimulated synaptic vesicle recycling measured by FM4-64 dye uptake.;In order to gain insight into how CAPS functions to promote regulated exocytosis, chemical mutagenesis was performed on C. elegans to isolate loss-of-function mutations in UNC-31/CAPS. Two loss-of-function missense mutations were isolated in the C2 domain of UNC-31. These conserved residues (L468 and G476) were mutated in rat CAPS and subsequently shown to be similarly non-functional proteins. One of these mutations, G476E, exhibited hyper-oligomerization when subjected to native gel analysis, which led us to hypothesize that the C2 domain may be an oligomerization domain and that oligomerization might be important for the function of CAPS.;CAPS was originally purified as a dimer of ∼300kda, but the significance of its oligomeric state has not been determined. Using native-PAGE, we confirmed that CAPS exists primarily as a dimer, however a small fraction also exists as a tetramer. We find that the tetramer is the active form of CAPS and represented the major species of CAPS that bound to t-SNAREs, both in purified proteoliposomes and on the plasma membrane of permeable PC12 cells. Furthermore, deletion of the C2 domain impaired tetramer formation and completely abolished CAPS activity, without affecting dimer formation. We conclude that CAPS acts as a tetramer to promote regulated exocytosis and that the C2 domain of CAPS is required for optimal tetramerization. |
