Novel Ca2+-binding Protein (CAPS) Related to UNC-31 Required for Ca2+-activated Exocytosis

doi:10.1074/jbc.272.32.19637

Novel Ca²⁺-binding Protein (CAPS) Related to UNC-31 Required for Ca²⁺-activated Exocytosis*

From the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

Abstract

Exocytotic secretion in neuroendocrine cells is activated by cytoplasmic Ca²⁺ increases. Late post-docking events in dense core vesicle exocytosis in permeable PC12 cells require cytosolic factors for sequential ATP-dependent priming and Ca²⁺-dependent triggering steps. The cytosolic proteins phosphatidylinositol transfer protein and phosphatidylinositol (4)-phosphate 5-kinase, as well as membrane-boundN-ethylmaleimide-sensitive factor, are required for the ATP-dependent priming step. Following priming, the Ca²⁺-dependent triggering of vesicle fusion requires an additional cytosolic factor, CAPS, which was purified as a 145-kDa protein. To clarify late Ca²⁺-dependent events in vesicle fusion, the sequence of rat CAPS cDNA was determined and found to encode a novel protein that is the vertebrate homologue of the Caenorhabditis elegans UNC-31 protein shown genetically to be required for neurosecretion. Recombinant CAPS substituted for cytosol in the Ca²⁺ triggering step in permeable PC12 cells and exhibited moderate affinity (K _d = 270 μm) Ca²⁺binding (2 mol Ca²⁺/mol CAPS dimer), consistent with a role at a Ca²⁺-regulated step in exocytosis.

Footnotes

↵* This work was supported by National Institutes of Health Grants DK25861 and DK40428 (to T. F. J. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U16802.
↵‡ To whom correspondence should be addressed: Dept. of Biochemistry, University of Wisconsin, 420 Henry Mall, Madison, Wisconsin, 53706. Tel.: 608-263-2427; Fax: 608-262-3453; E-mail:tfmartin{at}facstaff.wisc.edu.
↵1 The abbreviations used are: NE, norepinephrine; kb, kilobase(s); BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid; PAGE, polyacrylamide gel electrophoresis.
↵2 B. Berwin, personal communication.
- Received May 9, 1997.