Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion

Neil J. Daily,Thomas F.J. Martin,Kristin L. Boswell,Declan J. James

doi:10.1074/jbc.m110.145169

Neil J. Daily, Thomas F.J. Martin + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m110.145169

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Abstract

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca(2+)-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.

Highlights

Plasma membrane t-SNARE syntaxin-1, and two helices contributed by the plasma membrane t-SNARE SNAP-25 (3)
We suggest that CAPS promotes the priming of vesicle exocytosis by driving trans SNARE complex formation through direct interactions with three SNARE motifs
CAPS is essential for optimal Ca2ϩ-triggered vesicle exocytosis where it functions in priming reactions that precede triggered membrane fusion (19, 38, 39)

Summary

EXPERIMENTAL PROCEDURES

Materials—1,2-dioleoyl phosphatidylcholine (DOPC), 1,2dioleoyl phosphatidylserine (DOPS), N-(7-nitro-2–1,3benzoxadiazol-4-yl)-1,2-dipalmitoyl phosphatidylethanolamine (NBD-PE), N-(lissamine rhodamine B sulfonyl)-1,2-dipalmitoyl phosphatidylethanolamine (Rh-PE), L-␣-phosphatidylinositol4,5-bisphosphate (PI 4,5-P2), and 1,2-dioleoyl-sn-glycero3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (MPB-PE) were purchased from Avanti Polar Lipids, Inc. PET10 plasmids encoding His-tagged VAMP-2-(1–94) and pET28a plasmids encoding His-tagged complexin were used to express and purify proteins by Ni-NTA chromatography with elution into reconstitution buffer without glycerol. Preparation of Proteoliposomes—For liposome fusion assays, proteoliposomes were formed by co-micellization in the presence of either VAMP-2, co-expressed syntaxin-1 and SNAP25B or syntaxin-(183–288) and SNAP-25B as previously described (2, 22). A lipid film containing 1.5 ␮mol of DOPC: DOPS in an 85:15 mole ratio was resuspended with 500 ␮l of SNARE proteins in elution buffer (25 mM HEPES-KOH pH 7.4, 400 mM KCl, 500 mM imidazole-OAc, pH 7.4, 10% glycerol (w/v), 1.0% ␤-octylglucoside). The dried lipid film containing a DOPC:DOPS:Rh-PE:NBD-PE lipid mix in an 82:15:1.5:1.5 mol ratio was resuspended with 500 ␮l of 280 ␮g/ml VAMP-2 protein diluted in elution buffer. His-tagged CAPS was immobilized via Ni2ϩ-NTA chelation, and proteins were examined for binding by flowing over the chip at a concentration of 20 ␮M

RESULTS

CAPS interacts with a subset of exocytic syntaxin isoforms by binding

DISCUSSION