SNIP, a Novel SNAP-25-interacting Protein Implicated in Regulated Exocytosis

doi:10.1074/jbc.275.2.1191

SNIP, a Novel SNAP-25-interacting Protein Implicated in Regulated Exocytosis*

From the Departments of Pharmacology and Physiology, Bowles Center for Alcohol Studies, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599

Abstract

Synaptosome-associated protein of 25 kDa (SNAP-25) is a presynaptic membrane protein that has been clearly implicated in membrane fusion in both developing and mature neurons, although its mechanisms of action are unclear. We have now identified a novel SNAP-25-interacting protein named SNIP. SNIP is a hydrophilic, 145-kDa protein that comprises two predicted coiled-coil domains, two highly charged regions, and two proline-rich domains with multiple PPXY and PXXP motifs. SNIP is selectively expressed in brain where it co-distributes with SNAP-25 in most brain regions. Biochemical studies have revealed that SNIP is tightly associated with the brain cytoskeleton. Subcellular fractionation and immunofluorescence localization studies have demonstrated that SNIP co-localizes with SNAP-25 as well as the cortical actin cytoskeleton, suggesting that SNIP serves as a linker protein connecting SNAP-25 to the submembranous cytoskeleton. By using deletion analysis, we have mapped the binding domains of SNIP and SNAP-25, and we have demonstrated that the SNIP-SNAP-25 association is mediated via coiled-coil interactions. Moreover, we have shown that overexpression of SNIP or its SNAP-25-interacting domain inhibits Ca²⁺-dependent exocytosis from PC12 cells. These results indicate that SNIP is involved in regulation of neurosecretion, perhaps via its interaction with SNAP-25 and the cytoskeleton.

Footnotes

↵* This work was supported by National Institutes of Health Grant NS37939, University of North Carolina Junior Faculty Development award, and grants from the University of North Carolina Research Council and the Foundation of Hope (to L. L.).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) and .
↵‡ To whom correspondence should be addressed: Dept. of Pharmacology, Rm. 1025A Thurston-Bowles, University of North Carolina, Chapel Hill, NC 27599-7178. Tel.: 919-966-0503; Fax: 919-966-5679; E-mail: LianLi@med.unc.edu.
Abbreviations:

SNAP-25

synaptosome-associated protein of 25 kDa

SNIP

SNAP-25-interacting protein

SNAP

soluble N-ethylmaleimide-sensitive fusion attachment protein

SNARE

SNAP receptor

GST

glutathioneS-transferase

GH

growth hormone

HA

hemagglutinin

NGF

nerve growth factor

PAGE

polyacrylamide gel electrophoresis

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- Received June 10, 1999.
- Revision received October 22, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.