ABSTRACT
Intracellular membrane fusion is mediated by the formation of a four-helix bundle comprised of SNARE proteins. Every cell expresses a large number of SNARE proteins that are localized to particular membrane compartments, suggesting that the fidelity of vesicle trafficking might in part be determined by specific SNARE pairing. However, the promiscuity of SNARE pairing in vitro suggests that the information for membrane compartment organization is not encoded in the inherent ability of SNAREs to form complexes. Here, we show that exocytosis of norepinephrine from PC12 cells is only inhibited or rescued by specific SNAREs. The data suggest that SNARE pairing does underlie vesicle trafficking fidelity, and that specific SNARE interactions with other proteins may facilitate the correct pairing.
Subject(s)
Membrane Fusion/physiology , Membrane Proteins/physiology , Vesicular Transport Proteins , Amino Acid Sequence/genetics , Animals , Botulinum Toxins/pharmacology , Exocytosis/drug effects , Membrane Fusion/drug effects , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/pharmacology , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/physiology , Norepinephrine/antagonists & inhibitors , Norepinephrine/metabolism , PC12 Cells/metabolism , Peptide Fragments/pharmacology , Qa-SNARE Proteins , R-SNARE Proteins , Rats , SNARE Proteins , Solubility , Substrate Specificity , Synaptosomal-Associated Protein 25ABSTRACT
Neurotransmitter exocytosis, a process mediated by a core complex of syntaxin, SNAP-25, and VAMP (SNAREs), is inhibited by SNARE-cleaving neurotoxins. Botulinum neurotoxin E inhibition of norepinephrine release in permeabilized PC12 cells can be rescued by adding a 65 aa C-terminal fragment of SNAP-25 (S25-C). Mutations along the hydrophobic face of the S25-C helix result in SNARE complexes with different thermostabilities, and these mutants rescue exocytosis to different extents. Rescue depends on the continued presence of both S25-C and Ca2+ and correlates with complex formation. The data suggest that Ca2+ triggers S25-C binding to a low-affinity site, initiating trans-complex formation. Pairing of SNARE proteins on apposing membranes leads to bilayer fusion and results in a high-affinity cis-SNARE complex.
Subject(s)
Calcium/metabolism , Membrane Fusion/physiology , Membrane Proteins/metabolism , Vesicular Transport Proteins , Amino Acid Sequence , Animals , Base Sequence , Botulinum Toxins/toxicity , DNA Primers/genetics , Exocytosis/drug effects , Exocytosis/physiology , Macromolecular Substances , Mice , Molecular Sequence Data , Mutation , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , PC12 Cells , Rats , SNARE Proteins , Synaptosomal-Associated Protein 25ABSTRACT
Soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins of the vesicle-associated membrane protein (VAMP) and syntaxin families play a central role in vesicular trafficking through the formation of complexes between proteins present on vesicle and target membranes. Formation of these complexes is proposed to mediate aspects of the specificity of vesicle trafficking and to promote fusion of the lipid bilayers. In order to further understand the molecular mechanisms that organize membrane compartments, we have characterized seven new mammalian proteins of the VAMP and syntaxin families. The proteins are broadly expressed; however, syntaxin 13 is enriched in brain and VAMP 8 in kidney. The seven novel SNAREs localize in distinct patterns overlapping with Golgi, endosomal, or lysosomal markers. Our studies support the hypothesis that evolutionary radiation of these two gene families gave rise to sets of proteins whose differential expression and combinatorial associations define and organize the membrane compartments of cells.
