rbSec1A and B colocalize with syntaxin 1 and SNAP-25 throughout the axon, but are not in a stable complex with syntaxin.
rbSec1 is a mammalian neuronal protein homologous to the yeast SEC1 gene product which is required for exocytosis. Mutations in Sec1 homologues in the nervous systems of C. elegans and D. melanogaster lead to defective neurotransmitter secretion. Biochemical studies have shown that recombinant rbSec1 binds syntaxin 1 but not SNAP-25 or synaptobrevin/VAMP, the two proteins which together with syntaxin 1 form the synaptic SNARE complex. In this study we have examined the subcellular localization of rbSec1 and the degree of interaction between rbSec1 and syntaxin 1 in situ. rbSec1, which we show here to be represented by two alternatively spliced isoforms, rbSec1A and B, has a widespread distribution in the axon and is not restricted to the nerve terminal. This distribution parallels the localization of syntaxin 1 and SNAP-25 along the entire axonal plasmalemma. rbSec1 is found in a soluble and a membrane-associated form. Although a pool of rbSec1 is present on the plasmalemma, the majority of membrane-bound rbSec1 is not associated with syntaxin 1. We also show that rbSec1 is not part of the synaptic SNARE complex or of the syntaxin 1/SNAP-25 complex we show to be present in non-synaptic regions of the axon. Thus, in spite of biochemical studies demonstrating the high affinity interaction of rbSec1 and syntaxin 1, our results indicate that rbSec1 and syntaxin 1 are not stably associated. They also suggest that the function of rbSec1, syntaxin 1, and SNAP-25 is not restricted to synaptic vesicle exocytosis at the synapse.
Garcia
E P
EP
Department of Cell Biology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
McPherson
P S
PS
Chilcote
T J
TJ
Takei
K
K
De Camilli
P
P
D017398:Alternative Splicing; D000818:Animals; D000954:Antigens, Surface / Q000032:analysis* / Q000096:biosynthesis; D001369:Axons / Q000378:metabolism / Q000648:ultrastructure*; D001483:Base Sequence; D001921:Brain / Q000166:cytology* / Q000378:metabolism; D001923:Brain Chemistry; D017931:DNA Primers; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000032:analysis* / Q000096:biosynthesis*; D046911:Macromolecular Substances; D008297:Male; D008565:Membrane Proteins / Q000032:analysis* / Q000096:biosynthesis; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000096:biosynthesis; D009474:Neurons / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D016716:PC12 Cells; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis; D012689:Sequence Homology, Nucleic Acid; D013569:Synapses / Q000378:metabolism / Q000648:ultrastructure; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D017931:DNA Primers; D005656:Fungal Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C494210:Snap25 protein, rat; C494213:Stx1a protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins