Abstract
Synaptophysin is the major integral membrane protein of small synaptic vesicles. Its primary structure deduced from rat and human complementary DNA sequences predicts that synaptophysin contains four transmembrane regions and a carboxyl-terminal domain having a novel repetitive structure. To elucidate the transmembrane organization of this protein in the synaptic vesicle, five antipeptide antibodies were raised. The site-specific antibodies were used to map the cognate sequences to the cytoplasmic or intravesicular side of the synaptic vesicle membrane by determining the susceptibility of the epitopes to proteolysis. The results confirm a topographic model for synaptophysin in which the protein spans the vesicle membrane four times, with both the amino and carboxyl terminus being cytoplasmic. In addition, the evolutionary conservation of the synaptophysin domains was addressed as a function of their membrane localization. To this end the primary structure of bovine synaptophysin was determined. Sequence comparisons between bovine, rat, and human synaptophysin revealed that only the intravesicular loops showed a significant number of amino acid substitutions (22%), while the transmembrane regions and cytoplasmic sequences were highly conserved (3% substitutions). These results depict synaptophysin as a protein with multiple membrane spanning regions whose functional site is likely to reside in highly conserved intramembranous and cytoplasmic sequences.
Highlights
Synaptophysin is the major integral membrane protein of small synaptic vesicles
Synaptophysin is a comparatively abundant synaptic vesicle membrane protein of unknown function. In neurons it is found on all small synaptic vesicles independent of neurotransmitter content ajusdged byimmunocytochemistry (Wiedemann and Franke, 1985;Navone et al, 1986).Based on the deduced structure of the protein an unusual transmembrane model with possible functional implications has been proposed for synaptophysin (Sudhof et al, 1987 a and b)
The present study reports the production of several sitespecific peptide antibodies against synaptophysin, which is the major intrinsic synapticvesicle membrane protein
Summary
Synuptophysin-Protein A Fusion Protein-A DNA fragment from the ratsynaptophysin cDNA (nucleotides 740-1021) which codesfor the carboxyl-terminal 73 amino acids was cloned into the filled-in SalI site of pRIT2T (Pharmacia LKB Biotechnology Inc.). The fusion protein was constitutively expressed in Escherichia coli HBlOl cells and purified by IgG-Sepharose chromatography according to the manufacturer's suggestions. CDNA Cloning and Sequencing-A bovine brain cDNA library (kind gift of Dr Paul Greengard, Rockefeller University) was screened with uniformly 32P-labeledprobes as described (Sudhof et d,1985). Three hybridization positive clones (frequency one per 50,000)were isolated and found to be identical by restriction mapping and sequencing. One clone was sequenced entirely using the dideoxy chain termination method and single-stranded M13 subclones of the cDNA as described (Sanger et al, 1977; Sudhof et d,1987a).
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