Abstract
The synaptic vesicle protein synaptotagmin has been implicated in the docking and subsequent calcium-regulated exocytosis of synaptic vesicles. We demonstrate that synaptotagmin is a major constituent of synaptic vesicle membranes, comprising 7-8% of the total vesicle protein. A proteolytic fragment of synaptotagmin, containing two repeats homologous to the C2-domain of protein kinase C, bound to a variety of natural membranes in a calcium-dependent manner (EC50 approximately 30 microM calcium). Binding was insensitive to proteolysis of the acceptor membranes suggesting an interaction with the lipid constituents. This interaction was confirmed using a recombinant fusion protein, containing both C2-like domains of synaptotagmin, that bound to artificial liposomes in a calcium-dependent manner. Phospholipid binding properties were preserved in a 114-amino acid domain corresponding to the first C2-like repeat of the protein and represents the shortest functional cassette yet reported. Furthermore, deletion of a highly conserved 9-amino acid motif, within this region, was sufficient to abolish the calcium-dependent phospholipid binding properties of this domain. This mutation may provide a means to selectively disrupt individual C2-domains in order to assess their relative contributions to function.
Highlights
From the Howard Hughes Medical Institute and Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Hauen, Connecticut 06510
We demonstrate core vesicles occurs in PC12 cell lines lacking synaptotagmin that synaptotagmin is a major constituent of synaptic (Shoji-Kasai et al, 1992)
- tein kinase C, bound to a varietyof natural membranes fective synaptic transmission. These studies indicate that synin a calcium-dependent manne(rECso 30 p~calcium). aptotagmin is not an absolute requirement for exocytosis to Binding was insensitive to proteolysis of the acceptor occur but suggest that the protein plays a crucial modulatory membranessuggestingan interaction with the lipid con- role in regulated secretion. This role has been examined by stituents.This interaction was confirmuedsing a recom- Littleton et al (1993) who reported that Drosophila larva, with binant fusion protein, containing both C2-like domains partial lack-of-function synaptotagmin mutationse, xhibited an of synaptotagmin, that bountdo artificialliposomesin a increase in miniature excitatory potentials and a decrease in calcium-dependent manner
Summary
Phate-buffered saline and the immunoreactive bands visualized with 0.5 mg/ml 4-chloro-1-napthol and 0.05% HzOz in phosphate-buffered. Digest and the vesicle membranes were removed by centrifugation as primer used to prepare C2A were used These PCR products were gel described above. The full-length deletion mutant was thengenwas mixed with other membrane preparations (described below) and erated by carrying out PCR between the annealedregion and the5' and assayed for binding, again, by co-sedimentation. In these experiments, 3' end primers used to prepare C2A. The high degree of calcium independent binding of liposomes comprised of PE and P I P C is likely due t o nonspecific ionic and hydrophobic interactions with theimmobilized synaptotagmin fusion protein To eliminate these variables we measured the calciumdependent component of the binding directly by elution with EGTA. Densitometry-Densitometry was carried out using a Visage 2000 scanner (Bio Image Products, MilligenEIioresearch Division of Millipore)
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