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Intracellular membrane traffic is governed by a conserved set of proteins, including Syts (synaptotagmins). The mammalian Syt family includes 15 isoforms. Syts are membrane proteins that possess tandem C2 domains (C2AB) implicated in calcium-dependent phospholipid binding. We performed a pair-wise amino acid sequence comparison, together with functional studies of rat Syt C2ABs, to examine common and divergent properties within the mammalian family. Sequence analysis indicates three different C2AB classes, the members of which share a high degree of sequence similarity. All the other C2ABs are highly divergent in sequence. Nearly half of the Syt family does not exhibit calcium/phospholipid binding in comparison to Syt I, the major brain isoform. Syts do, however, possess a more conserved function, namely calcium-independent binding to target SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) heterodimers. All tested isoforms, except Syt XII and Syt XIII, bound the target SNARE heterodimer comprising syntaxin 1 and SNAP-25 (25 kDa synaptosome-associated protein). Our present study suggests that many Syt isoforms can function in membrane trafficking to interact with the target SNARE heterodimer on the pathway to calcium-triggered membrane fusion.

Synaptotagmin is a synaptic vesicle protein that has been proposed to be the calcium sensor responsible for fast neurotransmitter release at synapses. Synaptotagmin's two C2 domains, C2A and C2B, each provide a calcium binding pocket lined with negative charges contributed by five conserved aspartates. We find that even when all of C2A's conserved aspartates are neutralized by replacement with asparagines, neurotransmitter release still occurs at hippocampal synapses in culture. Because exocytosis continues to be dependent on extracellular calcium concentration, the C2A domain cannot represent the entire calcium sensor. C2A does appear to be part of the calcium sensor, however, because substitution of D232 alters the calcium dependence of release, perhaps by reducing the number of calcium ions that must bind to trigger exocytosis. We conclude that neutralization of the negative charge at D232 by coordination of a calcium ion is necessary--but not sufficient--for fast neurotransmission at mammalian CNS synapses.

Synaptotagmin I is the leading candidate for the calcium sensor that triggers exocytosis at conventional synapses. However, physiological characterization of the calcium sensor for phasic release at the ribbon-style synapses of the goldfish Mb1 bipolar cell demonstrates a lower than predicted affinity for calcium, suggesting that a modified or different sensor triggers exocytosis at this synapse. We examined synaptotagmin immunolabeling in goldfish retina using two different antibodies directed against synaptotagmin epitopes that specifically labeled the expected 65-kDa protein on western blots of goldfish and mouse retinal membranes. The first antiserum strongly labeled conventional synapses in the inner plexiform layer (IPL), but did not label the ribbon-style synapse-containing synaptic terminals of goldfish Mb1 bipolar cells or photoreceptors. The second antibody also specifically labeled the expected 65-kDa protein on western blots but did not label any synapses in the goldfish retina. A third synaptotagmin antibody that performed poorly on western blots selectively labeled goldfish photoreceptor terminals. These results suggest that synaptotagmin may exist in at least three distinct "forms" in goldfish retinal synapses. These forms, which are differentially localized to conventional synapses, bipolar cell, and photoreceptor terminals, may represent differences in isoform, posttranslational modifications, epitope availability, and protein-binding partners. Labeling with these antibodies in the salamander and mouse retina revealed species-specific differences, indicating that synaptotagmin epitopes can vary across species as well as among synapses.

Synaptotagmins represent a family of putative vesicular trafficking proteins. With synaptotagmin 13, we have now identified a novel synaptotagmin, making this one of the largest families of trafficking proteins. Similar to synaptotagmins 3, 4, 6, 7, 9, and 11, synaptotagmin 13 is expressed at highest levels in brain but is also detectable at lower levels in non-neuronal tissues. Synaptotagmin 13 is composed of the canonical domains of synaptotagmins that include an N-terminal transmembrane region and two C-terminal cytoplasmic C2-domains (C2A- and C2B-domain) and a connecting sequence between the transmembrane region and the C2-domains. Different from most other synaptotagmins, however, synaptotagmin 13 does not have an N-terminal sequence preceding the transmembrane region, and features an unusually long connecting sequence that is proline-rich. Furthermore, the C2-domains of synaptotagmin are degenerate and lack almost all of the residues involved in Ca2+ binding, suggesting that synaptotagmin 13 is not a Ca2+-binding protein unlike most other synaptotagmins. Our data demonstrate that synaptotagmins represent a larger and more complex gene family than previously envisioned.

The synaptotagmins now constitute a large family of membrane proteins characterized by one transmembrane region and two C2 domains. Dimerization of synaptotagmin (Syt) I, a putative low affinity Ca(2+) sensor for neurotransmitter release, is thought to be important for expression of function during exocytosis of synaptic vesicles. However, little is known about the self-dimerization properties of other isoforms. In this study, we demonstrate that a subclass of synaptotagmins (III, V, VI, and X) (Ibata, K., Fukuda, M., and Mikoshiba, K. (1998) J. Biol. Chem. 273, 12267-12273) forms beta-mercaptoethanol-sensitive homodimers and identify three evolutionarily conserved cysteine residues at the N terminus (N-terminal cysteine motif, at amino acids 10, 21, and 33 of mouse Syt III) that are not conserved in other isoforms. Site-directed mutagenesis of these cysteine residues and co-immunoprecipitation experiments clearly indicate that the first cysteine residue is essential for the stable homodimer formation of Syt III, V, or VI, and heterodimer formation between Syts III, V, VI, and X. We also show that native Syt III from mouse brain forms a beta-mercaptoethanol-sensitive homodimer. Our results suggest that the cysteine-based heterodimerization between Syt III and Syt V, VI, or X, which have different biochemical properties, may modulate the proposed function of Syt III as a putative high affinity Ca(2+) sensor for neurotransmitter release.

NSP1 is a nuclear pore protein (nucleoporin) essential for cell growth. To identify the components that functionally interact with NSP1 in the living cell, we developed a genetic screen for mutants that are lethal in a genetic background of mutated, but not wild type NSP1. Fourteen synthetic lethal mutants were obtained, belonging to at least four different complementation groups. The genes of two complementation groups, NSP116 and NSP49, were cloned. Like the previously described nucleoporins, these genes encode proteins with many repeat sequences. NSP116 and NSP49, however, contain a new repetitive sequence motif 'GLFG', which classifies them as a subclass of nucleoporins. NSP116 and NSP49, tagged with the IgG binding domain of protein A and expressed in yeast, are located at the nuclear envelope. These data provide in vivo evidence that distinct subclasses of nucleoporins physically interact or share overlapping function in nuclear pore complexes.