Abstract
Synaptotagmins I and II are essential for Ca2+-regulated exocytosis of synaptic vesicles from neurons, probably serving as Ca2+ sensors. This Ca2+-sensing function is thought to be disrupted by binding of an inositol 1,3,4,5-tetrakisphosphate (IP4) to the C2B domain of synaptotagmin I or II (Fukuda, M., Moreira, J. E., Lewis, F. M. T., Sugimori, M., Niinobe, M., Mikoshiba, K., and Llinás, R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10708-10712). Recently, several synaptotagmin isoforms, expressed outside the nervous system, have been identified in rats and proposed to be involved in constitutive vesicle traffic. To test whether the inositol high polyphosphates also regulate constitutive vesicle traffic by binding to the non-neuronal synaptotagmins, we examined the IP4 binding properties of the recombinant C2 domains of both neuronal (III, V, X, and XI) and non-neuronal (VI-VIII and IX) synaptotagmins. The C2B domains of synaptotagmins VII-IX and XI had strong IP4 binding activity, but the C2B domain of synaptotagmin VI showed very weak IP4 binding activity. In contrast, there was no significant IP4 binding activity of the C2B domains of synaptotagmins III, V, and X or any of the C2A domains. A phylogenetic tree of the C2 domains of 11 isoforms revealed that synaptotagmins III, V, VI, and X (IP4-insensitive or very weak IP4-binding isoforms) belong to the same branch. Based on the sequence comparison between the IP4-sensitive and -insensitive isoforms, we performed site-directed mutagenesis of synaptotagmin III and identified several amino acid substitutions that abolish IP4 binding activity. Our data suggest that the inositol high polyphosphates might also regulate constitutive vesicle traffic via binding to the IP4-sensitive non-neuronal synaptotagmins.
Highlights
Synaptotagmins I and II are essential for Ca2؉-regulated exocytosis of synaptic vesicles from neurons, probably serving as Ca2؉ sensors
We showed that neuronal synaptotagmins I, II, and IV, but not synaptotagmin III, are IP4- or inositol high polyphosphatebinding proteins [20, 22, 23]
To further examine whether other neuronal and non-neuronal isoforms of synaptotagmins are regulated by inositol high polyphosphates like synaptotagmin I, we prepared Glutathione S-transferase (GST) fusion proteins of C2 domains of synaptotagmins V–XI and tested for their IP4 binding activity (Fig. 1 and Table I)
Summary
Synaptotagmins I and II are essential for Ca2؉-regulated exocytosis of synaptic vesicles from neurons, probably serving as Ca2؉ sensors. Most of the proteins involved in Ca2ϩ-regulated exocytosis in neurons (e.g. synaptobrevin or syntaxin) have been reported to have homologues involved in constitutive membrane trafficking; and it has been suggested that the same protein family governs both constitutive and regulated vesicle traffic [11,12,13] Based on this idea, the ubiquitous isoforms of synaptotagmin are thought to be involved in constitutive vesicle trafficking because synaptotagmin I (the best characterized neuronal type) is essential for Ca2ϩ-regulated exocytosis in neurons and some endocrine cells (probably functioning as a Ca2ϩ sensor) (reviewed in Ref. 1). IP5 is suggested to function as a fusion clamp for exocytosis because IP5 is rapidly accumulated after depolarizing stimulation [17] These observations raised the possibility that inositol high polyphosphates may regulate other types of vesicle traffic (e.g. constitutive) via binding to ubiquitous members of the synaptotagmin family [24].
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