Selective Photoaffinity Labeling of the Inositol Polyphosphate Binding C2B Domains of Synaptotagmins

Abstract

. Although the CD spectrum of this 32-mer at two pH values showed a random coil, the photoaffinity analogue of IP 6 appeared to induce a binding-compatible structure in the short peptide.The synaptotagmins (Syts) 1 are synaptic vesicle proteins that play essential roles in nucleating the clathrin coat during endocytosis and in acting as Ca 2ϩ sensors (1-3) and phosphoinositide sensors (4) during exocytosis. They are a critical part of a complex machinery of intracellular protein transport (5, 6) and the synaptic vesicle cycle (7). In addition to a short Nterminal intravesicular region and single transmembrane domain, Syts have two copies of highly conserved repeats, known as the C2A and C2B domains, which are homologous to the C2 regulatory region of protein kinase C (8). In particular, the C2B domain appears to play several roles. First, the C2B domain of mouse Syt II shows specific binding to high polyphosphate inositols (IP n s) (9), a feature that is not shared by the highly homologous C2A domain nor with the C2 domains of other proteins such as rabphilin. Moreover, the C2B domain is necessary but not sufficient for IP n binding. Although Syt II and IV show high affinity binding of IP 4 and IP 6 , the Syt III-C2B domain shows negligible binding, despite a high sequence identity with the Syt II-C2B domain, including the 32-residue region examined by mutational analysis (10). Inhibition of C2B function in the squid giant axon disrupts synaptic vesicle release and recycling (11). Similarly, Caenorhabditis elegans mutants lacking Syt or simply the C2B domain cannot recycle synaptic vesicles (12), and the presence of Syt I in cerebrospinal fluid of Alzheimer's patients (13) may provide clues to the disruption of synaptic function in humans. The C2B domain acts as a high affinity receptor for clathrin assembly protein AP-2 (14), and Ca 2ϩ appears to mediate Syt dimerization via the C2B domain (15).Synaptotagmin II isolated from mouse cerebellum shows high affinity binding to Ins(1,3,4,5)P 4 with a K D of 30 M (16) and 117 nM for the GST-Syt II-C2B construct (9). Curiously, the rank order of IP n s binding for native Syt II was Ins-(1,3,4,5,6)-P 5 Ͼ (1,3,4,5)-P 4 Ͼ Ins(1,2,3,4,5,6)-P 6 Ͼ Ins(1,4,5)-P 3 , whereas for the GST-Syt II-C2B domain the order was IP 6 Ͼ IP 5 Ͼ IP 4 Ͼ IP 3 . Deletion mutants allowed mapping of the IP n binding site to the central region of the mouse Syt II-C2B domain, specifically residues 315-346 (IHLMQNGKRLKKKK-TTVKKKTLNPYFNESFSF) (9). In order to obtain direct evidence for the binding site of IP n s in the C2B domain, to examine the InsP n selectivity of the domains, to explore the Ca 2ϩ dependence of binding, to determine the role of the central 315-346 peptide, and to evaluate the reason for the failure of Syt III-C2B domain to bind IP n s, we undertook a series of photoaffinity labeling experiments using four tritium-labeled, benzophenone-containing derivatives of IP 3 , IP 4 , and IP 6 (17). EXPERIMENTAL PROCEDURES Chemicals-P-1-Tethered

Specific Interaction of Golgi Coatomer Protein α-COP with Phosphatidylinositol 3,4,5-Trisphosphate

“…PtdIns (3,4,5) (36). The advantages of benzophenone over the classical arylazide photochemistry include improved chemically stability of ligands and adducts, stability in ambient light, low background from nonspecific labeling, and the efficient C-H insertion of the triplet diradicaloid intermediate formed by irradiation at 360 nm (39).…”

“…Moreover, binding of coatomer complexes to phosphatidylinositol polyphosphates (PtdInsP n s) remains unreported. To address the InsP n and PtdInsP n binding specificities of individual COPI subunits, and to obtain evidence to support the roles of these high affinity interactions in vesicular trafficking, we employed a photoaffinity labeling approach with benzophenone-containing InsP n and PtdInsP n analogs (36). The benzophenone photophore allows handling in ambient light, activation at wavelengths Ͼ320 nm, and covalent labeling of active site residues in hydrophobic regions of proteins with high efficiency (37,38).…”

Regulation of AMP Deaminase by Phosphoinositides

“…A variety of inositides and phospholipids were tested for displacement of the photolabel. PtdIns(4,5)P 2 and PtdIns(4)P were the most potent inhibitors of photolabeling, with 50% displacement of the label (IC 50 ) effected by addition of 250 nM (Fig. 2, A and B).…”

Inositol 1,3,4,5-Tetrakisphosphate Binding Activities of Neuronal and Non-neuronal Synaptotagmins

“…Among them, synaptotagmins are apparently distinguished from other proteins in that they have a single transmembrane region and tandem C2 domains (C2A and C2B domains) with a short spacer. In our previous studies, we showed that neuronal synaptotagmins I, II, and IV, but not synaptotagmin III, are IP 4 -or inositol high polyphosphatebinding proteins (20,22,23). To further examine whether other neuronal and non-neuronal isoforms of synaptotagmins are also regulated by inositol high polyphosphates like synaptotagmin I, we prepared GST fusion proteins of C2 domains of synaptotagmins V-XI and tested for their IP 4 binding activity ( Fig.…”