Synaptotagmin Is an Inositol Polyphosphate Binding Protein: Isolation and Characterization as an Ins 1,3,4,5-P4 Binding Protein

Niinobe, Michio; Yamaguchi, Yoshihide; Fukuda, Mitsunori; Mikoshiba, Katsuhiko

doi:10.1006/bbrc.1994.2770

Cited by 61 publications

(48 citation statements)

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“…The dissociation constants (K d ) were within the nanomolar range and are therefore similar to those for other Ca 2q -dependent membrane binding proteins such as annexins (Tait et al, 1989) and typical C2 domain-containing proteins, such as PKC (Bazzi and Nelsestuen, 1987), synaptotagmin (Niinobe et al, 1994) and phospholipase A2 (Kim et al, 1997). This observation underlines the role of phospholipids as potential binding partners of activated calpain at the cellular membranes, a function once attributed exclusively to membrane-associated proteins (Inomata et al, 1990).…”

Section: Interaction Of Calpain With Membrane Lipids Is Nonselectivesupporting

confidence: 75%

μ-Calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation

Fernández-Montalván

Assfalg-Machleidt²,

Pfeiler³

et al. 2006

Biological Chemistry

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active-site mutated heterodimeric human m-calpain with phospholipid bilayers was studied in vitro using proteinto-lipid fluorescence resonance energy transfer and surface plasmon resonance. Binding to liposomes was Ca 2q -dependent, but not selective for specific phospholipid head groups. wCa 2q x 0.5 for association with lipid bilayers was not lower than that required for the exposure of hydrophobic surface (detected by TNS fluorescence) or for enzyme activity in the absence of lipids. Deletion of domain V reduced the lipid affinity of the isolated small subunit (600-fold) and of the heterodimer (10-to 15-fold), thus confirming the proposed role of domain V for membrane binding. Unexpectedly, mutations in the acidic loop of the 'C2-like' domain III, a putative Ca 2q and phospholipid-binding site, did not affect lipid affinity. Taken together, these results support the hypothesis that in vitro membrane binding of m-calpain is due to the exposed hydrophobic surface of the active conformation and does not reduce the Ca 2q requirement for activation.

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Section: Interaction Of Calpain With Membrane Lipids Is Nonselectivesupporting

confidence: 75%

μ-Calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation

Fernández-Montalván

Assfalg-Machleidt²,

Pfeiler³

et al. 2006

Biological Chemistry

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“…The Binding of the C 2 Domains of RIM to Syt-I Is Sensitive to Inositol Polyphosphates-Synaptotagmins are known to bind inositol polyphosphates (15). The binding site has been shown to overlap the domain required for self-oligomerization of synaptotagmins (46).…”

Section: Rim Binds To Ca 2ϩ Channels Snap-25 and Synaptotagminmentioning

confidence: 99%

“…Although most of these interactions are influenced by both C 2 domains, Ca 2ϩ -dependent binding to syntaxin-1 and anionic phospholipids is predominantly associated with C 2 A (11-13). In contrast, Ca 2ϩ -dependent oligomerization of synaptotagmins and several Ca 2ϩ -independent interactions such as the binding to the II-III cytoplasmic loop or "synprint" (synaptic protein interaction) motif of the pore-forming subunit of Ca 2ϩ channels (14) to inositol polyphosphates (15) and to SNAP-25 (16) are mediated predominantly by C 2 B.…”

mentioning

confidence: 99%

Direct Interaction of the Rab3 Effector RIM with Ca2+Channels, SNAP-25, and Synaptotagmin

Coppola¹,

Magnin-Lüthi²,

Perret-Menoud³

et al. 2001

Journal of Biological Chemistry

203

191

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To define the role of the Rab3-interacting molecule RIM in exocytosis we searched for additional binding partners of the protein. We found that the two C 2 domains of RIM display properties analogous to those of the C 2 B domain of synaptotagmin-I. Thus, RIM-C 2 A and RIM-C 2 B bind in a Ca 2؉ -independent manner to ␣1B, the pore-forming subunit of N-type Ca 2؉ channels (EC 50 ‫؍‬ ϳ20 nM). They also weakly interact with the ␣1C but not the ␣1D subunit of L-type Ca 2؉ channels. In addition, the C 2 domains of RIM associate with SNAP-25 and synaptotagmin-I. The binding affinities for these two proteins are 203 and 24 nM, respectively, for RIM-C 2 A and 224 and 16 nM for RIM-C 2 B. The interactions of the C 2 domains of RIM with SNAP-25 and synaptotagmin-I are modulated by Ca 2؉ . Thus, in the presence of Ca 2؉ (EC 50 ‫؍‬ ϳ75 M) the interaction with synaptotagmin-I is increased, whereas SNAP-25 binding is reduced. Synaptotagmin-I binding is abolished by mutations in two positively charged amino acids in the C 2 domains of RIM and by the addition of inositol polyphosphates. We propose that the Rab3 effector RIM is a scaffold protein that participates through its multiple binding partners in the docking and fusion of secretory vesicles at the release sites.Secretion of neurotransmitters, polypeptide hormones, and a variety of other proteins occurs by exocytosis, a multistage process including targeting, docking, and, finally, fusion of secretory vesicles with the plasma membrane. During the last few years, a combination of genetic and biochemical approaches has lead to the identification of several proteins involved in this complex cascade of events. Most of these proteins turned out to be specialized components of the evolutionary conserved machinery that governs intracellular vesicular trafficking in eukaryotic cells (1). Exocytosis was found to necessitate the assembly of a ternary complex between the vesicular SNARE 1 VAMP, associated with the secretory vesicle, and the target SNAREs syntaxin-1 and SNAP-25, localized at the plasma membrane (2). The SNARE complex was initially proposed to ensure the docking of secretory vesicles at the plasma membrane (3). However, it is unlikely that SNARE assembly constitutes the sole determinant for the targeting of secretory vesicles at the release sites because SNARE pairing is rather promiscuous (4, 5), and the localization of syntaxin-1 and SNAP-25 is not restricted to active zones (6). A current hypothesis, supported by biochemical and structural data, proposes that the assembly of the heterotrimeric complex between VAMP, SNAP-25, and syntaxin-1 provides the driving force for membrane fusion (7).In most secretory systems, the exocytotic process is initiated by an increase in the intracellular Ca 2ϩ concentration. In some cells, such as neurons, the elevation of Ca 2ϩ ions is due to opening of voltage-gated calcium channels that are clustered at the release sites, whereas in others, Ca 2ϩ ions are mobilized from intracellular stores. Biochemical and genetic studies indicate...

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“…The pleckstrin homology domain of dynamin has been found to bind to Ins(1,4,5)P 3 and PtdIns(4,5)P 2 (18). Synaptotagmin, a synaptic vesicle membrane protein, has been found to bind to inositol polyphosphates and inositol lipids (19,20), and antibodies that block this binding have been found to inhibit transmission at the squid giant synapse (21). Synaptojanin (p145/dephosphin) is a nerve terminal enriched inositide 5-phosphatase, which inter-* This work was supported by National Institutes of Health NINDS Grant NS29051 (to E. M. L.), NIGMS Grant GM31278 (to J. R. F.), and NINDS Grant NS29632 (to G. D. P.), and by NEN Life Sciences.…”

Section: D-myo-[mentioning

confidence: 99%

Regulation of AP-3 Function by Inositides

Hao

Tan

Prasad

et al. 1997

Journal of Biological Chemistry

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As part of the growing effort to understand the role inositol phosphates and inositol lipids play in the regulation of vesicle traffic within nerve terminals, we determined whether or not the synapse-specific clathrin assembly protein AP-3 can interact with inositol lipids. We found that soluble dioctanoyl-phosphatidylinositol 3,4,5-trisphosphate (DiC 8 PtdIns(3,4,5)P 3 ) was only 7.5-fold weaker a ligand than D-myo-inositol hexakisphosphate in assays that measured the displacement of D-myo-[3 H]inositol hexakisphosphate. In functional assays we found that both of these ligands inhibited clathrin assembly, but DiC 8 -PtdIns(3,4,5)P 3 was more potent and exhibited a larger maximal effect. We also examined the structural features of DiC 8 -PtdIns(3,4,5)P 3 that establish specificity. Dioctanoyl-phosphatidylinositol 3,4-bisphosphate, which does not have a 5-phosphate, and 4,5-O-bisphosphoryl-D-myo-inosityl 1-O-(1,2-O-diundecyl)-sn-3-glycerylphosphate, which does not have a 3-phosphate, were, respectively, 2-fold and 4-fold less potent than DiC 8 -PtdIns(3,4,5)P 3 as inhibitors of clathrin assembly. Deacylation of DiC 8 -PtdIns(3,4,5)P 3 reduced its affinity for AP-3 almost 20-fold, and also dramatically lowered its ability to inhibit clathrin assembly. The deacylated products of the soluble derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 4,5-bisphosphate were both not significant inhibitors of clathrin assembly. It therefore appears that the interactions of inositides with AP-3 should not be considered simply in terms of electrostatic effects of the highly charged phosphate groups. Ligand specificity appears also to be mediated by hydrophobic interactions with the fatty-acyl chains of the inositol lipids.There is increasing evidence that certain inositides (members of a large family of inositol phosphates and inositol lipids) act at several levels to regulate both the recruitment and the activation of proteins involved in vesicular transport. PtdIns(3,4,5)P 3 , 1 and the polyphosphoinositide 3-kinase that synthesizes this lipid both feature prominently in this area (1, 2). For example, intracellular trafficking of tyrosine kinase receptors depends upon interactions between the internalized receptors and the 3-kinase (3). Additionally, PtdIns(3,4,5)P 3 interacts either directly or indirectly with several GTPases that play important roles in vesicle traffic (1). Finally, in yeast, the Vps34 protein that is essential for Golgi to vacuole transport is a phosphoinositide 3-kinase (4).Interest in this field is not restricted to the inositol lipids. Other studies into the roles of the inositides in protein traffic have ascertained that certain inositol polyphosphates can bind with high affinity to the clathrin assembly proteins, AP-2 and AP-3 (5-9). AP-2 is believed to play a general role in endocytosis, since it has been found on vesicles budding from the plasma membranes of all types of cells (10). AP-3 is believed to play a more specialized role in synaptic vesicle biogenesis and recycling...

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Synaptotagmin Is an Inositol Polyphosphate Binding Protein: Isolation and Characterization as an Ins 1,3,4,5-P4 Binding Protein

Cited by 61 publications

References 0 publications

μ-Calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation

μ-Calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation

Direct Interaction of the Rab3 Effector RIM with Ca2+Channels, SNAP-25, and Synaptotagmin

Regulation of AP-3 Function by Inositides

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