Synexin facilitates fusion of specific phospholipid membranes at divalent cation concentrations found intracellularly.

Hong, Keelung; Düzgüneş, Nejat; Ekerdt, Roland; Papahadjopoulos, Demetrios

doi:10.1073/pnas.79.15.4642

Cited by 65 publications

(23 citation statements)

References 19 publications

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“…Isolation and purification of chromaffin granules were performed as described by Brocklehurst and Pollard (61). Large unilamellar PS liposomes ("LUV") were prepared by the swelling method (62) and fusion analyzed (55,56 (1996) tated synexin was then assayed for bound nucleotides, as described (68).…”

Section: Methodsmentioning

confidence: 99%

Membrane fusion protein synexin (annexin VII) as a Ca2+/GTP sensor in exocytotic secretion.

Caohuy

Srivastava

Pollard

1996

Proc. Natl. Acad. Sci. U.S.A.

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Exocytotic membrane fusion and secretion are promoted by the concerted action of GTP and Ca2 , although the precise site(s) of action in the process are not presently known. However, the calcium-dependent membrane fusion reaction driven by synexin (annexin VII) is an in vitro model for this process, which we have now found to be further activated by GTP. The mechanism of fusion activation depends on the unique ability of synexin to bind and hydrolyze GTP in a calcium-dependent manner, both in vitro and in vivo in streptolysin 0-permeabilized chromaffin cells. The required [Ca2+] for GTP binding by synexin is in the range of 50-200 ,AM, which is known to occur at exocytotic sites in chromaffin cells, neurons, and other cell types. Previous immunolocalization studies place synexin at exocytotic sites in chromaffin cells, and we conclude that synexin is an atypical G protein that may be responsible for both detecting and mediating the Ca2+/GTP signal for exocytotic membrane fusion.GTP and its nonhydrolyzable analogue guanosine 5'- [y-thio]triphosphate (GTP[,yS]) are known to promote Ca2+-dependent exocytotic secretion from many cell types by a mechanism thought to involve as yet unknown proteins in the GTPase superfamily (1-4). Specific members of this superfamily have been considered as mediators of these GTP effects on exocytosis, including heterotrimeric G proteins (5-9) and low molecular weight ras-like proteins such as Rab (10-15) and ARF (16). The current "fusion machine" hypothesis (17-19) envisions a core complex formed between plasma membrane syntaxin and SNAP-25 and the synaptic vesicle protein synaptobrevin/VAMP (20), with vesicular synaptotagmin putatively identified as a low-affinity calcium sensor that interacts with regulatory syntaxin 1 (21,22). However, none of the proteins presently identified in the hypothetical fusion machine have actually been shown to be activated by GTP or, indeed, even to fuse membranes (17,(23)(24)(25). Therefore, it has been suggested that other GTP-binding proteins, as yet unidentified, might control the activity of the fusion complex (19,25).The site of GTP action in exocytosis has been hypothesized to be closely associated with the site of calcium action in a common pathway (2,5,26,27). The most recent consensus on the nature of this calcium binding site is that it is involved in docking and fusion and that the affinity of the site for Ca2+ may be in the range of 50-200 ,tM (28-36

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Section: Methodsmentioning

confidence: 99%

Membrane fusion protein synexin (annexin VII) as a Ca2+/GTP sensor in exocytotic secretion.

Caohuy

Srivastava

Pollard

1996

Proc. Natl. Acad. Sci. U.S.A.

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“…Several years ago Papahadjopoulos and colleagues [13][14][15][16] showed that synexin could fuse liposomes prepared from phosphatidylethanolamine (PE) and phosphatidylserine (PS). However, synexin could only bind to and aggregate phosphatidylinositol (PI) liposomes.…”

Section: Introductionmentioning

confidence: 99%

Synexin (annexin VII): A cytosolic calcium-binding protein which promotes membrane fusion and forms calcium channels in artificial bilayer and natural membranes

1990

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“…One fruitful approach to this problem has been to study proteins such as synexin, which fuse certain natural and artificial membranes in a calciumdependent manner (2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Recently, bovine synexin has been shown to generate classical capacitative gating currents in isolated acidic phospholipid bilayers (12) and to exhibit exquisitely selective, voltage-sensitive calcium channel activity in similar membranes (13).…”

mentioning

confidence: 99%

Calcium channel activity of purified human synexin and structure of the human synexin gene.

Burns

Magendzo

Shirvan

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

135

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Synexin is a calcium-dependent membrane binding protein that not only fuses membranes but also acts as a voltage-dependent calcium channel. We have isolated and sequenced a set ofoverlapping cDNA clones for human synexin. The derived amino acid sequence of synexin reveals strong homology in the C-terminal domain with a previously identified class of calcium-dependent membrane binding proteins. (18,19). cDNA inserts of the three positive recombinants or restriction fragments of the largest cDNA (L4a) were subcloned into M13 for DNA sequencing with M13-or synexinspecific primers (20). Specific oligonucleotide or cDNA probes were used to screen additional cDNA libraries: retina from Jeremy Nathans (Genentech) (Fig. 2); B-cell, adrenal, and lung from Clontech; and fibroblast from H. Okayama (National Institutes of Health) (data not shown) (20).Single Channel Current Measurements. Calcium channel activity of human synexin was measured by methods previously described (refs. 13, 21; Fig. 1 B and C). Briefly, bilayers of phosphatidylserine (PtdSer) or phosphatidylinositol (PtdIns) (Avanti Polar Lipids) were prepared at the tips of patch pipets by a double dip method. Upon forming the bilayer the open tip resistance rose from 15-20 MQ to 3-5 GQl, and channel activity was acquired only after addition of synexin to the bath. The compositions of the various solutions are stated either in the legend to Fig. 1 or in the text.

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Synexin facilitates fusion of specific phospholipid membranes at divalent cation concentrations found intracellularly.

Cited by 65 publications

References 19 publications

Membrane fusion protein synexin (annexin VII) as a Ca2+/GTP sensor in exocytotic secretion.

Membrane fusion protein synexin (annexin VII) as a Ca2+/GTP sensor in exocytotic secretion.

Synexin (annexin VII): A cytosolic calcium-binding protein which promotes membrane fusion and forms calcium channels in artificial bilayer and natural membranes

Calcium channel activity of purified human synexin and structure of the human synexin gene.

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