Vesicle Membrane Order Controls Fusion by Determining Synaptobrevin's Conformation

The highly specific binding and uptake of BoNTs (botulinum neurotoxins; A–G) into peripheral cholinergic motoneurons turns them into the most poisonous substances known. Interaction with gangliosides accumulates the neurotoxins on the plasma membrane and binding to a synaptic vesicle membrane protein leads to neurotoxin endocytosis. SV2 (synaptic vesicle glycoprotein 2) mediates the uptake of BoNT/A and /E, whereas Syt (synaptotagmin) is responsible for the endocytosis of BoNT/B and /G. The Syt-binding site of the former was identified by co-crystallization and mutational analyses. In the present study we report the identification of the SV2-binding interface of BoNT/E. Mutations interfering with SV2 binding were located at a site that corresponds to the Syt-binding site of BoNT/B and at an extended surface area located on the back of the conserved ganglioside-binding site, comprising the N- and C-terminal half of the BoNT/E-binding domain. Mutations impairing the affinity also reduced the neurotoxicity of full-length BoNT/E at mouse phrenic nerve hemidiaphragm preparations demonstrating the crucial role of the identified binding interface. Furthermore, we show that a monoclonal antibody neutralizes BoNT/E activity because it directly interferes with the BoNT/E–SV2 interaction. The results of the present study suggest a novel mode of binding for BoNTs that exploit SV2 as a cell surface receptor.

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Polyunsaturated Lipids Regulate Membrane Domain Stability by Tuning Membrane Order

Biophysical Journal ◽

10.1016/j.bpj.2016.03.012 ◽

2016 ◽

Vol 110 (8) ◽

pp. 1800-1810 ◽

Cited By ~ 90

Author(s):

Kandice R. Levental ◽

Joseph H. Lorent ◽

Xubo Lin ◽

Allison D. Skinkle ◽

Michal A. Surma ◽

...

Keyword(s):

Membrane Domain ◽

Membrane Order ◽

Polyunsaturated Lipids ◽

Domain Stability

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The Orientation of Cytochrome c Oxidase in Coupled Phospholipid Membrane Vesicles—A Spin-Label Study

Canadian Journal of Biochemistry ◽

10.1139/o75-050 ◽

1975 ◽

Vol 53 (3) ◽

pp. 364-370 ◽

Cited By ~ 18

Author(s):

J. A. Kornblatt ◽

W. L. Chen ◽

J. C. Hsia ◽

G. R. Williams

Keyword(s):

Molecular Weight ◽

Binding Site ◽

Cytochrome Oxidase ◽

Spin Label ◽

Resonance Spectrum ◽

Membrane Vesicles ◽

Phospholipid Membrane ◽

Vesicle Membrane ◽

Exterior Surface ◽

Label Study

Cytochrome oxidase, an enzyme containing six different subunits, has been shown to span the inner mitochrondrial membrane. The arrangement of the subunits within the membrane is unknown. We have specifically labeled the 25 000 molecular weight subunit with a spin-label derivative of N-ethylmaleimide, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (NEM-SL(5)). NEM-SL(5)-labeled cytochrome oxidase can be incorporated into phospholipid membranes to form coupled vesicles of the Hinkle, Kim &Racker ((1972) J. Biol. Chem. 247, 1338–1339) type. The resonance spectrum of NEM-SL(5) is similar in both soluble and vesicular cytochrome oxidase. Since ascorbate has been shown to reduce only spin label that is exposed to the exterior surface of a closed vesicle, we have used ascorbate to determine the NEM-SL(5)-binding site in the coupled vesicles. NEM-SL(5)-labeled cytochrome oxidase vesicles are reduced by 10 mM ascorbate with [Formula: see text] of 1 min at 22 °C. The rate of reduction is relatively independent of temperature. We conclude that (1) cytochrome oxidase is unidirectionally or preferentially oriented in the vesicle membrane, and (2) the NEM-SL(5)-binding site on the 25 000 molecular weight subunit is exposed to the external aqueous medium.

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S. Aureus Adapt to Growth Conditions by Changing Membrane Order

Biophysical Journal ◽

10.1016/j.bpj.2013.11.3216 ◽

2014 ◽

Vol 106 (2) ◽

pp. 580a ◽

Cited By ~ 2

Author(s):

Maria I. Perez ◽

Steven M. Trier ◽

Adriana Bernal ◽

Juan Camilo Vargas ◽

Cornelia Herrfurth ◽

...

Keyword(s):

Growth Conditions ◽

Membrane Order

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Inhibition of ester hydrolysis by a lipid vesicle membrane

The Journal of Organic Chemistry ◽

10.1021/jo00159a020 ◽

1983 ◽

Vol 48 (11) ◽

pp. 1886-1890 ◽

Cited By ~ 5

Author(s):

Alim A. Fatah ◽

L. M. Loew

Keyword(s):

Ester Hydrolysis ◽

Lipid Vesicle ◽

Vesicle Membrane

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The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis

The Journal of Cell Biology ◽

10.1083/jcb.150.5.1125 ◽

2000 ◽

Vol 150 (5) ◽

pp. 1125-1136 ◽

Cited By ~ 91

Author(s):

Radhika C. Desai ◽

Bimal Vyas ◽

Cynthia A. Earles ◽

J. Troy Littleton ◽

Judith A. Kowalchyck ◽

...

Keyword(s):

Synaptic Vesicle ◽

Pc12 Cells ◽

Conformational Changes ◽

Cytoplasmic Domain ◽

Dominant Negative ◽

Fusion Pore ◽

Vesicle Membrane ◽

C2 Domains ◽

Essential Step ◽

Synaptotagmin I

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca2+ ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca2+-sensing module. Here, we report that Ca2+ drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca2+ are mediated by a set of conserved acidic Ca2+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca2+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.

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Partial purification of presynaptic plasma membrane by immunoadsorption.

The Journal of Cell Biology ◽

10.1083/jcb.94.1.88 ◽

1982 ◽

Vol 94 (1) ◽

pp. 88-96 ◽

Cited By ~ 23

Author(s):

G P Miljanich ◽

A R Brasier ◽

R B Kelly

Keyword(s):

Plasma Membrane ◽

Synaptic Vesicle ◽

Nerve Terminal ◽

Specific Activity ◽

Electric Organ ◽

Partial Purification ◽

Vesicle Membrane ◽

Specific Activities ◽

Active Zones ◽

Presynaptic Plasma Membrane

During transmitter release, synaptic vesicle membrane is specifically inserted into the nerve terminal plasma membrane only at specialized sites or "active zones." In an attempt to obtain a membrane fraction enriched in active zones, we have utilized the electric organ of the marine ray. From this organ, a fraction enriched in nerve terminals (synaptosomes) was prepared by conventional means. These synaptosomes were bound to microscopic beads by an antiserum to purified electric organ synaptic vesicles (anti-SV). The success of this immunoadsorption procedure was demonstrated by increased specific activities of bead-bound nerve terminal cytoplasmic markers and decreased specific activities of markers for contaminating membranes. To obtain a presynaptic plasma membrane (PSPM) fraction, we lysed the bead-bound synaptosomes by hypoosmotic shock and sonication, resulting in complete release of cytoplasmic markers. When the synaptosomal fraction was surface-labeled with iodine before immunoadsorption, 10% of this label remained bead-bound after lysis, compared with 2% of the total protein, indicating an approximately fivefold enrichment of bead-bound plasma membrane. Concomitantly, the specific activity of bead-bound anti-SV increased approximately 30-fold, indicating an enrichment of plasma membrane which contained inserted synaptic vesicle components. This PSPM preparation is not simply synaptic vesicle membrane since two-dimensional electrophoresis revealed that the polypeptides of the surface-iodinated PSPM preparation include both vesicle and numerous nonvesicle components. Secondly, antiserum to the PSPM fraction is markedly different from anti-SV and binds to external, nonvesicle, nerve terminal components.

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Presence of the vesicular inhibitory amino acid transporter in GABAergic and glycinergic synaptic terminal boutons

Journal of Cell Science ◽

10.1242/jcs.112.6.811 ◽

1999 ◽

Vol 112 (6) ◽

pp. 811-823

Author(s):

A. Dumoulin ◽

P. Rostaing ◽

C. Bedet ◽

S. Levi ◽

M.F. Isambert ◽

...

Keyword(s):

Spinal Cord ◽

Amino Acid ◽

Synaptic Vesicle ◽

Glycine Receptor ◽

Primary Cultures ◽

Ultrastructural Level ◽

Amino Acid Transporter ◽

Vesicle Membrane ◽

Hybridization Data ◽

Acid Transporter

The characterization of the Caenorhabditis elegans unc-47 gene recently allowed the identification of a mammalian (gamma)-amino butyric acid (GABA) transporter, presumed to be located in the synaptic vesicle membrane. In situ hybridization data in rat brain suggested that it might also take up glycine and thus represent a general Vesicular Inhibitory Amino Acid Transporter (VIAAT). In the present study, we have investigated the localization of VIAAT in neurons by using a polyclonal antibody raised against the hydrophilic N-terminal domain of the protein. Light microscopy and immunocytochemistry in primary cultures or tissue sections of the rat spinal cord revealed that VIAAT was localized in a subset (63-65%) of synaptophysin-immunoreactive terminal boutons; among the VIAAT-positive terminals around motoneuronal somata, 32.9% of them were also immunoreactive for GAD65, a marker of GABAergic presynaptic endings. Labelling was also found apposed to clusters positive for the glycine receptor or for its associated protein gephyrin. At the ultrastructural level, VIAAT immunoreactivity was restricted to presynaptic boutons exhibiting classical inhibitory features and, within the boutons, concentrated over synaptic vesicle clusters. Pre-embedding detection of VIAAT followed by post-embedding detection of GABA or glycine on serial sections of the spinal cord or cerebellar cortex indicated that VIAAT was present in glycine-, GABA- or GABA- and glycine-containing boutons. Taken together, these data further support the view of a common vesicular transporter for these two inhibitory transmitters, which would be responsible for their costorage in the same synaptic vesicle and subsequent corelease at mixed GABA-and-glycine synapses.

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Effect of tumour angiogenesis factors on the membrane of capillary and aortic endothelial cells: an e.s.r. spin-label study

Journal of Cell Science ◽

10.1242/jcs.68.1.153 ◽

1984 ◽

Vol 68 (1) ◽

pp. 153-162

Author(s):

N.J. Dodd ◽

S. Kumar

Keyword(s):

Endothelial Cells ◽

Spin Label ◽

Plasma Membranes ◽

Detectable Effect ◽

Aortic Endothelial Cells ◽

Whole Cells ◽

Membrane Order ◽

Migration Factor ◽

Aortic Endothelial

Two distinct factors have been separated from an angiogenic extract of a rat Walker 256 carcinoma, one inducing proliferation and the other migration of capillary endothelial cells in vitro, but having no detectable effect on aortic endothelial cells. The influence of these factors on the order of plasma membranes of these cells was examined by electron spin resonance, using the lipophilic spin label 5-doxyl stearic acid. No detectable effect was observed on treating whole cells or isolated membranes with proliferation factor. In contrast, exposure of capillary endothelial cell membranes to migration factor caused a reduction of membrane order, particularly at temperatures above 30 degrees C. The migration factor had no detectable effect on membrane order of aortic endothelial cells.

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