scholarly journals Author response: Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

2020 ◽  
Author(s):  
Jason D Vevea ◽  
Edwin R Chapman
Keyword(s):  
Membrane Protein ◽  
Synaptic Vesicle ◽  
Hippocampal Neurons ◽  
Author Response ◽  
Vesicle Membrane ◽  
Synaptotagmin 1

scholarly journals Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jason D Vevea ◽  
Edwin R Chapman
Keyword(s):  
Membrane Protein ◽  
Synaptic Vesicle ◽  
Protein Function ◽  
Neurotransmitter Release ◽  
Cell Biology ◽  
Hippocampal Neurons ◽  
Cell Types ◽  
Spontaneous Release ◽  
Vesicle Membrane ◽  
Synaptotagmin 1

The success of comparative cell biology for determining protein function relies on quality disruption techniques. Long-lived proteins, in postmitotic cells, are particularly difficult to eliminate. Moreover, cellular processes are notoriously adaptive; for example, neuronal synapses exhibit a high degree of plasticity. Ideally, protein disruption techniques should be both rapid and complete. Here, we describe knockoff, a generalizable method for the druggable control of membrane protein stability. We developed knockoff for neuronal use but show it also works in other cell types. Applying knockoff to synaptotagmin 1 (SYT1) results in acute disruption of this protein, resulting in loss of synchronous neurotransmitter release with a concomitant increase in the spontaneous release rate, measured optically. Thus, SYT1 is not only the proximal Ca2+ sensor for fast neurotransmitter release but also serves to clamp spontaneous release. Additionally, knockoff can be applied to protein domains as we show for another synaptic vesicle protein, synaptophysin 1.

scholarly journals Identification and characterization of SV31, a novel synaptic vesicle membrane protein and potential transporter

2007 ◽  
Vol 0 (0) ◽  
pp. 070710052154005-???
Author(s):  
Jacqueline Burré ◽  
Herbert Zimmermann ◽  
Walter Volknandt
Keyword(s):  
Membrane Protein ◽  
Synaptic Vesicle ◽  
Vesicle Membrane ◽  
Identification And Characterization

scholarly journals Synaptophysin I Controls the Targeting of VAMP2/Synaptobrevin II to Synaptic Vesicles

2003 ◽  
Vol 14 (12) ◽  
pp. 4909-4919 ◽  
Author(s):  
Maria Pennuto ◽  
Dario Bonanomi ◽  
Fabio Benfenati ◽  
Flavia Valtorta
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Synaptic Vesicle ◽  
Cell Body ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Direct Interaction ◽  
Diffuse Component ◽  
Synaptotagmin I ◽  
Dose Dependent

Synaptic vesicle (SV) proteins are synthesized at the level of the cell body and transported down the axon in membrane precursors of SVs. To investigate the mechanisms underlying sorting of proteins to SVs, fluorescent chimeras of vesicle-associated membrane protein (VAMP) 2, its highly homologous isoform VAMP1 and synaptotagmin I (SytI) were expressed in hippocampal neurons in culture. Interestingly, the proteins displayed a diffuse component of distribution along the axon. In addition, VAMP2 was found to travel in vesicles that constitutively fuse with the plasma membrane. Coexpression of VAMP2 with synaptophysin I (SypI), a major resident of SVs, restored the correct sorting of VAMP2 to SVs. The effect of SypI on VAMP2 sorting was dose dependent, being reversed by increasing VAMP2 expression levels, and highly specific, because the sorting of the SV proteins VAMP1 and SytI was not affected by SypI. The cytoplasmic domain of VAMP2 was found to be necessary for both the formation of VAMP2-SypI hetero-dimers and for VAMP2 sorting to SVs. These data support a role for SypI in directing the correct sorting of VAMP2 in neurons and demonstrate that a direct interaction between the two proteins is required for SypI in order to exert its effect.

scholarly journals Synaptic vesicle membrane proteins interact to form a multimeric complex.

1992 ◽  
Vol 116 (3) ◽  
pp. 761-775 ◽  
Author(s):  
M K Bennett ◽  
N Calakos ◽  
T Kreiner ◽  
R H Scheller
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Synaptic Vesicle ◽  
Protein Interactions ◽  
Synaptic Vesicles ◽  
Spatial Organization ◽  
Gel Filtration ◽  
Vesicle Membrane ◽  
Multicomponent Complex ◽  
Membrane Components

Potential interactions between membrane components of rat brain synaptic vesicles were analyzed by detergent solubilization followed by size fractionation or immunoprecipitation. The behavior of six synaptic vesicle membrane proteins as well as a plasma membrane protein was monitored by Western blotting. Solubilization of synaptic vesicle membranes in CHAPS resulted in the recovery of a large protein complex that included SV2, p65, p38, vesicle-associated membrane protein, and the vacuolar proton pump. Solubilization in octylglucoside resulted in the preservation of interactions between SV2, p38, and rab3A, while solubilization of synaptic vesicles with Triton X-100 resulted in two predominant interactions, one involving p65 and SV2, and the other involving p38 and vesicle-associated membrane protein. The multicomponent complex preserved with CHAPS solubilization was partially reconstituted following octylglucoside solubilization and subsequent dialysis against CHAPS. Reduction of the CHAPS concentration by gel filtration chromatography resulted in increased recovery of the multicomponent complex. Examination of the large complex isolated from CHAPS-solubilized vesicles by negative stain EM revealed structures with multiple globular domains, some of which were specifically labeled with gold-conjugated antibodies directed against p65 and SV2. The protein interactions defined in this report are likely to underlie aspects of neurotransmitter secretion, membrane traffic, and the spatial organization of vesicles within the nerve terminal.

A synaptic vesicle membrane protein is conserved from mammals to Drosophila

Neuron ◽  
1989 ◽  
Vol 2 (5) ◽  
pp. 1475-1481 ◽  
Author(s):  
Thomas C. Südhof ◽  
Marion Baumert ◽  
Mark S. Perin ◽  
Reinhard Jahn
Keyword(s):  
Membrane Protein ◽  
Synaptic Vesicle ◽  
Vesicle Membrane

scholarly journals Glycosylation Is Dispensable for Sorting of Synaptotagmin 1 but Is Critical for Targeting of SV2 and Synaptophysin to Recycling Synaptic Vesicles

2012 ◽  
Vol 287 (42) ◽  
pp. 35658-35668 ◽  
Author(s):  
Sung E. Kwon ◽  
Edwin R. Chapman
Keyword(s):  
Synaptic Vesicle ◽  
Post Translational Modification ◽  
Vesicle Membrane ◽  
Knock Out ◽  
Synaptic Localization ◽  
Glycosylation Sites ◽  
Synaptotagmin 1 ◽  
Knock Out Mice ◽  
And Function

Glycosylation is a major form of post-translational modification of synaptic vesicle membrane proteins. For example, the three major synaptic vesicle glycoproteins, synaptotagmin 1, synaptophysin, and SV2, represent ∼30% of the total copy number of vesicle proteins. Previous studies suggested that glycosylation is required for the vesicular targeting of synaptotagmin 1, but the role of glycosylation of synaptophysin and SV2 has not been explored in detail. In this study, we analyzed all glycosylation sites on synaptotagmin 1, synaptophysin, and SV2A via mutagenesis and optical imaging of pHluorin-tagged proteins in cultured neurons from knock-out mice lacking each protein. Surprisingly, these experiments revealed that glycosylation is completely dispensable for the sorting of synaptotagmin 1 to SVs whereas the N-glycans on SV2A are only partially dispensable. In contrast, N-glycan addition is essential for the synaptic localization and function of synaptophysin. Thus, glycosylation plays distinct roles in the trafficking of each of the three major synaptic vesicle glycoproteins.

scholarly journals Synaptotagmin-1 drives synchronous Ca2+-triggered fusion by C2B-domain-mediated synaptic-vesicle-membrane attachment

2017 ◽  
Vol 21 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Shuwen Chang ◽  
Thorsten Trimbuch ◽  
Christian Rosenmund
Keyword(s):  
Synaptic Vesicle ◽  
Vesicle Membrane ◽  
Synaptotagmin 1 ◽  
Membrane Attachment
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