scholarly journals Biogenesis of synaptic vesicles in vitro.

1995 ◽  
Vol 130 (5) ◽  
pp. 1041-1049 ◽  
Author(s):  
C Desnos ◽  
L Clift-O'Grady ◽  
R B Kelly
Keyword(s):  
Membrane Proteins ◽  
Cell Surface ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Vesicle Membrane ◽  
Synaptic Vesicle Protein ◽  
Vesicle Biogenesis ◽  
Vesicle Protein ◽  
Correct Size

Synaptic vesicles are synthesized at a rapid rate in nerve terminals to compensate for their rapid loss during neurotransmitter release. Their biogenesis involves endocytosis of synaptic vesicle membrane proteins from the plasma membrane and requires two steps, the segregation of synaptic vesicle membrane proteins from other cellular proteins, and the packaging of those unique proteins into vesicles of the correct size. By labeling an epitope-tagged variant of a synaptic vesicle protein, VAMP (synaptobrevin), at the cell surface of the neuroendocrine cell line PC12, synaptic vesicle biogenesis could be followed with considerable precision, quantitatively and kinetically. Epitope-tagged VAMP was recovered in synaptic vesicles within a few minutes of leaving the cell surface. More efficient targeting was obtained by using the VAMP mutant, del 61-70. Synaptic vesicles did not form at 15 degrees C although endocytosis still occurred. Synaptic vesicles could be generated in vitro from a homogenate of cells labeled at 15 degrees C. The newly formed vesicles are identical to those formed in vivo in their sedimentation characteristics, the presence of the synaptic vesicle protein synaptophysin, and the absence of detectable transferrin receptor. Brain, but not fibroblast cytosol, allows vesicles of the correct size to form. Vesicle formation is time and temperature-dependent, requires ATP, is calcium independent, and is inhibited by GTP-gamma S. Thus, two key steps in synaptic vesicle biogenesis have been reconstituted in vitro, allowing direct analysis of the proteins involved.

scholarly journals The AP-3 Complex Required for Endosomal Synaptic Vesicle Biogenesis is Associated with a Casein Kinase Ια-Like Isoform

2000 ◽  
Vol 11 (8) ◽  
pp. 2591-2604 ◽  
Author(s):  
Victor V. Faundez ◽  
Regis B. Kelly
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Enzymatic Reaction ◽  
Casein Kinase ◽  
Small Gtpase ◽  
Casein Kinase I ◽  
Vesicle Membranes ◽  
Vesicle Biogenesis ◽  
Hinge Domain

The formation of small vesicles is mediated by cytoplasmic coats the assembly of which is regulated by the activity of GTPases, kinases, and phosphatases. A heterotetrameric AP-3 adaptor complex has been implicated in the formation of synaptic vesicles from PC12 endosomes ( Faundez et al., 1998 ). When the small GTPase ARF1 is prevented from hydrolyzing GTP, we can reconstitute AP-3 recruitment to synaptic vesicle membranes in an assembly reaction that requires temperatures above 15°C and the presence of ATP suggesting that an enzymatic step is involved in the coat assembly. We have now found an enzymatic reaction, the phosphorylation of the AP-3 adaptor complex, that is linked with synaptic vesicle coating. Phosphorylation occurs in the β3 subunit of the complex by a kinase similar to casein kinase 1α. The kinase copurifies with neuronal-specific AP-3. In vitro, purified casein kinase I selectively phosphorylates the β3A and β3B subunit at its hinge domain. Inhibiting the kinase hinders the recruitment of AP-3 to synaptic vesicles. The same inhibitors that prevent coat assembly in vitro also inhibit the formation of synaptic vesicles in PC12 cells. The data suggest, therefore, that the mechanism of AP-3-mediated vesiculation from neuroendocrine endosomes requires the phosphorylation of the adaptor complex at a step during or after AP-3 recruitment to membranes.

scholarly journals Synaptic Vesicle Protein 2 (SV2) does not hydrolyze ATP

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 209
Author(s):  
Jia Yao ◽  
Sandra M. Bajjalieh
Keyword(s):  
Atpase Activity ◽  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Endocrine Cells ◽  
Essential Role ◽  
Adenine Nucleotides ◽  
Regulated Secretion ◽  
Synaptic Vesicle Protein ◽  
Vesicle Protein

Synaptic vesicle protein 2 (SV2) is a transporter-like protein specifically expressed in endocrine cells and neurons, where it is localized to vesicles that undergo regulated secretion and plays an essential role in regulating neurotransmitter release. SV2 binds adenine nucleotides including ATP. Analysis of ATP transport revealed that SV2 is not an ATP transporter, nor does it affect ATP transport. As a further step toward understanding how ATP binding contributes to SV2 function, we investigated whether SV2 is an ATPase using an in vitro measure of ATPase activity. The study reported here indicates that SV2 does not have ATPase activity. Thus, binding to adenine nucleotides likely modulates other actions of SV2.

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.

scholarly journals Roles of BLOC-1 and Adaptor Protein-3 Complexes in Cargo Sorting to Synaptic Vesicles

2009 ◽  
Vol 20 (5) ◽  
pp. 1441-1453 ◽  
Author(s):  
Karen Newell-Litwa ◽  
Gloria Salazar ◽  
Yoland Smith ◽  
Victor Faundez
Keyword(s):  
Membrane Proteins ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Adaptor Protein ◽  
Protein Composition ◽  
Organelle Biogenesis ◽  
Vesicle Membrane ◽  
Early Endosomes ◽  
Lysosomal Sorting ◽  
Lysosome Related Organelles

Neuronal lysosomes and their biogenesis mechanisms are primarily thought to clear metabolites and proteins whose abnormal accumulation leads to neurodegenerative disease pathology. However, it remains unknown whether lysosomal sorting mechanisms regulate the levels of membrane proteins within synaptic vesicles. Using high-resolution deconvolution microscopy, we identified early endosomal compartments where both selected synaptic vesicle and lysosomal membrane proteins coexist with the adaptor protein complex 3 (AP-3) in neuronal cells. From these early endosomes, both synaptic vesicle membrane proteins and characteristic AP-3 lysosomal cargoes can be similarly sorted to brain synaptic vesicles and PC12 synaptic-like microvesicles. Mouse knockouts for two Hermansky–Pudlak complexes involved in lysosomal biogenesis from early endosomes, the ubiquitous isoform of AP-3 (Ap3b1−/−) and muted, defective in the biogenesis of lysosome-related organelles complex 1 (BLOC-1), increased the content of characteristic synaptic vesicle proteins and known AP-3 lysosomal proteins in isolated synaptic vesicle fractions. These phenotypes contrast with those of the mouse knockout for the neuronal AP-3 isoform involved in synaptic vesicle biogenesis (Ap3b2−/−), in which the content of select proteins was reduced in synaptic vesicles. Our results demonstrate that lysosomal and lysosome-related organelle biogenesis mechanisms regulate steady-state synaptic vesicle protein composition from shared early endosomes.

scholarly journals Biogenesis of synaptic vesicle-like structures in a pheochromocytoma cell line PC-12.

1990 ◽  
Vol 110 (5) ◽  
pp. 1693-1703 ◽  
Author(s):  
L Clift-O'Grady ◽  
A D Linstedt ◽  
A W Lowe ◽  
E Grote ◽  
R B Kelly
Keyword(s):  
Membrane Proteins ◽  
Rat Brain ◽  
Synaptic Vesicle ◽  
Cell Line ◽  
Pc12 Cells ◽  
Synaptic Vesicles ◽  
Protein Composition ◽  
Major Protein ◽  
Vesicle Membrane ◽  
Pheochromocytoma Cell

The presence of unique proteins in synaptic vesicles of neurons suggests selective targeting during vesicle formation. Endocrine, but not other cells, also express synaptic vesicle membrane proteins and target them selectively to small intracellular vesicles. We show that the rat pheochromocytoma cell line, PC12, has a population of small vesicles with sedimentation and density properties very similar to those of rat brain synaptic vesicles. When synaptophysin is expressed in nonneuronal cells, it is found in intracellular organelles that are not the size of synaptic vesicles. The major protein in the small vesicles isolated from PC12 cells is found to be synaptophysin, which is also the major protein in rat brain vesicles. At least two of the minor proteins in the small vesicles are also known synaptic vesicle membrane proteins. Synaptic vesicle-like structures in PC12 cells can be shown to take up an exogenous bulk phase marker, HRP. Their proteins, including synaptophysin, are labeled if the cells are surface labeled and subsequently warmed. Although the PC12 vesicles can arise by endocytosis, they seem to exclude the receptor-mediated endocytosis marker, transferrin. We conclude that PC12 cells contain synaptic vesicle-like structures that resemble authentic synaptic vesicles in physical properties, protein composition and endocytotic origin.

scholarly journals A new mechanism for antiepileptic drug action: vesicular entry may mediate the effects of levetiracetam

2011 ◽  
Vol 106 (3) ◽  
pp. 1227-1239 ◽  
Author(s):  
Anna L. Meehan ◽  
Xiaofeng Yang ◽  
Brian D. McAdams ◽  
LiLian Yuan ◽  
Steven M. Rothman
Keyword(s):  
Binding Site ◽  
Synaptic Vesicle ◽  
Pyramidal Neurons ◽  
Synaptic Activity ◽  
Dependent Manner ◽  
Prolonged Incubation ◽  
Synaptic Vesicle Protein ◽  
Vesicle Protein ◽  
Synaptic Vesicle Protein 2A

Levetiracetam (LEV) is one of the most commonly prescribed antiepileptic drugs, but its mechanism of action is uncertain. Based on prior information that LEV binds to the vesicular protein synaptic vesicle protein 2A and reduces presynaptic neurotransmitter release, we wanted to more rigorously characterize its effect on transmitter release and explain the requirement for a prolonged incubation period for its full effect to manifest. During whole cell patch recordings from rat hippocampal pyramidal neurons in vitro, we found that LEV decreased synaptic currents in a frequency-dependent manner and reduced the readily releasable pool of vesicles. When we manipulated spontaneous activity and stimulation paradigms, we found that synaptic activity during LEV incubation alters the time at which LEV's effect appears, as well as its magnitude. We believe that synaptic activity and concomitant vesicular release allow LEV to enter recycling vesicles to reach its binding site, synaptic vesicle protein 2A. In support of this hypothesis, a vesicular “load-unload” protocol using hypertonic sucrose in the presence of LEV quickly induced LEV's effect. The effect rapidly disappeared after unloading in the absence of LEV. These findings are compatible with LEV acting at an intravesicular binding site to modulate the release of transmitter and with its most marked effect on rapidly discharging neurons. Our results identify a unique neurobiological explanation for LEV's highly selective antiepileptic effect and suggest that synaptic vesicle proteins might be appropriate targets for the development of other neuroactive drugs.

scholarly journals Quantitative Comparison of Glutamatergic and GABAergic Synaptic Vesicles Unveils Selectivity for Few Proteins Including MAL2, a Novel Synaptic Vesicle Protein

2010 ◽  
Vol 30 (1) ◽  
pp. 2-12 ◽  
Author(s):  
M. Gronborg ◽  
N. J. Pavlos ◽  
I. Brunk ◽  
J. J. E. Chua ◽  
A. Munster-Wandowski ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Quantitative Comparison ◽  
Synaptic Vesicle Protein ◽  
Vesicle Protein

scholarly journals Rab3C is a synaptic vesicle protein that dissociates from synaptic vesicles after stimulation of exocytosis

1994 ◽  
Vol 269 (15) ◽  
pp. 10971-10974
Author(s):  
G. Fischer von Mollard ◽  
B. Stahl ◽  
A. Khokhlatchev ◽  
T.C. Südhof ◽  
R. Jahn
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Synaptic Vesicle Protein ◽  
Vesicle Protein ◽  
Stimulation Of
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