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 Cross-linking mass spectrometry uncovers protein interactions and functional assemblies in synaptic vesicle membranes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sabine Wittig ◽  
Marcelo Ganzella ◽  
Marie Barth ◽  
Susann Kostmann ◽  
Dietmar Riedel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Synaptic Vesicle ◽  
Protein Interactions ◽  
Synaptic Vesicles ◽  
Large Scale ◽  
Cross Linking ◽  
Synaptic Membrane ◽  
Biophysical Parameters ◽  
Vesicle Membrane ◽  
Pass Through

AbstractSynaptic vesicles are storage organelles for neurotransmitters. They pass through a trafficking cycle and fuse with the pre-synaptic membrane when an action potential arrives at the nerve terminal. While molecular components and biophysical parameters of synaptic vesicles have been determined, our knowledge on the protein interactions in their membranes is limited. Here, we apply cross-linking mass spectrometry to study interactions of synaptic vesicle proteins in an unbiased approach without the need for specific antibodies or detergent-solubilisation. Our large-scale analysis delivers a protein network of vesicle sub-populations and functional assemblies including an active and an inactive conformation of the vesicular ATPase complex as well as non-conventional arrangements of the luminal loops of SV2A, Synaptophysin and structurally related proteins. Based on this network, we specifically target Synaptobrevin-2, which connects with many proteins, in different approaches. Our results allow distinction of interactions caused by ‘crowding’ in the vesicle membrane from stable interaction modules.

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 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 microfluidic strategy for the detection of membrane protein interactions

Lab on a Chip ◽  
2020 ◽  
Vol 20 (17) ◽  
pp. 3230-3238
Author(s):  
Yuewen Zhang ◽  
Therese W. Herling ◽  
Stefan Kreida ◽  
Quentin A. E. Peter ◽  
Tadas Kartanas ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Interactions ◽  
Native Solution ◽  
Exchange Of Information ◽  
Extracellular Environment

Membrane proteins are gatekeepers for exchange of information and matter between the intracellular and extracellular environment. This paper opens up a route to probe membrane protein interactions under native solution conditions using microfluidics.

scholarly journals Botulinum Neurotoxin a Blocks Synaptic Vesicle Exocytosis but Not Endocytosis at the Nerve Terminal

1999 ◽  
Vol 147 (6) ◽  
pp. 1249-1260 ◽  
Author(s):  
Elaine A. Neale ◽  
Linda M. Bowers ◽  
Min Jia ◽  
Karen E. Bateman ◽  
Lura C. Williamson
Keyword(s):  
Synaptic Vesicle ◽  
Nerve Terminal ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Botulinum Neurotoxin ◽  
Vesicle Membrane ◽  
Botulinum Neurotoxin A ◽  
Synaptic Vesicle Exocytosis ◽  
Vesicle Exocytosis ◽  
Botulinum Neurotoxins

The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K+-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K+ depolarization, in the presence of Ca2+, triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A–blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca2+ is required for synaptic vesicle membrane retrieval.

scholarly journals Interactions of synapsin I with phospholipids: possible role in synaptic vesicle clustering and in the maintenance of bilayer structures.

1993 ◽  
Vol 123 (6) ◽  
pp. 1845-1855 ◽  
Author(s):  
F Benfenati ◽  
F Valtorta ◽  
M C Rossi ◽  
F Onofri ◽  
T Sihra ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
High Surface ◽  
Resonance Spectroscopy ◽  
Synapsin I ◽  
Hydrophobic Core ◽  
Membrane Phospholipids ◽  
Head Region ◽  
Vesicle Membrane ◽  
Liposome Fusion

Synapsin I is a synaptic vesicle-specific phosphoprotein composed of a globular and hydrophobic head and of a proline-rich, elongated and basic tail. Synapsin I binds with high affinity to phospholipid and protein components of synaptic vesicles. The head region of the protein has a very high surface activity, strongly interacts with acidic phospholipids and penetrates the hydrophobic core of the vesicle membrane. In the present paper, we have investigated the possible functional effects of the interaction between synapsin I and vesicle phospholipids. Synapsin I enhances both the rate and the extent of Ca(2+)-dependent membrane fusion, although it has no detectable fusogenic activity per se. This effect, which appears to be independent of synapsin I phosphorylation and localized to the head region of the protein, is attributable to aggregation of adjacent vesicles. The facilitation of Ca(2+)-induced liposome fusion is maximal at 50-80% of vesicle saturation and then decreases steeply, whereas vesicle aggregation does not show this biphasic behavior. Association of synapsin I with phospholipid bilayers does not induce membrane destabilization. Rather, 31P-nuclear magnetic resonance spectroscopy demonstrated that synapsin I inhibits the transition of membrane phospholipids from the bilayer (L alpha) to the inverted hexagonal (HII) phase induced either by increases in temperature or by Ca2+. These properties might contribute to the remarkable selectivity of the fusion of synaptic vesicles with the presynaptic plasma membrane during exocytosis.

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