Faculty Opinions recommendation of Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling.

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.

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Identification of synaptotagmin effectors via acute inhibition of secretion from cracked PC12 cells

The Journal of Cell Biology ◽

10.1083/jcb.200302060 ◽

2003 ◽

Vol 162 (2) ◽

pp. 199-209 ◽

Cited By ~ 84

Author(s):

Ward C. Tucker ◽

J. Michael Edwardson ◽

Jihong Bai ◽

Hyun-Jung Kim ◽

Thomas F.J. Martin ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Pc12 Cells ◽

Functional Significance ◽

Synaptic Vesicles ◽

Membrane Traffic ◽

Binding Activity ◽

C2 Domains ◽

Expression Levels ◽

Target Molecules

T he synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the Ca2+-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative Ca2+–syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I–XI to interfere with endogenous syt–effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin–SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PIP2)–binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their Ca2+-regulated interactions with t-SNAREs and PIP2, target molecules known to play critical roles in exocytosis.

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Biogenesis of synaptic vesicles in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.130.5.1041 ◽

1995 ◽

Vol 130 (5) ◽

pp. 1041-1049 ◽

Cited By ~ 52

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.

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Visualization of the Dynamics of Synaptic Vesicle and Plasma Membrane Proteins in Living Axons

The Journal of Cell Biology ◽

10.1083/jcb.140.3.659 ◽

1998 ◽

Vol 140 (3) ◽

pp. 659-674 ◽

Cited By ~ 201

Author(s):

Takao Nakata ◽

Sumio Terada ◽

Nobutaka Hirokawa

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Synaptic Vesicle ◽

Synaptic Vesicles ◽

Fluorescent Protein ◽

Plasma Membrane Proteins ◽

Trans Golgi Network ◽

Direction Of Movement ◽

Green Fluorescent ◽

Golgi Network

Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles. However, their transporting vesicles have not yet been identified. We have successfully visualized the transporting vesicles of plasma membrane proteins, synaptic vesicle proteins, and the trans-Golgi network residual proteins in living axons at high resolution using laser scan microscopy of green fluorescent protein-tagged proteins after photobleaching. We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement. These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e08-05-0456 ◽

2009 ◽

Vol 20 (5) ◽

pp. 1441-1453 ◽

Cited By ~ 61

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.

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