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 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 UNC-11, a Caenorhabditis elegans AP180 Homologue, Regulates the Size and Protein Composition of Synaptic Vesicles

1999 ◽  
Vol 10 (7) ◽  
pp. 2343-2360 ◽  
Author(s):  
Michael L. Nonet ◽  
Andrea M. Holgado ◽  
Faraha Brewer ◽  
Craig J. Serpe ◽  
Betty A. Norbeck ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Adaptor Protein ◽  
Protein Composition ◽  
Mammalian Brain ◽  
Snare Protein ◽  
Vesicle Biogenesis ◽  
Clathrin Adaptor

The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second,unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.

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 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.

Identification of synapsin I peptides that insert into lipid membranes

2001 ◽  
Vol 354 (1) ◽  
pp. 57-66 ◽  
Author(s):  
James J. CHEETHAM ◽  
Sabine HILFIKER ◽  
Fabio BENFENATI ◽  
Thomas WEBER ◽  
Paul GREENGARD ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Molecular Mechanisms ◽  
Phospholipid Bilayer ◽  
Synapsin I ◽  
Affinity Binding ◽  
Hydrophobic Core ◽  
Vesicle Membrane ◽  
High Affinity Binding ◽  
High Affinity

The synapsins constitute a family of synaptic vesicle-associated phosphoproteins essential for regulating neurotransmitter release and synaptogenesis. The molecular mechanisms underlying the selective targeting of synapsin I to synaptic vesicles are thought to involve specific protein–protein interactions, while the high-affinity binding to the synaptic vesicle membrane may involve both protein–protein and protein–lipid interactions. The highly hydrophobic N-terminal region of the protein has been shown to bind with high affinity to the acidic phospholipids phosphatidylserine and phosphatidylinositol and to penetrate the hydrophobic core of the lipid bilayer. To precisely identify the domains of synapsin I which mediate the interaction with lipids, synapsin I was bound to liposomes containing the membrane-directed carbene-generating reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and subjected to photolysis. Isolation and N-terminal amino acid sequencing of 125I-labelled synapsin I peptides derived from CNBr cleavage indicated that three distinct regions in the highly conserved domain C of synapsin I insert into the hydrophobic core of the phospholipid bilayer. The boundaries of the regions encompass residues 166–192, 233–258 and 278–327 of bovine synapsin I. These regions are surface-exposed in the crystal structure of domain C of bovine synapsin I and are evolutionarily conserved among isoforms across species. The present data offer a molecular explanation for the high-affinity binding of synapsin I to phospholipid bilayers and synaptic vesicles.

scholarly journals AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway

2012 ◽  
Vol 23 (18) ◽  
pp. 3612-3623 ◽  
Author(s):  
Michael R. Dores ◽  
May M. Paing ◽  
Huilan Lin ◽  
William A. Montagne ◽  
Adriano Marchese ◽  
...  
Keyword(s):  
Adaptor Protein ◽  
Kinase Substrate ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Early Endosomes ◽  
Lysosomal Sorting ◽  
Protease Activated Receptor 1 ◽  
Signaling Receptors ◽  
G Protein Coupled ◽  
Hepatocyte Growth

The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor–regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein–coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail–localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting.

scholarly journals Retrieval and recycling of synaptic vesicle membrane in pinched-off nerve terminals (synaptosomes).

1978 ◽  
Vol 78 (3) ◽  
pp. 685-700 ◽  
Author(s):  
R C Fried ◽  
M P Blaustein
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Plasma Membranes ◽  
Large Diameter ◽  
Small Diameter ◽  
Physiological Saline ◽  
Nerve Terminals ◽  
Vesicle Membrane ◽  
Thorium Dioxide ◽  
Vesicular Exocytosis

The morphological features of pinched-off presynaptic nerve terminals (synaptosomes) from rat brain were examined with electron microscope techniques; in many experiments, an extracellular marked (horseradish peroxidase or colloidal thorium dioxide) was included in the incubation media. When incubated in physiological saline, most terminals appeared approximately spherical, and were filled with small (approximately 400-A diameter) "synaptic vesicles"; mitochondria were also present in many of the terminals. In a number of instances the region of synaptic contact, with adhering portions of the postsynaptic cell membrane and postsynaptic density, could be readily discerned. Approximately 20--30% of the terminals in our preparations exhibited clear evidence of damage, as indicated by diffuse distribution of extracellular markers in the cytoplasm; the markers appeared to be excluded from the intraterminal vesicles under these circumstances. The markers were excluded from the cytoplasm in approximately 70--80% of the terminals, which may imply that these terminals have intact plasma membranes. When the terminals were treated with depolarizing agents (veratridine or K-rich media), in the presence of Ca, many new, large (600--900-A diameter) vesicles and some coated vesicles and new vacuoles appeared. When the media contained an extracellular marker, the newly formed structures frequently were labeled with the marker. If the veratridine-depolarized terminals were subsequently treated with tetrodotoxin (to repolarize the terminals) and allowed to "recover" for 60--90 min, most of the large marker-containing vesicles disappeared, and numerous small (approximately 400-A diameter) marker-containing vesicles appeared. These observations are consistent with the idea that pinched-off presynaptic terminals contain all of the machinery necessary for vesicular exocytosis and for the retrieval and recycling of synaptic vesicle membrane. The vesicle membrane appears to be retrieval primarily in the form of large diameter vesicles which are subsequently reprocessed to form new "typical" small-diameter synaptic vesicles.

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