Protein Quantification at the Single Vesicle Level Reveals That a Subset of Synaptic Vesicle Proteins Are Trafficked with High Precision

Mutch, Sarah; Kensel-Hammes, Patricia; Gadd, Jennifer C.; Fujimoto, Bryant S.; Allen, Richard; Schiro, Perry G.; Lorenz, Robert M.; Kuyper, Christopher L.; Kuo, Jason S.; Bajjalieh, Sandra M.; Chiu, Daniel T.

doi:10.1523/jneurosci.3805-10.2011

Cited by 162 publications

(162 citation statements)

References 39 publications

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“…Synapses lacking synaptobrevin 2 display <1% of wild-type release when stimulated by action potential (AP)-mediated calcium influx (6). Proteomic studies have shown that synaptobrevin 2 is a highly abundant SV protein (7) that is exoendocytically sorted with very high precision (8). Similar observations have been made for other SV proteins, including synaptotagmin and vesicular glutamate transporters (VGLUTs).…”

mentioning

confidence: 80%

SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors clathrin assembly lymphoid myeloid leukemia (CALM) and AP180 at synapses

Koo

Marković²,

Puchkov³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

124

177

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Neurotransmission depends on the exo-endocytosis of synaptic vesicles at active zones. Synaptobrevin 2 [also known as vesicleassociated membrane protein 2 (VAMP2)], the most abundant synaptic vesicle protein and a major soluble NSF attachment protein receptor (SNARE) component, is required for fast calcium-triggered synaptic vesicle fusion. In contrast to the extensive knowledge about the mechanism of SNARE-mediated exocytosis, little is known about the endocytic sorting of synaptobrevin 2. Here we show that synaptobrevin 2 sorting involves determinants within its SNARE motif that are recognized by the ANTH domains of the endocytic adaptors AP180 and clathrin assembly lymphoid myeloid leukemia (CALM). Depletion of CALM or AP180 causes selective surface accumulation of synaptobrevin 2 but not vGLUT1 at the neuronal surface. Endocytic sorting of synaptobrevin 2 is mediated by direct interaction of the ANTH domain of the related endocytic adaptors CALM and AP180 with the N-terminal half of the SNARE motif centered around M46, as evidenced by NMR spectroscopy analysis and site-directed mutagenesis. Our data unravel a unique mechanism of SNARE motif-dependent endocytic sorting and identify the ANTH domain proteins AP180 and CALM as cargo-specific adaptors for synaptobrevin endocytosis. Defective SNARE endocytosis may also underlie the association of CALM and AP180 with neurodevelopmental and cognitive defects or neurodegenerative disorders.clathrin-mediated endocytosis | structure N eurotransmission in the brain depends on the calcium-triggered fusion and recycling of neurotransmitter-filled synaptic vesicles (SVs) with the presynaptic membrane at active zones (1). Following their exocytic insertion into the presynaptic membrane, SV proteins need to be retrieved at a precisely defined stoichiometry by endocytosis, a process involving clathrin, adaptors, and other endocytic proteins (2). Fast calcium-triggered SV fusion critically depends on the SV arginine (R)-soluble NSF attachment protein receptor (SNARE) synaptobrevin [or vesicle-associated membrane protein (VAMP)], which by forming a complex with the plasma membrane glutamine (Q)-SNAREs syntaxin and synaptosomal-associated protein (SNAP)-25 (3) drives neuroexocytosis (4, 5). Synapses lacking synaptobrevin 2 display <1% of wild-type release when stimulated by action potential (AP)-mediated calcium influx (6). Proteomic studies have shown that synaptobrevin 2 is a highly abundant SV protein (7) that is exoendocytically sorted with very high precision (8). Similar observations have been made for other SV proteins, including synaptotagmin and vesicular glutamate transporters (VGLUTs). How such precise sorting of synaptobrevin 2 is achieved has remained enigmatic. Synaptobrevin lacks recognizable linear sorting motifs (9), and unlike other SNARE proteins does not contain a folded N-terminal domain that serves as a targeting determinant in other VAMP family members (10-12).Genetic data have linked synaptobrevin sorting to the function of the AP180 N-terminal homology (...

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mentioning

confidence: 80%

SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors clathrin assembly lymphoid myeloid leukemia (CALM) and AP180 at synapses

Koo

Marković²,

Puchkov³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

124

177

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“…At the synapse, tight exo-endocytic coupling maintains a pool of neurotransmitter-filled synaptic vesicles. Here, transmembrane cargo recognition is critical as synaptic vesicles have a highly defined protein composition (Takamori et al 2006), and there is a remarkably conserved packaging stoichiometry for some transmembrane proteins within reforming synaptic vesicles (Takamori et al 2006;Mutch et al 2011). Synaptotagmin 1 is a vesicle-associated, Ca 2þ -responsive bilayer fusion regulator and therefore necessarily packaged into each regenerating synaptic vesicle following exocytosis.…”

Section: Distant Relations: M-homology Domain Claspsmentioning

confidence: 99%

Cargo Recognition in Clathrin-Mediated Endocytosis

Traub

Bonifacino

2013

Cold Spring Harbor Perspectives in Biology

271

290

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The endosomal system is expansive and complex, characterized by swift morphological transitions, dynamic remodeling of membrane constituents, and intracellular positioning changes. To properly navigate this ever-altering membrane labyrinth, transmembrane protein cargoes typically require specific sorting signals that are decoded by components of protein coats. The best-characterized sorting process within the endosomal system is the rapid internalization of select transmembrane proteins within clathrin-coated vesicles. Endocytic signals consist of linear motifs, conformational determinants, or covalent modifications in the cytosolic domains of transmembrane cargo. These signals are interpreted by a diverse set of clathrin-associated sorting proteins (CLASPs) that translocate from the cytosol to the inner face of the plasma membrane. Signal recognition by CLASPs is highly cooperative, involving additional interactions with phospholipids, Arf GTPases, other CLASPs, and clathrin, and is regulated by large conformational changes and covalent modifications. Related sorting events occur at other endosomal sorting stations.

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“…1F). Second, in a recent study that quantified copy number variability of SV proteins within SV populations, SV2 was found to have the most consistent copy number, with nearly all SVs containing five copies of this protein (Mutch et al, 2011). Third, all experiments were performed at least 2 weeks from infection, at which time EGFP:SV2A expression would have been nearly uniform.…”

Section: Measuring Changes In Sv Pools Sizesmentioning

confidence: 99%

Use Dependence of Presynaptic Tenacity

Fisher-Lavie

Zeidan²,

Stern³

et al. 2011

J. Neurosci.

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Recent studies indicate that synaptic vesicles (SVs) are continuously interchanged among nearby synapses at very significant rates. These dynamics and the lack of obvious barriers confining synaptic vesicles to specific synapses would seem to challenge the ability of synapses to maintain a constant amount of synaptic vesicles over prolonged time scales. Moreover, the extensive mobilization of synaptic vesicles associated with presynaptic activity might be expected to intensify this challenge. Here we examined the ability of individual presynaptic boutons of rat hippocampal neurons to maintain their synaptic vesicle content, and the degree to which this ability is affected by continuous activity. We found that the synaptic vesicle content of individual boutons belonging to the same axons gradually changed over several hours, and that these changes occurred independently of activity. Intermittent stimulation for 1 h accelerated rates of vesicle pool size change. Interestingly, however, following stimulation cessation, vesicle pool size change rates gradually converged with basal change rates. Over similar time scales, active zones (AZs) exhibited substantial remodeling; yet, unlike synaptic vesicles, AZ remodeling was not affected by the stimulation paradigms used here. These findings indicate that enhanced activity levels can increase synaptic vesicle redistribution among nearby synapses, but also highlight the presence of forces that act to restore particular set points in terms of SV contents, and support a role for active zones in preserving such set points. These findings also indicate, however, that neither AZ size nor SV content set points are particularly stable, questioning the long-term tenacity of presynaptic specializations.

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Protein Quantification at the Single Vesicle Level Reveals That a Subset of Synaptic Vesicle Proteins Are Trafficked with High Precision

Cited by 162 publications

References 39 publications

SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors clathrin assembly lymphoid myeloid leukemia (CALM) and AP180 at synapses

SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors clathrin assembly lymphoid myeloid leukemia (CALM) and AP180 at synapses

Cargo Recognition in Clathrin-Mediated Endocytosis

Use Dependence of Presynaptic Tenacity

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