Endocytosis of Synaptotagmin 1 Is Mediated by a Novel, Tryptophan‐Containing Motif

Jarousse, Nadine; Wilson, Joshua D.; Araç, Demet; Rizo, Josep; Kelly, Regis B.

doi:10.1034/j.1600-0854.2003.00101.x

Cited by 28 publications

(28 citation statements)

References 71 publications

(92 reference statements)

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“…In this motif, it has been suggested that the hydroxyl group of tyrosine is essential for recognition by AP-2 m2 (Owen and Evans, 1998). However, the neonatal Fc receptor and synaptotagmin 1 have been found to internalize through direct binding of their tryptophan-containing motif, WxxW, to AP-2 m2 (Jarousse et al, 2003;Wernick et al, 2005;Wu and Simister, 2001). These studies suggest that aromatic amino acids in this sequence are sufficient for AP-2 m2 recognition.…”

Section: Discussionmentioning

confidence: 99%

“…Tryptophan is an aromatic amino acid, as is tyrosine, and isoleucine is a canonical hydrophobic amino acid. Importantly, the neonatal Fc receptor and the synaptic vesicle membrane protein, synaptotagmin 1, have been reported to use a WxxW motif instead of the YxxW sequence for AP-2 m2 binding and internalization (Jarousse et al, 2003;Wernick et al, 2005;Wu and Simister, 2001). Therefore, the ability of the M1-mAChR Cterminal WxxW motif to bind to the AP-2 m2 subunit was examined in a GST pulldown assay.…”

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confidence: 99%

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Intracellular localization of M1 muscarinic acetylcholine receptor through clathrin-dependent constitutive internalization via a C-terminal tryptophan-based motif

Uwada¹,

Yoshiki²,

Masuoka³

et al. 2014

Journal of Cell Science

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The M1 muscarinic acetylcholine receptor (M1-mAChR, encoded by CHRM1) is a G-protein-coupled membrane receptor that is activated by extracellular cholinergic stimuli. Recent investigations have revealed the intracellular localization of M1-mAChR. In this study, we observed constitutive internalization of M1-mAChR in mouse neuroblastoma N1E-115 cells without agonist stimulation. Constitutive internalization depended on dynamin, clathrin and the adaptor protein-2 (AP-2) complex. A WxxI motif in the M1-mAChR C-terminus is essential for its constitutive internalization, given that replacement of W 442 or I 445 with alanine residues abolished constitutive internalization. This WxxI motif resembles YxxW, which is the canonical binding motif for the m2 subunit of the AP-2 complex. The M1-mAChR C-terminal WxxI motif interacted with AP-2 m2. W442A and I445A mutants of the M1-mAChR C-terminal sequence lost AP-2-m2-binding activity, whereas the W442Y mutant bound more effectively than wild type. Consistent with these results, W442A and I445A M1-mAChR mutants selectively localized to the cell surface. By contrast, the W442Y receptor mutant was found only at intracellular sites. Our data indicate that the cellular distribution of M1-mAChR is governed by the C-terminal tryptophanbased motif, which mediates constitutive internalization.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Intracellular localization of M1 muscarinic acetylcholine receptor through clathrin-dependent constitutive internalization via a C-terminal tryptophan-based motif

Uwada¹,

Yoshiki²,

Masuoka³

et al. 2014

Journal of Cell Science

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“…In addition, a body of evidence implicates synaptotagmin as a regulator of endocytosis (14)(15)(16)(17). Caenorhabditis elegans lacking a functional SytI gene show a depletion of synaptic vesicles at nerve terminals, consistent with a defect in endocytosis (14).…”

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confidence: 99%

Kinetic efficiency of endocytosis at mammalian CNS synapses requires synaptotagmin I

Nicholson-Tomishima

Ryan

2004

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

135

111

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At nerve terminals, synaptic vesicle components are retrieved from the cell surface and recycled for local reuse soon after exocytosis. The kinetics of this coupling is critical for the proper functioning of synapses during repetitive action potential firing, because deficiencies in this process lead to abnormal depletion of the releasable vesicle pool. Although the molecular basis of this coupling is poorly understood, numerous biochemical data point to a role for synaptotagmin I (SytI), an essential synaptic vesicle protein required for fast calcium-dependent exocytosis. Here, using synapto-pHluorin in an approach that allows the dissection of endocytosis and exocytosis into separate components during periods of stimulation, we examined exocytic-endocytic coupling in synapses from SytI knockout mice and their WT littermates. We show that endocytosis is significantly impaired in the absence of SytI with the relative rates of endocytosis compared with exocytosis reduced Ϸ3-fold with respect to WT. Thus, in addition to regulating exocytosis, SytI also controls the kinetic efficiency of endocytosis at nerve terminals.

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“…Liposome studies suggest a direct fusogenic role for SytI, in which shallow insertion of the C 2 region into target membranes induces curvature to destabilize the lipid bilayer and form the fusion pore opening (1,3). Studies in mice, Drosophila, and C. elegans show that SytI also regulates the kinetics of endocytosis at nerve terminals, apparently in a clathrin-mediated manner (1,4).…”

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confidence: 99%

Arabidopsis synaptotagmin SYTA regulates endocytosis and virus movement protein cell-to-cell transport

Lewis

Lazarowitz

2010

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

214

233

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Synaptotagmins are calcium sensors that regulate synaptic vesicle exo/endocytosis. Thought to be exclusive to animals, they have recently been characterized in plants. We show that Arabidopsis synaptotagmin SYTA regulates endosome recycling and movement protein (MP)-mediated trafficking of plant virus genomes through plasmodesmata. SYTA localizes to endosomes in plant cells and directly binds the distinct Cabbage leaf curl virus (CaLCuV) and Tobacco mosaic virus (TMV) cell-to-cell movement proteins. In a SYTA knockdown line, CaLCuV systemic infection is delayed, and cell-to-cell spread of TMV and CaLCuV movement proteins is inhibited. A dominant-negative SYTA mutant causes depletion of plasma membrane-derived endosomes, produces large intracellular vesicles attached to plasma membrane, and inhibits cell-to-cell trafficking of TMV and CaLCuV movement proteins, when tested in an Agrobacterium-based leaf expression assay. Our studies show that SYTA regulates endocytosis, and suggest that distinct virus movement proteins transport their cargos to plasmodesmata for cell-tocell spread via an endocytic recycling pathway. S ynaptotagmins (Syts) are a large family of Ca 2+ /lipid binding proteins widely studied in animals due to their role in neurotransmitter release. They are also found in Drosophila and Caenorhabditis elegans and were recently described in plants (1, 2). Syts have a conserved domain structure: a short uncleaved N-terminal signal peptide that overlaps a transmembrane (TM) domain, followed by a cytosolic variable region and two C-terminal C 2 domains, C 2 A and C 2 B. Whereas C 2 A and C 2 B each bind phospholipids in a Ca 2+ -dependent manner, fold independently and act synergistically, C 2 B is essential for activity (1). SytI, the best studied Syt, is proposed to act as a Ca 2+ sensor to regulate rapid and synchronous synaptic vesicle exocytosis (1). Whether it regulates SNARE complex formation in a temporal and spatial manner, or is itself fusogenic, is unclear. Studies in PC12 cells, and of mouse and Drosophila sytI mutants, suggest that the SNARE complex VAMP1/SNAP25/syntaxin-1 targets the synaptic vesicle to the plasma membrane to create a metastable fusion intermediate. SytI on the vesicle membrane, and perhaps a distinct partner Syt on the plasma membrane, would then interact with phospholipids and the SNARE complex to accelerate SNARE-mediated fusion pore dilation. Liposome studies suggest a direct fusogenic role for SytI, in which shallow insertion of the C 2 region into target membranes induces curvature to destabilize the lipid bilayer and form the fusion pore opening (1, 3). Studies in mice, Drosophila, and C. elegans show that SytI also regulates the kinetics of endocytosis at nerve terminals, apparently in a clathrin-mediated manner (1, 4).Plant virus movement proteins (MPs) mediate the transport of progeny genomes across the cell wall for local and systemic infection. Despite diverse strategies for cell-to-cell movement, two common features have emerged: movement proteins alter plasmodes...

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Endocytosis of Synaptotagmin 1 Is Mediated by a Novel, Tryptophan‐Containing Motif

Cited by 28 publications

References 71 publications

Intracellular localization of M1 muscarinic acetylcholine receptor through clathrin-dependent constitutive internalization via a C-terminal tryptophan-based motif

Intracellular localization of M1 muscarinic acetylcholine receptor through clathrin-dependent constitutive internalization via a C-terminal tryptophan-based motif

Kinetic efficiency of endocytosis at mammalian CNS synapses requires synaptotagmin I

Arabidopsis synaptotagmin SYTA regulates endocytosis and virus movement protein cell-to-cell transport

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