Article |
Address correspondence to Regis B. Kelly, Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-0448. Tel.: (415) 476-4095. Fax: (415) 502-5145. E-mail: rkelly{at}biochem.ucsf.edu
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Abstract |
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Key Words: synaptotagmin; endocytosis; internalization signal; AP2 adaptor; synaptic vesicle
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Introduction |
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One additional family of proteins that participates in the endocytotic process are the synaptotagmins, a family of proteins primarily implicated in calcium-regulated exocytosis. Up to 13 synaptotagmin isoforms have been isolated (Matthew et al., 1981; Li et al., 1995; Babity et al., 1997; von Poser et al., 1997; von Poser and Sudhof, 2000; Fukuda and Mikoshiba, 2001). The best studied member of the family is synaptotagmin 1, a neuronal isoform present on the membranes of synaptic vesicles and secretory granules (Matthew et al., 1981). Genetic evidence in Drosophila, C. elegans, and mouse have implicated synaptotagmin 1 as an essential component of fast calcium-dependent neurotransmitter release (Littleton et al., 1993; Nonet et al., 1993; DiAntonio and Schwarz, 1994; Geppert et al., 1994; Littleton et al., 1994; Fernandez-Chacon et al., 2001; Littleton et al., 2001). Biochemical properties of synaptotagmin 1 support the idea that synaptotagmin 1 forms part of the calcium sensor in fast exocytosis. Synaptotagmins are integral membrane proteins that contain two calcium binding domains, C2A and C2B, in their large cytoplasmic region (Perin et al., 1991; Sudhof and Rizo, 1996). Calcium triggers binding of the C2A domain to phospholipids (Brose et al., 1992; Davletov and Sudhof, 1993; Chapman and Jahn, 1994; Chapman and Davis, 1998) as well as the clustering of synaptotagmin via the C2B domain (Chapman et al., 1996; Sugita et al., 1996). The two C2 domains cooperate to form complexes with components of the SNARE machinery (Li et al., 1995; Davis et al., 1999; Gerona et al., 2000), suggesting a model in which synaptotagmin triggers exocytosis through its interactions with membranes and the SNARE complex.
A role for synaptotagmin in endocytosis as well as exocytosis was first suggested by the high affinity, calcium-independent binding of the C2B domain to AP2 (Zhang et al., 1994), one of the most conserved properties among the synaptotagmin family (Li et al., 1995). Synaptotagmin 1 binds most tightly to a region of the µ chain of AP2 that is distinct from the AP2 region that binds tyrosine-based internalization signals (Owen and Evans, 1998; Haucke et al., 2000). Transfection experiments have confirmed that the synaptotagmins play a major role in receptor-mediated endocytosis. In one set of experiments, expression of the transmembrane domain of synaptotagmins reduced clathrin-coated pit formation and inhibited LDL uptake (von Poser et al., 2000). This effect was dependent on the presence of two cysteines involved in synaptotagmin oligomerization. In a second set, disrupting the C2BAP2 interactions in vivo by expressing the synprint region of calcium channels inhibited transferrin receptor internalization (Haucke et al., 2000). Moreover, interaction among cargo proteins, AP2, and synaptotagmin appears to be cooperative, since the presence of a peptide containing a tyrosine-based internalization signal enhanced AP2 binding to synaptotagmin in vitro (Haucke and De Camilli, 1999). Therefore, synaptotagmins play a critical role in nucleating a coated pitcontaining cargo, AP2, and clathrin, even in nonneuronal cells.
The brain-specific synaptotagmin 1 member of the synaptotagmin family appears to exhibit tissue-specific endocytosis, a relatively rare phenomenon. Although synaptotagmin 1 is readily internalized by the neuronal-like PC12 cells, in transfected fibroblasts it was found mostly on the cell surface (Feany et al., 1993). This suggested that cells of the neuroendocrine lineage possess an endocytotic mechanism lacking in nonneuronal cells. To try to understand this mechanism, we have examined the internalization of synaptotagmin 1 and its chimeras into neuronal and nonneuronal cell lines. Surprisingly, the AP2 binding site in the C2B domain is not needed for internalization of chimeras. By contrast, we find evidence for an internalization signal in the COOH-terminal domain of synaptotagmin 1. The internalization signal is sufficient to mediate internalization of chimeras in the absence of the C2B domain, and can be recognized by the endocytotic machinery even in nonneuronal cells. The C2B domain acts as a regulator of internalization since in fibroblast cells it normally conceals the synaptotagmin 1 internalization signal. In PC12 cells, little inhibition of the COOH-terminal internalization signal by the C2B domain is seen unless mutations are made in the C2B domain, in particular in a dilysine motif that has already been implicated in endocytosis by the synprint experiments of Haucke and De Camilli (1999). Endocytosis of synaptotagmin 1 appears to be controlled by a latent internalization signal that is activated by a neuroendocrine-specific mechanism acting via the C2B domain. Thus, synaptotagmin 1 internalization resembles ligand-activated endocytosis of surface receptors, except that the activation is intracellular.
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Results |
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We then examined whether the motifs that conformed to known internalization signals in the COOH-terminal domain were responsible for synaptotagmin 1 internalization. At present, two major groups of internalization motifs have been described (Mellman, 1996; Kirchhausen et al., 1997), tyrosine-based motifs and dileucinebased motifs in which one of the leucines can be substituted by isoleucine, methionine, or valine (Bremnes et al., 1994). The COOH-terminal domain in our CD4-CT is composed of 29 amino acids, starting with an isoleucineleucine motif (synaptotagmin 1 residues 393,394; Fig. 4 a). In a study of synaptic vesicle targeting signals (Blagoveshchenskaya et al., 1999), internalization of a CD4-CT construct lacking the isoleucineleucine motif was observed, showing that the motif is unlikely to be part of the internalization signal. The COOH-terminal domain contains a methioneleucine (ML) motif (synaptotagmin 1 residues 416,417) that was shown to mediate part of the synaptotagmin 1 targeting to synaptic-like microvesicles (Blagoveshchenskaya et al., 1999). The authors reported that this motif was not acting at the step of synaptotagmin 1 internalization. The ML motif is preceded by a cluster of acidic residues. Acidic residues have been shown to be involved in targeting events, either by themselves or in combination with tyrosine or dileucine-based motifs (Matter et al., 1994; Pond et al., 1995; Voorhees et al., 1995; Piguet et al., 2000). To assess the function of these putative internalization signals, we substituted them for alanine residues in our CD4-C2AB-CT construct. Mutation of one (D414A) or three (E410,411,412A) of the acidic residues did not affect internalization of the CD4-C2AB-CT construct (Fig. 4 b). Simultaneous mutation of M416A, L417A also had no effect on internalization of the CD4-C2AB-CT construct, thus confirming that the synaptic vesicle targeting signal was not affecting synaptotagmin 1 internalization. These results suggest the existence of an unconventional internalization signal in the COOH-terminal region of synaptotagmin 1.
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Discussion |
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Our data suggest that the tissue specificity of synaptotagmin 1 endocytosis is due to a regulatory mechanism that is only activated in PC12 cells. Although synaptotagmins bind AP2 (Zhang et al., 1994; Li et al., 1995), AP2 by itself cannot explain the neuroendocrine specificity that we observe. Indeed, AP2 is present in the cytosols of both fibroblasts and PC12 cells and can bind to immobilized synaptotagmin tails (Fig. 7). Moreover, the AP2 binding site does not act as an internalization motif since its deletion did not inhibit internalization in PC12 cells (Fig. 3 b). Instead, the internalization signal was found in the COOH-terminal region of the protein (Fig. 3). In absence of the C2B domain, the COOH-terminus internalization signal could promote endocytosis in both PC12 and CHO cells (Figs. 3 b and 5 b). Thus, the tissue specificity of synaptotagmin 1 internalization is not due to a neuron-specific internalization motif, but to differences in the accessibility of the internalization signal. This accessibility appears to be controlled by the C2B domain, which totally inhibits internalization in CHO cells (Fig. 5 a) and also in PC12 cells when the oligomerization domain is inactivated by the K326,327A mutation (Fig. 6). Internalization in PC12 cells is also influenced, albeit more weakly, by the calcium binding region of the C2B domain. A mutation that inhibited calcium-mediated conformational changes (Y311N) reduced endocytosis, while one that partially neutralizes the charge (D363,365N) enhanced it (Fig. 6 a). The D363,365N was reported to cause the C2B domain to dimerize as if it had calcium bound (Desai et al., 2000).
C2 domains are usually thought to regulate membrane traffic by their ability to sense changes in intracellular environment, often with calcium levels, then trigger association with membranes. Synaptic vesicle recycling has been shown to be crucially dependent on the presence of synaptotagmin (Jorgensen et al., 1995), particularly its C2B domain (Fukuda et al., 1995b; Littleton et al., 2001). We propose that the C2B domain acts as a sensor of intracellular changes in neurons or neuroendocrine cells and couples these changes to endocytosis by activating a novel internalization signal. A plausible change that the C2B domain might sense is that a calcium-mediated exocytotic event has occurred. The C2B domain could detect such an event in several ways. The K326,327 motif has been implicated in calcium-dependent dimerization of synaptotagmin 1 (Chapman et al., 1998), which in turn is thought to be required for exocytosis (Desai et al., 2000). If calcium-mediated dimerization were also to expose the COOH-terminal internalization domain, then endocytosis would be elegantly coupled to exocytosis. Calcium-mediated dimerization of synaptotagmin 1 cannot fully explain the regulation of its endocytosis, however, since the D363,365N mutation, which gives constitutive dimerization (Desai et al., 2000), did not restore internalization in CHO cells (data not shown). An alternative to calcium-mediated dimerization activating internalization of synaptotagmin 1 is binding via its dilysine motif to calcium channels (Chapman et al., 1998), to phosphorylated phosphatidyl inositides (Fukuda et al., 1995a), or to an unknown protein on the plasma membrane. If any such interaction activates the internalization signal, synaptotagmin 1 would only bind coats after its arrival at the cell surface, not while a component of an intracellular organelle. Since AP2 also binds to the dilysine motif, an unattractive feature of these conjectures is that they require the absence of AP2 in order to activate a coating step. AP2 binding could still play a significant role if the neuroendocrine-specific factor activated endocytosis when it interacted with AP2 bound to the C2B domain. Candidates for such AP2 binding factors abound and include AP180 and the nerve-specific forms of endophilin, syndapin, dynamin, and intersectin. Several of these factors have been shown to cluster at "hot spots" on the plasma membrane of nerve terminals (Estes et al., 1996; Gonzalez-Gaitan and Jackle, 1997; Roos and Kelly, 1998) that are sometimes adjacent to sites of exocytosis (Roos and Kelly, 1999). If they released an inhibition, this would provide a mechanism for restricting synaptotagmin 1 internalization to these specialized endocytotic zones. However, in preliminary experiments, cotransfection of CHO cells with synaptotagmin and AP180 or dynamin 1 has not activated the internalization of synaptotagmin (data not shown). However, an AP2 binding protein need not be cytoplasmic. A complex of AP2 with a synaptic vesicle cargo protein could trigger internalization of synaptotagmin 1.
An earlier study suggested that oligomerization of synaptotagmin via its transmembrane domain plays a role in formation of clathrin-coated pits (von Poser et al., 2000). Since our first series of constructs (Fig. 2) lacked this domain, we generated a second series of chimeras (Fig. 8 a) to verify that our observations were still true in the presence of the transmembrane domain of synaptotagmin 1. Our results clearly show that the COOH-terminal region acts as an internalization signal even in presence of the transmembrane domain, and that the K326,327A mutation abolishes internalization. A puzzling observation, though, is that the tailess construct containing the synaptotagmin transmembrane domain was internalized to a higher extent than the one containing the CD4 transmembrane region (CD4-TM tailless and CD4 tailless, respectively; Fig. 8 b). A possible mechanism is that the synaptotagmin transmembrane region forms oligomers (von Poser et al., 2000) with the endogenous synaptotagmin in PC12 cells, thus resulting in internalization of the CD4-TM tailless via a piggy back mechanism. Alternatively, the transmembrane and/or the short proximal cytoplasmic domain (to residue 94) may contain an additional weak internalization signal. Both transmembrane and COOH-terminal signals are inhibited by the K326,327A mutation (Fig. 8 b).
Important questions remain about the role of the synaptotagmins in nonneuronal cells. The COOH-terminal internalization signal of synaptotagmin 1 is recognized in fibroblasts by a machinery that cannot be cell typespecific. A peptide that blocks the binding of AP2 to the C2B domain of an endogenous synaptotagmin inhibited transferrin uptake in fibroblasts (Haucke et al., 2000). One possible interpretation is that the peptide used in this study inhibited an activating region required for exposure of an internalization signal. The synaptotagmins are now known to regulate exocytosis in neuronal cells as well as nonneuronal ones (Martinez et al., 2000). Our data hint that they might also be widespread regulators of endocytosis.
Our analysis reveals an unexpected complexity in synaptotagmin 1 internalization, which is controlled by a mixture of internalization and regulatory signals to give tissue-specific internalization. Another example of tissue-specific internalization was reported for a specific isoform of the Fc receptor family, FcRII-B1. In this case, a B cellspecific in frame insertion into an internalization signal prevented the lymphocytes from executing FcR-dependent antigen presentation (Miettinen et al., 1989; Amigorena et al., 1992). Exposure of latent endocytotic signals has been reported for receptor tyrosine kinases or G proteincoupled receptors that undergo ligand-stimulated endocytosis (Ferguson, 2001). The presence of a latent signal in synaptotagmin 1 and the involvement of the synaptotagmins in both compensatory endocytosis in the nerve terminals (Jorgensen et al., 1995) and constitutive endocytosis in fibroblasts (Haucke et al., 2000; von Poser et al., 2000) implies that these other forms of endocytosis might also be regulated, perhaps via the C2B domain of synaptotagmins. In contrast to ligand-stimulated endcoytosis, however, the signals controlling endocytosis rates are intracellular.
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Materials and methods |
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Reagents and antibodies
The cDNA for rat synaptotagmin 1 was provided by Dr. Kathleen M. Buckley (Harvard Medical School, Boston, MA). The pGEXsynaptotagmin 1 plasmid encoding the GST protein fused to the cytoplasmic region of rat synaptotagmin 1 was a gift from Dr. Thomas C. Südhof (University of Texas Southwestern Medical Center, Dallas, TX). The pcDNA3-CD4 and pBMN-Z-I Neo plasmids were provided by Dr. Don Ganem (University of California at San Francisco, San Francisco, CA).
Monoclonal antibody against the lumenal domain of synaptotagmin 1 (clone 604.1) was provided by Dr. R. Jahn (Goettingen, Germany). Monoclonal antibody against the external domain of the human CD4 (clone Q4120) was obtained from the Medical Research Council AIDS Reagents Program (National Institute for Biological Standards and Control). Anti-adaptin antibody was purchased from Transduction Laboratories.
Constructs
The rat synaptotagmin 1 ORF was subcloned in the pcDNA3hygro vector (Invitrogen).
For the CD4-synaptotagmin constructs, a CD4 fragment (corresponding to residues 1426) encoding the lumenal, transmembrane, and 12 amino acids of the cytoplasmic region of the human CD4 was amplified by PCR from pcDNA3-CD4. The primers were chosen so that a SmaI restriction site followed by a stop codon was added to the 3' end of the CD4 fragment. The PCR product was subcloned in the pCR3.1 vector (T/A cloning kit; Stratagene), resulting in the pCR3.1-CD4 tailless plasmid. The cytoplasmic domains of synaptotagmin 1 were amplified by PCR from the pGEXsynaptotagmin 1 plasmid to generate the following fragments: C2AB-CT (encoding for residues 95421), C2AB (residues 95392), C2B-CT (residues 266421), and CT (residues 393421). The forward primers were flanked with a SmaI restriction site. The PCR products were subcloned in the pCR3.1 vector. The synaptotagmin 1encoding regions were then cut out as SmaI-XhoI fragments and ligated to the corresponding sites of the pCR3.1-CD4 tailless to generate in-frame CD4/synaptotagmin 1 chimeras. The resultant constructs were sequenced and the CD4/synaptotagmin 1 ORFs were introduced as BamHI-XhoI fragments in the pBMN-Z-I-Neoderived vector. The K326,327A mutants were generated the same way, except that the synaptotagmin 1 fragments were amplified from pGEXsynaptotagmin 1-KK/AA plasmid, in which the K326,327A had been introduced using a QuikChange site-directed mutagenesis kit (Stratagene). The other point mutations in the CD4-C2AB-CT and CD4-C2B-CT constructs were generated by PCR using standard procedures. A synaptotagmin 1 fragment containing the transmembrane and proximal cytoplasmic domain (corresponding to amino acids 5394) was amplified by PCR from the pcDNA3synaptotagmin 1 vector and fused in frame by PCR to the lumenal domain of the CD4 molecule (CD4 residues 1394). The resulting chimeric fragment was flanked with a BamH1 and a SmaI site at the 5' and 3' ends, respectively. The PCR product was subcloned as a BamH1-SmaI fragment in the PCR3.1 plasmids either upstream of a stop codon to generate the CD4-TM tailless construct (synaptotagmin 1 residues 5394), or upstream of the synaptotagmin 1 cytoplasmic domains to give the following constructs: CD4-TM-C2AB-CT (synaptotagmin 1 residues 53421), CD4-TM-C2AB (synaptotagmin 1 residues 53392) and CD4-TM-CT (synaptotagmin 1 residues 5394 and 393421). The SmaI restriction site used to generate the TM-containing constructs resulted in an insertion of two amino acids (P, G) between amino acid 94 and 95 of synaptotagmin 1. The constructs were sequenced and a subsequent subcloning in the pBMN-Z-I-Neoderived vector was done as described above.
Transfections and retroviral infections
CHO and HEK cells were stably transfected with the pcDNA3synaptotagmin 1 vector using the Fugene-6 product according to the manufacturer's instructions (Boehringer). Cells were selected in the presence of 400 µg/ml of hygromycin.
In initial experiments we tried to use transient transfectants to identify sorting motifs. However, FACS analysis revealed an extremely high variability in the level of expression from cell to cell. Since our assays measure the behavior of the transfected protein, they are heavily biased towards cells with high expression levels. Since we were concerned about saturation of sorting pathways in overexpressing cells, we chose instead the route of retroviral infection that gives rise to moderate and homogenous level of expression. For retroviral infections, we used a vector derived from pBMN-Z-I-Neo in which the LacZ gene was deleted (fragments BamH1-SalI) and replaced by our genes of interest (fragment BamHI-XhoI containing the CD4/synaptotagmin 1 ORFs). The vector contains the internal ribosome entry site of the encephalomyocarditis virus upstream of the neomycin resistance gene. This permits both the gene of interest and the neomycin resistance gene to be translated from a single bicistronic mRNA, resulting in nearly all surviving colonies stably expressing the gene of interest after selection with G418. Expression of the bicistronic mRNA is controlled by the 5' viral LTR promoter (Full length Moloney LTR). On transfection of the vectors, the Phoenix-packaging cell line produces replication-defective viral particles that were used for stable gene transfer and expression in PC12 and CHO cells. The Phoenix cells were transfected with the different CD4synaptotagmin 1 constructs using the Fugene-6 product. Virus-containing supernatants were filtered through a low binding protein 0.45-µm filter (Pall Corporation) 48 h posttransfection, diluted with 4 µg/ml of hexadimethrine bromide (Sigma-Aldrich tailess), and used to infect PC12 or CHO cells. After 12 h, the inoculum was replaced by fresh media; 2436 h later, 400 µg/ml of G418 was added. 10 d after infection, colonies were pooled and propagated in culture in the presence of 400 µg/ml of G418. Cells were treated for 20 h before the experiments with 500 nM of trichostatin A to enhance expression of the constructs.
Flow cytometry analysis
For internalization assays, cells were detached by using enzyme-free/PBS-based cell dissociation buffer (GIBCO BRL) according to the manufacturer's instructions. Cells were incubated with 2 µg/ml of 604.1 antibody in a saline solution (119 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM Hepes, pH 7.4, and 30 mM glucose) supplemented with 3% BSA for 60 min at 4°C. Cells were washed, incubated at 37°C for different periods of time, and returned to 4°C to stop endocytosis. The 604.1 antibody remaining at the surface was revealed by incubation at 4°C with a fluorescein-conjugated antimouse antibody. Cells were analyzed with a Becton Dickinson FACScalibur. The data were collected in a logarithmic mode, and the geometric mean of fluorescence intensity was calculated.
To measure the expression level of synaptotagmin 1, cells were dissociated in enzyme-free buffer and fixed in 4% paraformaldehyde in PBS. Antibody incubations and washings were performed in PBS 1% BSA in the presence of 0.02% saponin (Sigma-Aldrich). Cells were washed and stained with 604.1 antibody. After washing, bound antibody was visualized by addition of phycoerythrin-conjugated goat antimouse antibody. Cells incubated with the secondary antibody alone served as negative controls. Cells were analyzed by flow cytometry as mentioned above.
Internalization assays using iodinated antibodies
100 µg of 604.1 or 50 µg of Q4120 antibody was iodinated on iodogen-coated tubes (Pierce Chemical Co.) as described (Clift-O'Grady et al., 1998). Cells were plated on collagen and poly-D-lysinecoated 12-well plates 2 d before the assay. Cells were incubated for 1 h at 4°C with 2 µg/ml of 125I -604.1 antibody or 100 ng/ml of 125I-Q4120 in DMEH-21 media supplemented with 1% BSA and 10 mM Hepes, pH 7.4. Unbound antibody was removed by extensive washes. Cells were next incubated at 37°C to allow endocytosis and then returned to 4°C. Antibody remaining at the cell surface was removed by two 10-min washes at 4°C in PBS/BSA supplemented with 30 mM glycine and adjusted to pH 2.4. Acid-resistant antibody was collected by lysing the cells in 2 M NaOH. The fraction of antibody internalized was calculated by dividing the acid washresistant radioactive cpm by the sum of acid washresistant and accessible cpm. A background of acid-resistant counts in cells kept at 4°C was subtracted from each value.
GST fusion protein pull down assay
The pGEXsynaptotagmin 1 plasmid was used to express the fusion protein according to standard methods. GST control protein was generated by expression of the plasmid pGEX-3X (Amersham Pharmacia Biotech). GST fusion proteins were bound to glutathione-agarose chromatography and subjected to an extensive wash with intracellular buffer (38 mM potassium aspartate, 20 mM potassium MOPS, pH 7.2, 5 mM reduced glutathione, 5 mM sodium carbonate, 2.5 mM magnesium sulfate). The washed beads were used in a standard GST fusion protein binding assay. In brief, PC12 or CHO cell extracts were obtained by lysis with 1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, in the presence of proteases inhibitors. 40 µl of saturated beads were incubated with 500 µg of cell extracts overnight at 4°C. Beads were washed extensively in the Triton X-100containing buffer and eluted with 20 mM glutathione in 120 mM NaCl, 50 mM Tris, pH 8, for 30 min at room temperature. Proteins were then separated by SDS-PAGE and assayed by Western blotting with an anti-adaptin antibody used at 1:1,000.
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Footnotes |
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Acknowledgments |
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This work was funded by the Albert Bowers chair in Biochemistry, the National Institutes of Health grants NS09878 and DA10154 (to Dr. R.B. Kelly), and by Institut Pasteur, Association pour la Recherche sur le Cancer, National Alliance for Research on Schizophrenia and Depression, and Philippe Foundation (to Dr. N. Jarousse).
Submitted: 9 March 2001
Revised: 11 July 2001
Accepted: 13 July 2001
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References |
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Amigorena, S., C. Bonnerot, J.R. Drake, D. Choquet, W. Hunziker, J.G. Guillet, P. Webster, C. Sautes, I. Mellman, and W.H. Fridman. 1992. Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes. Science. 256:18081812.[Medline]
Babity, J.M., J.N. Armstrong, J.C. Plumier, R.W. Currie, and H.A. Robertson. 1997. A novel seizure-induced synaptotagmin gene identified by differential display. Proc. Natl. Acad. Sci. USA. 94:26382641.
Blagoveshchenskaya, A.D., E.W. Hewitt, and D.F. Cutler. 1999. Di-leucine signals mediate targeting of tyrosinase and synaptotagmin to synaptic-like microvesicles within PC12 cells. Mol. Biol. Cell. 10:39793990.
Bremnes, B., T. Madsen, M. Gedde-Dahl, and O. Bakke. 1994. An LI and ML motif in the cytoplasmic tail of the MHC-associated invariant chain mediate rapid internalization. J. Cell Sci. 107:20212032.
Brose, N., A.G. Petrenko, T.C. Sudhof, and R. Jahn. 1992. Synaptotagmin: a calcium sensor on the synaptic vesicle surface. Science. 256:10211025.[Medline]
Chapman, E.R., and A.F. Davis. 1998. Direct interaction of a Ca2+-binding loop of synaptotagmin with lipid bilayers. J. Biol. Chem. 273:1399514001.
Chapman, E.R., and R. Jahn. 1994. Calcium-dependent interaction of the cytoplasmic region of synaptotagmin with membranes. Autonomous function of a single C2-homologous domain. J. Biol. Chem. 269:57355741.
Chapman, E.R., S. An, J.M. Edwardson, and R. Jahn. 1996. A novel function for the second C2 domain of synaptotagmin. Ca2+-triggered dimerization. J. Biol. Chem. 271:58445849.
Chapman, E.R., R.C. Desai, A.F. Davis, and C.K. Tornehl. 1998. Delineation of the oligomerization, AP-2 binding, and synprint binding region of the C2B domain of synaptotagmin. J. Biol. Chem. 273:3296632972.
Clift-O'Grady, L., C. Desnos, Y. Lichtenstein, V. Faundez, J.T. Horng, and R.B. Kelly. 1998. Reconstitution of synaptic vesicle biogenesis from PC12 cell membranes. Methods. 16:150159.[Medline]
Davis, A.F., J. Bai, D. Fasshauer, M.J. Wolowick, J.L. Lewis, and E.R. Chapman. 1999. Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes. Neuron. 24:363376 (erratum published 24:1049).
Davletov, B.A., and T.C. Sudhof. 1993. A single C2 domain from synaptotagmin I is sufficient for high affinity Ca2+/phospholipid binding. J. Biol. Chem. 268:2638626390.
Desai, R.C., B. Vyas, C.A. Earles, J.T. Littleton, J.A. Kowalchyck, T.F. Martin, and E.R. Chapman. 2000. The C2B domain of synaptotagmin is a Ca2+-sensing module essential for exocytosis. J. Cell Biol. 150:11251136.
DiAntonio, A., and T.L. Schwarz. 1994. The effect on synaptic physiology of synaptotagmin mutations in Drosophila. Neuron. 12:909920.[Medline]
Estes, P.S., J. Roos, A. van der Bliek, R.B. Kelly, K.S. Krishnan, and M. Ramaswami. 1996. Traffic of dynamin within individual Drosophila synaptic boutons relative to compartment-specific markers. J. Neurosci. 16:54435456.
Feany, M.B., A.G. Yee, M.L. Delvy, and K.M. Buckley. 1993. The synaptic vesicle proteins SV2, synaptotagmin and synaptophysin are sorted to separate cellular compartments in CHO fibroblasts. J. Cell Biol. 123:575584.[Abstract]
Ferguson, S.S. 2001. Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol. Rev. 53:124.
Fernandez-Chacon, R., A. Konigstorfer, S.H. Gerber, J. Garcia, M.F. Matos, C.F. Stevens, N. Brose, J. Rizo, C. Rosenmund, T.C. Sudhof. 2001. Synaptotagmin I functions as a calcium regulator of release probability. Nature. 410:4149.[Medline]
Fukuda, M., and K. Mikoshiba. 2001. Characterization of KIAA1427 protein as an atypical synaptotagmin (Syt XIII). Biochem. J. 354:249257.[Medline]
Fukuda, M., T. Kojima, J. Aruga, M. Niinobe, and K. Mikoshiba. 1995a. Functional diversity of C2 domains of synaptotagmin family. Mutational analysis of inositol high polyphosphate binding domain. J. Biol. Chem. 270:2652326527.
Fukuda, M., J.E. Moreira, F.M. Lewis, M. Sugimori, M. Niinobe, K. Mikoshiba, and R. Llinas. 1995b. Role of the C2B domain of synaptotagmin in vesicular release and recycling as determined by specific antibody injection into the squid giant synapse preterminal. Proc. Natl. Acad. Sci. USA. 92:1070810712.[Abstract]
Geppert, M., Y. Goda, R.E. Hammer, C. Li, T.W. Rosahl, C.F. Stevens, and T.C. Sudhof. 1994. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell. 79:717727.[Medline]
Gerona, R.R., E.C. Larsen, J.A. Kowalchyk, and T.F. Martin. 2000. The C terminus of SNAP25 is essential for Ca2+-dependent binding of synaptotagmin to SNARE complexes. J. Biol. Chem. 275:63286336.
Gonzalez-Gaitan, M., and H. Jackle. 1997. Role of Drosophila -adaptin in presynaptic vesicle recycling. Cell. 88:767776.[Medline]
Grote, E., and R.B. Kelly. 1996. Endocytosis of VAMP is facilitated by a synaptic vesicle targeting signal. J. Cell Biol. 132:537547.[Abstract]
Haucke, V., and P. De Camilli. 1999. AP-2 recruitment to synaptotagmin stimulated by tyrosine-based endocytic motifs. Science. 285:12681271.
Haucke, V., M.R. Wenk, E.R. Chapman, K. Farsad, and P. De Camilli. 2000. Dual interaction of synaptotagmin with mu2- and -adaptin facilitates clathrin-coated pit nucleation. EMBO J. 19:60116019.
Jarousse, N., and R.B. Kelly. 2001. Endocytotic mechanisms in synapses. Curr. Opin. Cell Biol. In press.
Jorgensen, E.M., E. Hartwieg, K. Schuske, M.L. Nonet, Y. Jin, and H.R. Horvitz. 1995. Defective recycling of synaptic vesicles in synaptotagmin mutants of Caenorhabditis elegans. Nature. 378:196199.[Medline]
Kirchhausen, T. 1999. Adaptors for clathrin-mediated traffic. Annu. Rev. Cell Dev. Biol. 15:705732.[Medline]
Kirchhausen, T., J.S. Bonifacino, and H. Riezman. 1997. Linking cargo to vesicle formation: receptor tail interactions with coat proteins. Curr. Opin. Cell Biol. 9:488495.[Medline]
Li, C., B. Ullrich, J.Z. Zhang, R.G. Anderson, N. Brose, and T.C. Sudhof. 1995. Ca2+-dependent and -independent activities of neural and non-neural synaptotagmins. Nature. 375:594599.[Medline]
Linstedt, A.D., and R.B. Kelly. 1991. Synaptophysin is sorted from endocytotic markers in neuroendocrine PC12 cells but not transfected fibroblasts. Neuron. 7:309317.[Medline]
Littleton, J.T., M. Stern, K. Schulze, M. Perin, and H.J. Bellen. 1993. Mutational analysis of Drosophila synaptotagmin demonstrates its essential role in Ca2+-activated neurotransmitter release. Cell. 74:11251134.[Medline]
Littleton, J.T., M. Stern, M. Perin, and H.J. Bellen. 1994. Calcium dependence of neurotransmitter release and rate of spontaneous vesicle fusions are altered in Drosophila synaptotagmin mutants. Proc. Natl. Acad. Sci. USA. 91:1088810892.
Littleton, J.T., J. Bai, B. Vyas, R. Desai, A.E. Baltus, M.B. Garment, S.D. Carlson, B. Ganetzky, and E.R. Chapman. 2001. Synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo. J. Neurosci. 21:14211433.
Martinez, I., S. Chakrabarti, T. Hellevik, J. Morehead, K. Fowler, and N.W. Andrews. 2000. Synaptotagmin VII regulates Ca2+-dependent exocytosis of lysosomes in fibroblasts. J. Cell Biol. 148:11411149.
Matter, K., E.M. Yamamoto, and I. Mellman. 1994. Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells. J. Cell Biol. 126:9911004.[Abstract]
Matthew, W.D., L. Tsavaler, and L.F. Reichardt. 1981. Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue. J. Cell Biol. 91:257269.[Abstract]
Mellman, I. 1996. Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol. 12:575625.[Medline]
Miettinen, H.M., J.K. Rose, and I. Mellman. 1989. Fc receptor isoforms exhibit distinct abilities for coated pit localization as a result of cytoplasmic domain heterogeneity. Cell. 58:317327.[Medline]
Nonet, M.L., K. Grundahl, B.J. Meyer, and J.B. Rand. 1993. Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin. Cell. 73:12911305.[Medline]
Owen, D.J., and P.R. Evans. 1998. A structural explanation for the recognition of tyrosine-based endocytotic signals. Science. 282:13271332.
Pelchen-Matthews, A., J.E. Armes, G. Griffiths, and M. Marsh. 1991. Differential endocytosis of CD4 in lymphocytic and nonlymphocytic cells. J. Exp. Med. 173:575587.[Abstract]
Perin, M.S., N. Brose, R. Jahn, and T.C. Sudhof. 1991. Domain structure of synaptotagmin (p65) J. Biol. Chem. 266:623629 (erratum published 266:10018).
Piguet, V., L. Wan, C. Borel, A. Mangasarian, N. Demaurex, G. Thomas, and D. Trono. 2000. HIV-1 Nef protein binds to the cellular protein PACS-1 to downregulate class I major histocompatibility complexes. Nat. Cell Biol. 2:163167.[Medline]
Pond, L., L.A. Kuhn, L. Teyton, M.P. Schutze, J.A. Tainer, M.R. Jackson, and P.A. Peterson. 1995. A role for acidic residues in di-leucine motif-based targeting to the endocytic pathway. J. Biol. Chem. 270:1998919997.
Roos, J., and R.B. Kelly. 1998. Dap160, a neural-specific Eps15 homology and multiple SH3 domain-containing protein that interacts with Drosophila dynamin. J. Biol. Chem. 273:1910819119.
Roos, J., and R.B. Kelly. 1999. The endocytic machinery in nerve terminals surrounds sites of exocytosis. Curr. Biol. 9:14111414.[Medline]
Sudhof, T.C., and J. Rizo. 1996. Synaptotagmins: C2-domain proteins that regulate membrane traffic. Neuron. 17:379388.[Medline]
Sugita, S., Y. Hata, and T.C. Sudhof. 1996. Distinct Ca2+-dependent properties of the first and second C2-domains of synaptotagmin I. J. Biol. Chem. 271:12621265.
von Poser, C., and T.C. Sudhof. 2000. Synaptotagmin 13: structure and expression of a novel synaptotagmin. Eur. J. Cell Biol. 80:4147.
von Poser, C., K. Ichtchenko, X. Shao, J. Rizo, and T.C. Sudhof. 1997. The evolutionary pressure to inactivate. A subclass of synaptotagmins with an amino acid substitution that abolishes Ca2+ binding. J. Biol. Chem. 272:1431414319.
von Poser, C., J.Z. Zhang, C. Mineo, W. Ding, Y. Ying, T.C. Sudhof, and R.G. Anderson. 2000. Synaptotagmin regulation of coated pit assembly. J. Biol. Chem. 275:3091630924.
Voorhees, P., E. Deignan, E. van Donselaar, J. Humphrey, M.S. Marks, P.J. Peters, and J.S. Bonifacino. 1995. An acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-Golgi network localization and internalization from the cell surface. EMBO J. 14:49614975.[Abstract]
Zhang, J.Z., B.A. Davletov, T.C. Sudhof, and R.G. Anderson. 1994. Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling. Cell. 78:751760.[Medline]