Mints, Munc18-interacting Proteins in Synaptic Vesicle Exocytosis*

Munc18-1 is a neuronal protein that interacts with syntaxin 1 and is required for synaptic vesicle exocytosis. We have now identified two Munc18-1-interacting proteins called Mint1 and Mint2 that may mediate the function of Munc18-1. Mint proteins are detectable only in brain and are composed of an N-terminal sequence that binds Munc18-1, a middle phosphotyrosine-binding domain, and two C-terminal PDZ domains thought to attach proteins to the plasma membrane. In brain, Mint proteins are part of a multimeric complex containing Munc18-1 and syntaxin that likely functions as an intermediate in synaptic vesicle docking/fusion. The phosphotyrosine-binding domain specifically binds to phosphatidylinositol phosphates known to be produced during vesicle exocytosis (Hay, J. C., Fisette, P. L., Jenkins, G. H., Fukami, K., Takonawa, T., Anderson, R. A., and Martin, T. F. J. (1995) Nature 374, 173–177). Our data suggest a model whereby local production of phosphatidylinositol phosphates may trigger the binding of vesicles to the active zone via the Mint·Munc18-1 complex in conjunction with syntaxin 1.

Exocytosis is a universal process in eukaryotes with many functions. At the synapse, exocytosis of synaptic vesicles releases neurotransmitters and constitutes the first step in synaptic transmission. Synaptic vesicle exocytosis starts with the docking of the vesicles at the active zone. Docked vesicles are then primed for Ca 2ϩ in a complex reaction that may involve partial fusion of the vesicles. Finally, Ca 2ϩ rapidly triggers the release of neurotransmitters with a latency of a few hundred microseconds. Although several proteins that function in synaptic vesicle exocytosis have been identified, the mechanisms of docking and fusion are still incompletely understood (for reviews, see Refs. [1][2][3][4]. Munc18-1 is a 65-kDa protein originally identified as the major brain protein that binds to syntaxin 1, a synaptic vesicle fusion protein (5). Munc18-1 belongs to a family of membrane trafficking proteins related to the yeast sec1, sly1, and slp1 genes. Mammals express three highly homologous isoforms of Munc18. Munc18-1 is enriched in neurons, whereas Munc18-2 and Munc18-3 are expressed ubiquitously (alternative names: Munc18-1 ϭ rbsec1 and nsec1, Munc18-2 ϭ Munc18b, and Munc18-3 ϭ Munc18c) (5)(6)(7)(8)(9). Knockouts of Munc18-1 demonstrated that it is essential for synaptic vesicle exocytosis. 1 In addition to binding strongly to syntaxin 1, Munc18-1 binds to Doc2a and Doc2b, C 2 domain proteins that are associated peripherally with synaptic vesicles (10). Since the interactions of Munc18-1 seem insufficient to explain its function, we have now searched for additional binding proteins for Munc18-1. We have identified a family of Munc18-interacting proteins named Mint proteins with unusual properties, suggesting a new model for the docking and priming reactions that initiate exocytosis.

MATERIALS AND METHODS
Yeast Two-hybrid Screens-We screened a cDNA library constructed from poly(A) ϩ -enriched rat brain RNA from postnatal day 8 with a bait vector encoding full-length Munc18-1 fused to LexA (pBTMMunc18-1-1) (11,12). Of Ͼ1000 positive clones obtained from 2.8 ϫ 10 8 yeast colonies, 100 clones were rescued, retransformed into fresh L40 yeast cells, and confirmed by growth on plates lacking histidine and by activation of ␤-galactosidase. 55 of the 100 clones were then further analyzed by cotransformation with an irrelevant bait and by sequencing. 5 clones were lost, 10 clones exhibited only weak activation of ␤-galactosidase and were not analyzed further, 22 clones encoded Mint1 and 7 clones encoded Mint2 (see Fig. 1; several clones were isolated multiple times independently), 3 clones encoded PSD95, 2 clones encoded syntaxin 5, 2 clones encoded a kinesin-related transcript, and 4 clones were not identified in the data banks.
cDNA Cloning, Sequencing, and Sequence Analysis-Rat brain cDNA libraries in ZAPII (Stratagene) were screened with randomprimed DNA probes as described (13). The following human Mint cDNAs were obtained as expressed sequence tag clones from Research Genetics (Huntsville, AL): human Mint1, 279624; and human Mint2, 183680, 328119, 188448, and 139567. DNA sequencing was performed by the dideoxy nucleotide chain termination method using fluorescently labeled primers and an ABI370A DNA sequencer. The nucleotide sequences of the cDNA clones were deposited in the GenBank™/EMBL Data Bank.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF029105-AF029108.
‡ Supported by postdoctoral fellowships from the HFSP, the TOYOBO Biotechnology Foundation, and the Takeda Medical Foundation.
Affinity Chromatography on Immobilized GST Fusion Proteins-Six frozen rat brains were homogenized in 15 ml of 10 mM HEPES-NaOH, pH 7.4, and 100 mg/liter phenylmethylsulfonyl fluoride. 15 ml of 10 mM HEPES-NaOH, pH 7.4, 0.2 M NaCl, 100 mg/liter phenylmethylsulfonyl fluoride, 2 mM EDTA, and 2% Triton X-100 were added. Homogenates were extracted by end-over-end rotation for 4 h at 4°C and centrifuged for 1 h at 100,000 ϫ g in a Beckman Ti-45 rotor to obtain the total brain extract. Glutathione-agarose affinity columns (1-ml bed volume) with ϳ5 mg of GST-Mint1/MID, 5 mg of GST-Mint2/MID, 5 mg of GST, or 7 mg of GST-Munc18-1 were pre-equilibrated with core buffer (50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, and 2.5 mM MgCl 2 ) containing 2.5 mM CaCl 2 and 0.25% Triton X-100. The total brain extract was precleared by incubation for 6 h at 4°C with glutathione-agarose followed by centrifugation (800 ϫ g for 2 min). 13 ml of the recovered total brain extract were adjusted to 3.5 mM MgCl 2 and 3.5 mM CaCl 2 and loaded onto the columns by recirculation (4 -10 times) under gravity flow. The flow-through fraction from the last loading cycle was collected, and columns were washed with 20 ml of core buffer containing 2.5 mM CaCl 2 and 0.5% CHAPS and then sequentially eluted with 10 ml of core buffer containing 5 mM EGTA and 0.5% CHAPS (E1); 10 ml of core buffer containing 5 mM EDTA and 0.5% CHAPS (E2); 10 ml of core buffer containing 1 M NaCl, 5 mM EDTA, and 0.5% CHAPS (E3); and 4 ml of SDS-PAGE sample buffer (E4). For the binding experiments with recombinant proteins expressed in COS cells, only the fractions corresponding to E4 were used. Samples were analyzed by SDS-PAGE and immunoblotting.
Phospholipid Binding Assays-Phospholipids (phosphatidylcholine, phosphatidylserine, and phosphatidylinositol (Avanti Polar Lipids) or PIP and PIP 2 (Boehringer Mannheim)) were mixed in the appropriate ratios with 4 mg of total phospholipids/tube. The solvent was evaporated under nitrogen, 20 Ci of [ 3 H]phosphatidylcholine (1 mCi/ml, 37 MBq/ml; Amersham Corp.) was added as a tracer, and the remaining solvent was removed by N 2 . Dried phospholipids were resuspended in 10 ml of 10 mM HEPES, pH 7.4, and 0.1 M NaCl by vortexing for 1 min and sonicated for 15 s. After centrifugation at 10,000 ϫ g for 10 min, the supernatant was recovered and diluted twice with 10 mM HEPES, pH 7.4, and 0.1 M NaCl; kept at 4°C as a stock solution; and used within 2 weeks. For binding assays, 25 g of GST fusion proteins (GST-Mint1/ PTB, GST-SNAP-25, and GST-complexin I) bound to glutathione-agarose were incubated with 50 l of the 3 H-liposomes in 0.1 ml of binding buffer (50 mM Tris-HCl, pH 7.4, 0.1 M NaCl, 1 mM EDTA, 1 mM EGTA, 0.5 mM dithiothreitol, 0.1% bovine serum albumin, and 0.1% gelatin) for 10 min at room temperature in an Eppendorf shaker. After five washes with binding buffer, pellets were resuspended and quantitated in a scintillation counter.
Miscellaneous Procedures-SDS-PAGE and immunoblotting were performed essentially as described (5,10,14). Proteins were assayed with a Coomassie Blue-based assay kit (Bio-Rad). RNA blotting analysis was performed using commercially available blots (CLONTECH) containing total RNA from rat tissues.

RESULTS
Identification of Mint Proteins-We screened a rat brain cDNA library by yeast two-hybrid selection with full-length Munc18-1, a protein of 594 amino acids. Among 40 strongly positive clones, 29 clones were independent isolates of two closely related cDNAs (Fig. 1). The fact that we isolated multiple overlapping clones from two homologous proteins suggests that Munc18-1 specifically interacts with these proteins, which we therefore named Mint1 and Mint2. Two observations provided further support for the specificity of the Mint/Munc18-1 interaction. First, we did not isolate Mint clones in large yeast two-hybrid screens with several other baits. Second, we detected no interaction in yeast two-hybrid assays of Mint proteins with other proteins, such as Munc18-2, syntaxin, SNAP-25, synaptobrevin, and rabphilin (data not shown). Thus, Mint1 and Mint2 specifically bind to Munc18-1, but not to Munc18-2 or other trafficking proteins in yeast two-hybrid assays.
Since the yeast two-hybrid clones did not contain the complete coding sequence of Mint1 or Mint2, we isolated their full-length cDNAs. Their sequences revealed that Mint proteins have a multidomain structure composed of variable Nterminal and conserved C-terminal regions (Fig. 1B). Inspection of the different Mint prey clones localized the Munc18-1interacting domain to the N-terminal region. The C-terminal sequences of Mint proteins are composed of a PTB domain and two PDZ domains. PTB domains from several proteins bind phosphotyrosine-containing peptides and PIPs (15)(16)(17). PDZ domains are found in peripheral proteins of the plasma membrane, where they often bind to the cytoplasmic tails of intrinsic membrane proteins (18,19). Partial sequences of human Mint1 and mouse Mint2 were identified previously by positional cloning as candidate genes for Friedreich's ataxia, although they were later shown not to be involved in the disease (20,21). The genes were designated as human X11 and mouse X11 with the notion that they represent orthologs. Our fulllength sequences of both proteins from a single species, however, demonstrate that they are isoforms. Data bank searches revealed several Caenorhabditis elegans sequences corresponding to Mint proteins, suggesting that Mint proteins are conserved in invertebrates (data not shown).
Protein Binding of Mint Proteins and Munc18-1-Because yeast two-hybrid assays are prone to artifacts, we tested the interaction between Munc18-1 and Mint proteins by independent methods. For this purpose, we performed affinity chromatography experiments by binding rat brain proteins or recombinant proteins to immobilized GST-Mint fusion proteins or control GST proteins. We applied total rat brain homogenates solubilized with Triton X-100 to columns containing GST-Mint1, GST-Mint2, and GST and eluted bound proteins with high salt after extensive washing. Analysis of the eluates by SDS-PAGE followed by Coomassie Blue staining and immunoblotting revealed that we purified Munc18-1 on both Mint columns as the major component and syntaxin 1 as a minor component ( Fig. 2 and data not shown). Control GST columns were unable to enrich either protein. To ensure that the column purification of Munc18-1 and syntaxin 1 on immobilized Mint proteins was specific, we analyzed the eluates by immunoblotting for a number of known synaptic proteins (data not shown). Only Munc18-1 and syntaxin 1 were eluted. Control GST fusion proteins did not retain Munc18-1 or syntaxin 1 (data not shown). Thus, Munc18-1 and syntaxin 1 are purified on immobilized Mint proteins in a single step by a specific interaction. To ensure that Mint proteins and Munc18-1 also interact in brain, we performed immunoprecipitations with Mint1 and Mint2 antibodies from rat brain homogenates. Both revealed co-immunoprecipitation of Mint1 with Munc18-1 and syntaxin 1 (Fig. 3). Thus, three methods, yeast two-hybrid selection, affinity chromatography, and immunoprecipitation, show that Mint proteins are binding partners for Munc18-1.
These data demonstrate that syntaxin 1 and Munc18-1 are in a complex with Mint proteins, but do not tell us whether syntaxin 1 and Munc18-1 bind independently to Mint proteins or whether only one of the two proteins binds to Mint proteins and the other is purified piggyback on the first. To distinguish between these two possibilities, we expressed Munc18-1, syntaxin 1, and SNAP-25 individually in COS cells and tested their binding to Mint proteins singly or in combination (Fig. 4). Munc18-1 alone bound to Mint proteins. Syntaxin 1 bound only if Munc18-1 was also added, and SNAP-25 did not bind under any condition. These results agree well with the absence of an interaction of Mint proteins with syntaxin 1 in yeast twohybrid assays (see above) and suggest that syntaxin 1 interacts indirectly with Mint proteins via Munc18-1.
Tissue Distribution and Subcellular Localization of Mint Proteins-We hybridized RNA blots from multiple rat tissues with Mint probes at high stringency. In agreement with in situ hybridization data on X11 (20), the mRNAs for both Mint Residues in human Mint1 that differ from those in rat are shown above the rat Mint1 sequence, and residues in mouse Mint2 that differ from rat are shown below the rat Mint2 sequence. The human Mint2 sequence is identical to the rat sequence. Residues that are identical between Mint1 and Mint2 in any species combination are shown in black boxes. proteins were abundant in brain, but were not detectable in other tissues tested even after long exposures (data not shown). To investigate the localizations of Mint proteins, we performed subcellular fractionations. These showed that Mint proteins and Munc18-1 are largely membrane-bound and copurify with synaptic plasma membranes (data not shown). Although syntaxin 1 is primarily a plasma membrane protein, it is additionally present on synaptic vesicles (22,23). Thus, the Mint⅐Munc18-1⅐syntaxin 1 complex could potentially be on plasma membranes, on synaptic vesicles, or on both. To test this, we measured the relative enrichment of different proteins in synaptic vesicles compared with total brain (Fig. 5). Vesicle proteins such as synaptophysin 1, synaptogyrin, Rab3A, and synaptobrevin were greatly enriched in synaptic vesicles. By contrast, syntaxin 1 was de-enriched, and Munc18-1 and Mint proteins were barely detectable. These data indicate that Mint proteins and Munc18-1 are not components of synaptic vesicles.
Mint Proteins Bind to PIPs-Compared with other membrane trafficking proteins, Mint proteins have an unusual composition since they contain PTB and PDZ domains. Recent studies in permeabilized PC12 cells revealed that phosphatidylinositol-4-phosphate 5-kinase, an enzyme that generates PIPs such as PIP 2 , is required for exocytosis (24). Since the PTB domain of Shc binds PIP and PIP 2 (25), we investigated the possibility that the PTB domain of Mint proteins may bind PIP 2 . To test this, we studied the binding of 3 H-labeled liposomes composed of different phospholipids to GST-Mint1 and to control GST fusion proteins (Fig. 6). PIP and PIP 2 bound specifically to the PTB domain from Mint1, but not to other GST fusion proteins. Binding required only 0.7% PIP 2 , but much higher concentrations of PIP, suggesting that Mint proteins may prefer PIP 2 in vivo.

DISCUSSION
Exocytosis of synaptic vesicles starts with the docking of vesicles, proceeds by priming the vesicles for Ca 2ϩ -triggered release, and finishes with the release of neurotransmitters in a final Ca 2ϩ -dependent reaction. A number of proteins are known to function in priming and/or Ca 2ϩ -dependent release (e.g. CAPS, syntaxin 1, SNAP-25, synaptobrevin, synaptotagmin 1, Munc18-1, and Rab3), but few proteins involved in docking of vesicles have been described (1)(2)(3)(4).
We have now identified a family of proteins called Mint proteins that interact with Munc18-1, a protein essential for synaptic vesicle exocytosis. 1 The following evidence supports the notion that Mint proteins bind to Munc18-1 with high FIG. 2. Affinity purification of Munc18-1 on immobilized GST-Mint1. Total rat brain homogenate solubilized in Triton X-100 (L, load) was applied to a GST-Mint1 column (GST-Mint1/MID) in buffer containing Ca 2ϩ and 0.1 M NaCl. After collecting the flow-through fraction (F), the column was washed extensively with the loading buffer containing CHAPS instead of Triton X-100 (W, wash) and sequentially eluted with buffers containing 0.1 M NaCl and 5 mM EGTA (E1), 0.1 M NaCl and 5 mM EDTA (E2), and 1 M NaCl and 5 mM EDTA (E3). Equivalent amounts of all samples were analyzed by SDS-PAGE and Coomassie Blue staining. Immunoblotting identified the major 65-kDa band in the eluate as Munc18-1 and the minor 37-kDa protein as syntaxin 1. Parallel experiments with GST-Mint2 gave similar results; no protein bound to GST alone (not shown).
FIG. 3. Co-immunoprecipitation of Munc18-1 and syntaxin 1 with Mint proteins from rat brain homogenates. Total brain extracts (lane 1) were subjected to immunoprecipitations using polyclonal antibodies to Mint1 or Mint2 or the respective preimmune sera as shown. Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with monoclonal antibodies to Munc18-1 and syntaxin 1. IgG heavy chain was weakly stained in a nonspecific reaction because of the large amount of IgG in the sample. Together, these data suggest that in brain, munc18-1 and Mint proteins form a complex. Since the affinity chromatography experiments and immunoprecipitations also purified syntaxin 1 that binds to Munc18-1 but not to Mint proteins, it is likely that brain contains a complex of Munc18-1, syntaxin 1, and Mint proteins. Although Doc2 proteins also interact with Munc18-1 (10), Doc2 proteins are not copurified with Munc18-1 in the affinity chromatography experiments because the Doc2 interaction is relatively weak compared with the high affinity interaction of Mint proteins. Since both syntaxin 1 and Munc18-1 are essential for vesicle exocytosis, it seems likely that Mint proteins also function in exocytosis.
Mint proteins have an unusual structure that includes domains not previously identified in vesicular trafficking pro-teins, suggesting a novel function. The PTB and PDZ domains in Mint proteins provide them with the potential to unite signal transduction (PTB domain), localization to intercellular junctions (PDZ domains), and vesicular membrane traffic (Munc18-1 binding). The presence of a PTB domain suggests a connection between neurotransmitter release and tyrosine phosphorylation and/or PIPs. Binding to phosphotyrosines could play a potential role in synaptic plasticity (26). We showed that the PTB domain of Mint1 binds to PIPs in a specific reaction, an activity that could be important in view of the transient generation of PIPs during exocytosis (24). Finally, the presence of PDZ domains in Mint proteins indicates a possible role for Mint proteins in connecting synaptic vesicles to the sites of synaptic intercellular junctions. PDZ domains are known in other proteins to localize peripheral proteins to intercellular junctions (17,18) and may have an analogous function in Mint proteins.
Based on the properties of Mint proteins, we would like to propose a two-stage model for synaptic vesicle exocytosis that is meant as a framework for future experiments. In the first stage (docking), we propose that the Mint⅐Munc18-1 complex provides a linkage between the plasma membrane and synaptic vesicles that docks the vesicles. Mint proteins could bind synaptic vesicles via their interaction with PIPs, and Munc18-1 could bind vesicles via its interaction with Doc2 proteins (10). The association of Mint proteins with the plasma membrane could be mediated by the binding of its PDZ domains to the cytoplasmic tails of membrane proteins (17,18). In the second stage (priming), the Mint⅐Munc18-1 complex delivers the docked vesicles to the syntaxin 1⅐SNAP-25 complex by a direct interaction. As a result, synaptobrevin/vesicle-associated mem- FIG. 5. Absence of the Mint⅐Munc18-1⅐syntaxin 1 complex in synaptic vesicles. Aliquots of total rat brain homogenate (left lanes) or of purified synaptic vesicles (right lanes) were immunoblotted for the indicated proteins. The lower molecular mass band in the Mint2 blot (asterisk) probably corresponds to a breakdown product of this proteolytically sensitive protein. brane protein on the vesicles binds to the syntaxin 1⅐SNAP-25 complex to form the core complex during membrane fusion.
The model that we propose is highly speculative at present, but would provide potential explanations for two intriguing observations. First, Hay et al. (24) demonstrated an essential role for PIPs in Ca 2ϩ -regulated exocytosis from PC12 cells, suggesting that PIPs are important in exocytosis. A vesicledocking mechanism mediated by binding of Mint proteins to PIPs is attractive because the transient production of PIPs could provide a highly localized signal for docking. Second, the so-called SNAREs synaptobrevin/vesicle-associated membrane protein, SNAP-25, and syntaxin were shown to be essential for exocytosis, but not for the attachment of vesicles to the active zone (docking) (27)(28)(29). Our model explains why docking does not require syntaxin because we propose that docking is mediated by the binding of Mint proteins to the plasma membrane probably via its PDZ domains and by the binding of Mint proteins and Munc18-1 to synaptic vesicles via PIPs and Doc2 proteins. The proposed model also offers a potential explanation for how membrane traffic could be targeted to intercellular junctions by suggesting a role for PDZ domains in exocytosis. In view of the widespread occurrence of Munc18 homologues in cell types other than neurons, the model may also be applicable, at least in part, to the general process of exocytosis in non-neuronal cells.