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Originally published In Press as doi:10.1074/jbc.M208315200 on August 26, 2002

J. Biol. Chem., Vol. 277, Issue 46, 43973-43979, November 15, 2002
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Analysis of the Munc18b-Syntaxin Binding Interface

USE OF A MUTANT Munc18b TO DISSECT THE FUNCTIONS OF SYNTAXINS 2 AND 3*

Maria KauppiDagger , Gerd Wohlfahrt§**, and Vesa M. OlkkonenDagger ||

From the Dagger  Department of Molecular Medicine, National Public Health Institute, Biomedicum, P.O. Box 104, FIN-00251 Helsinki, Finland and the § Institute of Biochemistry, University of Cologne, Zülpicher Strasse 47, D-50674 Köln, Germany

Received for publication, August 14, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Munc18b is a mammalian Sec1-related protein that is abundant in epithelial cells and regulates vesicle transport to the apical plasma membrane. We constructed a homology model of Munc18b in complex with syntaxin 3 based on the crystal structure of the neuronal Sec1·syntaxin 1A complex. In this model we identified all residues in the interface between the two proteins that contribute directly to the interaction and mutagenized residues in Munc18b to alter its binding to syntaxins 1A, 2, and 3. The syntaxin-binding properties of the mutants were tested using an in vitro assay and by a co-immunoprecipitation approach employing Munc18b expressed in CHO-K1 cells. Three Munc18b variants, W28S, S42K, and E59K, were generated that are defective in binding to all three syntaxins. A fourth mutant protein, S48D, shows abolishment of syntaxin 3 interaction but binds syntaxin 2 at normal and syntaxin 1A at mildly reduced efficiency. Over-expression of Munc18b S48D inhibited transport of influenza hemagglutinin to the apical surface of Madin-Darby canine kidney II cells, which express syntaxin 2 abundantly, but not of Caco-2 cells, in which syntaxin 3 is the major apical target SNARE (soluble NSF (N-ethylmaleimide sensitive factor) attachment protein receptors). This suggests that, although syntaxin 3 is the main target SNARE operating in exocytic transport to the apical plasma membrane in certain epithelial cell types, syntaxin 2 may play an important role in this trafficking route in others.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Intracellular membrane trafficking in eukaryotic cells employs vesicular carriers that bud from one membrane compartment and fuse with another. This process is dependent on compartment-specific membrane-anchored proteins denoted collectively as SNAREs1 (soluble NSF (N-ethylmaleimide sensitive factor) attachment protein receptors) (1-3). The SNARE proteins present on the transport vesicles (v-SNAREs; related to the neuronal synaptobrevin/vesicle-associated membrane proteins) and the target membranes (t-SNAREs; homologues of the neuronal syntaxin (syn) and SNAP-25 proteins) are characterized by the presence of one or two "SNARE motifs," sequences capable of forming coiled-coil helix bundles upon assembly of v- and t-SNAREs into membrane-bridging complexes (called trans-SNARE complexes). Assembly of such trans-SNARE complexes results in a close apposition of the vesicle and target membranes and is suggested to indirectly or directly cause fusion of the membrane bilayers (4, 5). Syntaxins comprise a large family of t-SNAREs (6) that have a central role in SNARE complex assembly. They are type II membrane proteins anchored to the bilayer by a C-terminal transmembrane segment (7). Most syntaxins have an amino-terminal regulatory domain (Habc) folded as a three-helix bundle (helices a, b, and c), a linker region, and a membrane-proximal helix 3 (H3) that engages in coiled-coil SNARE complexes (8-11).

Sec1/Munc18 (SM) proteins (12-15) bind to specific syntaxins with high affinity, thus modulating the capability of these t-SNAREs to interact with their cognate SNARE partners. A given SM protein typically interacts with more than one syntaxin. The function of the mammalian Munc18 proteins (see below) involves contacts with both the Habc and the H3 regions of syntaxins (16-18). In vitro binding studies and over-expression of SM proteins have provided evidence for an inhibitory role of the proteins in SNARE complex formation and membrane trafficking. On the other hand, a wealth of evidence shows that SM action is essential for normal function of the intracellular trafficking pathways (see references in Refs. 13 and 14). Loss-of-function mutations in the Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster SM proteins lead to specific blocks in vesicle transport, and synaptic transmission was reported to be entirely absent in mice lacking the neuronal Sec1/Munc18a gene (19). The binding of SM proteins to syntaxins is suggested to protect syntaxins from promiscuous and harmful interactions during their intracellular transport (20, 21) and to provide a platform for SNARE complex assembly (11, 17). Three mammalian Sec1 homologues are suggested to control vesicle fusion at the plasma membrane. Munc18a/n-Sec1/rbSec1 is a predominantly neuronal protein, which binds the neuronal syntaxins 1A and 1B as well as syntaxins 2 and 3, and is essential for neurotransmission (19). Munc18b/Munc18-2 (22-24) interacts with the same syntaxins as Munc18a but is expressed mainly in epithelial cells, where it localizes at the apical plasma membrane (25-27). Munc18c, which is expressed ubiquitously, binds to syntaxins 2 and 4 and has been shown to control glucose transporter trafficking in adipocytes and skeletal muscle through regulation of syntaxin 4-based SNARE complexes, as well as platelet granule exocytosis (23, 28-33).

We recently showed that Munc18b, through its interaction with syntaxin 3, regulates biosynthetic transport to the apical plasma membrane in the epithelial Caco-2 cell line (34). In the present study we employed the three-dimensional structure of a complex between the neuronal Munc18a and syntaxin 1A (17) for molecular modeling of the Munc18b·syn3 complex. We used the model to identify residues in Munc18b that are essential for syntaxin interactions in general and ones that are predicted to contribute to the specificity of syntaxin binding. The residues were mutagenized, and the effects of the amino acid changes on Munc18b binding to different syntaxins were determined in vitro and in transfected cells. We succeeded in generating a mutant Munc18b (S48D) that binds at normal efficiency to syn2 but fails to interact with syn3. This mutant was then expressed in two epithelial cell lines, Caco-2 and MDCK II, which have different relative expression levels of syn2 and syn3, to assess the role of the two syntaxins in the transport of influenza virus hemagglutinin (HA) to the apical cell surface.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Homology Model of the Munc18b·Syntaxin3 Complex-- A homology model of MDCK II cell (dog) Munc18b (GenBankTM accession no. L41609) in complex with rat syntaxin 3 (GenBankTM accession no. L20820) was constructed based on the crystal structure of the neuronal Sec1·syntaxin 1A complex (17). Initial sequence alignments for the families of syntaxin and Sec1-related proteins were derived with ClustalW (35). Manual adjustment of the position of three gaps in the alignment was performed in non-interacting regions of the proteins. Loop regions were built with the BRAGI program (36, 37). Side chains were modeled with SCWRL (38), optimizing only the residues within the complex structure, which are not identical in template and target proteins. The final model was energy minimized with the AMBER 5.0 program (39) without explicit solvent using different distance-dependent di-electricity constants. The quality of the models generated was evaluated with PROCHECK (40) and ERRAT (41).

Cell Culture-- The human colon carcinoma cell line (Caco-2) was cultured in Eagle's minimum essential medium (Sigma) supplemented with 10% fetal bovine serum (Invitrogen), 1% non-essential amino acids, 100 IU/ml penicillin, and 100 µg/ml streptomycin. The Chinese hamster ovary cell line (CHO-K1) and Madin-Darby canine kidney cell line (MDCK, strain II) were cultured in Eagle's minimum essential medium (Sigma) supplemented with 10% fetal bovine serum (Invitrogen), 100 IU/ml penicillin, and 100 µg/ml streptomycin. The porcine kidney cell line LLC-PK1 was cultured in Medium 199 (Sigma) with Earle's salt supplemented with 10% fetal bovine serum (Invitrogen), 100 IU/ml penicillin, and 100 µg/ml streptomycin. For immunofluorescence studies Caco-2 and MDCK II cells were grown on Costar Transwell polycarbonate filters. Fresh medium was changed to the filters every day, and the polarized cells were used 7 days after reaching confluency or 5 days after transfection in suspension and plating at 100% confluency.

Antibodies-- The rabbit antiserum against Munc18b and the affinity purified anti-syn3 antibody have been characterized (25). Monoclonal anti-myc (9E10) was purchased from Santa Cruz and polyclonal rabbit antiserum against syn2 from Synaptic Systems. The polyclonal antiserum against influenza virus A H1N1 envelope glycoproteins was a gift from Dr. Ilkka Julkunen (National Public Health Institute, Helsinki, Finland).

Site-directed Mutagenesis-- The amino-terminally myc-tagged canine Munc18b cDNA in pBluescript SK(-) was mutagenized using the QuikChange system (Stratagene). The sequence changes were verified using a cycle-sequencing kit (BIGDYE) and an automated ABI377 sequencer (Applied Biosystems).

cDNA Constructs and Transfection-- Amino-terminally myc-tagged wild-type and mutant Munc18b cDNAs were transferred as BamHI fragments from pBluescript SK(-) to pcDNA3.1(+)(Invitrogen) for transfection experiments. CHO-K1 cells were transfected with LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. Transient expression of full-length mouse syn2A or rat syn3A cDNAs in pBK-CMV (Stratagene) was achieved by transfection of trypsinized Caco-2 or MDCK II cells using FuGENE 6 (Roche Molecular Biochemicals), followed by culture of the cells on polycarbonate filters for 5-6 days. cDNA fragments encoding the amino-terminal cytoplasmic domains of rat syntaxins 1A (GenBankTM accession no. AF217191, amino acids 1-265), 3 (L20820; amino acids 1-263), and 4 (L20821; amino acids 1-272) as well as of mouse syntaxin 2 (NM_007941; amino acids 1-265), were amplified by PCR and inserted in the EcoRI sites (syn2) or EcoRI-BamHI sites (syn3) of pGEX1lambda T or pGEX2T (syn4) (Amersham Biosciences) for Escherichia coli production of glutathione S-transferase (GST) fusion proteins, which were used in the in vitro binding assay.

In Vitro Assay for Munc18b-Syntaxin Interaction-- One µg/well of GST, GST-syn1A, GST-syn2, GST-syn3, or GST-syn4 in 50 mM NaHCO3 buffer, pH 9.6, was coated on MaxiSorb 96-well plates (Nunc) for 16 h at +4 °C. The binding of [35S]methionine labeled in vitro translated Munc18b to the immobilized GST-syntaxin fusion proteins was assayed essentially as in (34), with the exceptions that unspecific binding was now blocked with 1% bovine serum albumin, 0.05% Tween 20 in phosphate-buffered saline (PBS), and incubation of the in vitro translated radioactive Munc18b was carried out at +4 °C overnight.

Stability Test of the Mutagenized Munc18b Proteins-- CHO-K1 cells were grown on 3.5 cm dishes and transfected with Munc18b wild-type or mutant constructs for 24 h. The cells were pulse labeled with [35S]methionine and cysteine (Amersham Biosciences, AG0080; 30 µCi/dish) in Met- and Cys-free cell culture medium containing 1% dialyzed fetal bovine serum for 1 h. After PBS washes the cells were either lysed directly in immunoprecipitation (IP) buffer (10 mM Hepes, 1% Triton X-100, 140 mM KCl, 10 mM EDTA, 25 µg/ml each of chymostatin, leupeptin, antipain, and pepstatin A) or chased for 2 h with excess unlabeled Met and Cys. The myc-Munc18b proteins were immunoprecipitated by using the 9E10 anti-myc mAb and protein G-Sepharose (Amersham Biosciences) and analyzed by SDS-PAGE and fluorography.

Assay for Munc18b-Syntaxin Interaction in CHO Cells-- CHO-K1 cells were grown on 3.5 cm dishes and, after 24 h transfection with myc-Munc18b wt or mutant constructs, lysed in the above IP buffer and immunoprecipitated with anti-myc mAb. The immunoprecipitates were analyzed by SDS-PAGE and Western blotting using anti-syn2, anti-syn3, or anti-Munc18b antibodies and enhanced chemiluminescence detection (Amersham Biosciences).

Immunofluorescence Microscopy-- The filter-grown cells were fixed for 20 min at room temperature with 4% paraformaldehyde, 250 mM Hepes, pH 7.4, and permeabilized for 20 min with 0.1% Triton X-100 in PBS. The primary antibodies diluted in 5% fetal calf serum/PBS were incubated with the specimens overnight at 4 °C. The filters were washed with PBS on a shaker. The bound antibodies were detected with fluorescein-isothiocyanate (FITC)- or tetramethylrhodamine-isothiocyanate (TRITC)-conjugated goat anti-rabbit or anti-mouse F(ab')2 (Immunotech, Marseille, France). After extensive washes with PBS the specimens were mounted in Mowiol (Calbiochem), 50 µg/ml 1,4 diazabicyclo-[2.2.2]octane (Sigma), and investigated using a laser scanning confocal microscope (Leica SP1).

Generation of Recombinant SFVs and Viral Infections-- Recombinant SFVs expressing wt Munc18b, the S48D mutant, or human CLN3 (the control virus) and influenza virus WSN/33 hemagglutinin from two independent 26 S promoters were constructed using the strategy of (42) as described in (34). Caco-2 or MDCK II cells grown on polycarbonate filters were infected through the filter from the basal side as in Ref. 34. After the infection period the filter units were returned into complete medium and incubated at +37 °C in the presence of cycloheximide (20 µg/ml) to chase the expressed HA to the cell surface. Caco-2 cells were infected for 5 h and then chased for 4 h. For MDCK II the times were 7 h and 2 h, respectively.

HA Trafficking Assay by Confocal Immunofluorescence Microscopy-- Cells infected with recombinant SFVs as above were processed for immunofluorescence microscopy as decribed above and inspected with a Leica SP1 confocal microscope. The distribution of HA on the basal-apical axis was quantified in single infected cells with the Quantify function of the Leica confocal software by determining the mean fluorescence intensity at four z planes, the apical and basal surfaces plus two intermediate planes at equal intervals.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Model of the Munc18b·Syntaxin 3 Complex-- In the present study we generated, based on the published crystal structure of a complex of their neuronal homologues nSec1-Munc18a and syntaxin 1A (17), a homology model of the Munc18b·syntaxin 3 complex (Fig. 1). The overall sequence identity between Munc18a and Munc18b is 63%, the identity of syn1a and syn3 being 65%. The model shows only minor differences to the structure of the Munc18a·syn1a complex. All backbone deviations above 1 Å, with the exception of His-328 in Munc18b, are located in areas distant from the binding interface between the two proteins. The quality of the model minimized with a di-electricity constant of 4r was the best according to PROCHECK and ERRAT results. Consequently this model was used for planning mutants and further structural analysis.


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  		Fig. 1.   Ribbon representation of the structural model of the Munc18b·syntaxin 3 complex. The side chains of residues subjected to mutagenesis in the present study are shown in red (positive), blue (negative), violet (polar), and yellow (aromatic). The coordinates of the model are available on request from the authors.

Planning of Munc18b Mutants-- The program CONTACTS (55) was used to identify all residues in the interface between Munc18b and syn3 in our model that are in close proximity to atoms of the binding partner (< 4 Å). Multiple sequence alignment of the syntaxin family was used to analyze the conservation of interacting residues (Fig. 2). Their potential interaction partners in Munc18b were inspected in the structural model to see if they could be changed to amino acids, which would alter the binding of Munc18b to syntaxins, to (i) validate the model and (ii) to create Munc18b mutants with altered syntaxin-binding properties for functional studies. It is noteworthy here that the residues of Munc18b found to interact with syn3 are all fully conserved between mammalian species (rat, mouse, dog, human) (data not shown).


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  		Fig. 2.   Multiple sequence alignment of four syntaxin family proteins. All sequences are from rat with GenBankTM accession numbers: P32851 (syn1A), P50279 (syn2), Q08849 (syn3), and Q08850 (syn4). Conservation between the syntaxins is indicated in the consensus line: asterisk, identical or conserved residues in all sequences in the alignment; colon, conserved substitutions; period, semi-conserved substitutions. Residues predicted to interact with Munc18b are indicated on the top with H (hydrogen bond with side chain), H (hydrogen bond with backbone), V (hydrophobic contact), and x (long distance hydrogen bonds or hydrophobic contacts). Residues predicted to interact with side chains from Munc18b amino acids mutated in the present study are underlined and printed in bold. The Munc18b residues are indicated in color at the bottom, and the interacting syn3 residues are highlighted with the same colors.

To disable binding to all syntaxins, a highly conserved hydrophobic interaction with F36 in syn3 (Fig. 2) was removed in the mutant W28S. Glu-59 was changed to the oppositely charged lysine to create a repulsive interaction with Arg-114, which is also conserved in all syntaxins (Fig. 2). Additionally, we identified by sequence comparison those interacting residues that are not conserved in different syntaxins (Fig. 2). This information in combination with the structural model was used in efforts to disable Munc18b binding only to certain syntaxins. The variant S42K was intended to bind only syn2 and syn3 due to favorite interactions with Asn-233 in syn2 or Asn-231 in syn3, whereas the corresponding residue is arginine in syn1A, which is expected to prevent binding. The mutant S48D was predicted to interact only with syn2. S48D was expected to show reduced affinity for syn1A and syn3 because of a repulsion of the like charges of Asp-48 and Asp-231 in syn1A and Asp-230 in syn3. Syn2 binding should still be possible because of a more favorable interaction between Asn-48 in the mutant Munc18b and Asn-232 in the syntaxin.

In Vitro Interaction of the Munc18b Variants with Syntaxins-- To analyze the interaction of the Munc18b mutant proteins with different plasma membrane syntaxins we used a previously published (34) in vitro binding assay. This method was developed further to increase its sensitivity. In vitro translated [35S]methionine-labeled Munc18b or its variants were incubated on GST-syntaxin 1A, 2, 3, 4, or GST-coated 96-well plates, and the bound radioactivity was measured. Radioactivity bound to plain GST was used as background, which was subtracted from the GST-syntaxin-bound signal. The wild-type Munc18b showed most efficient binding to syn2, followed by syn3. Binding to syn1A was also clearly detectable, whereas no binding to syn4 was observed (Fig. 3A). The assay gave a linear response to the amount of radioactive Munc18b added in the concentration range tested.


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  		Fig. 3.   In vitro binding of myc-Munc18b variants to different Syntaxins. A, binding of wt Munc18b to syntaxin 1A, 2, 3, or 4. Increasing amounts of [35S]methionine-labeled Munc18b (x-axis) were incubated on GST-syntaxin or GST-coated 96-well plates. The radioactivity bound specifically (background radioactivity bound to GST-coated wells was subtracted) to syntaxins was measured (y-axis). The results (mean ± S.E.) represent three independent experiments, each carried out in triplicate. B, binding of Munc18b mutants to syntaxin 1A, 2, 3, or 4. [35S]methionine-labeled Munc18b variants (150,000 cpm/well) were incubated on GST-syntaxin or GST-coated plates. The radioactivity bound specifically to the syntaxins was measured. The results (mean ± S.E.) represent three independent experiments each carried out in triplicate.

In a previous study (34) we reported a Munc18b double mutant K314L/R315L that did not show significant binding to syn3. We investigated further the binding of K314L/R315L to syntaxins. With the more sensitive assay modification presently in use, binding of the mutant to syn3 was detectable even though the binding efficiency was low. The K314L/R315L mutant also showed markedly reduced but detectable affinity to syn1A and syn2. The new Munc18b mutants W28S and E59K were designed to block all interactions with syntaxins 1A, 2, and 3. The behavior of these mutated proteins in the in vitro binding assay was as expected. Also, the binding of S42K to all syntaxins was inhibited even though the mutation was originally designed to prevent only the interaction with syn1A. The S48D mutant was designed to selectively bind syn2. In the in vitro assay S48D showed normal binding to syn2 and reduced affinity to syn1A. Remarkably, the binding to syn3 was completely abolished. Neither the wt Munc18b nor the mutants detectably bound syn4 (Fig. 3B).

Interaction of the Munc18b Variants with Syntaxin 2 and 3 in CHO Cells-- The results of the in vitro binding assay were verified in terms of the syn2 and syn3 interactions by expressing the Munc18b variants in CHO-K1 cells, which were found to express both syn2 and syn3. First, the stability of the mutated proteins was tested by using a pulse-chase approach. The CHO cells were transiently transfected with myc-Munc18b constructs. After 24 h the cells were pulse labeled with [35S]methionine and lysed directly after pulse or after a 2 h chase. The myc-Munc18b in the lysates was immunoprecipitated with myc-antibody, and the precipitates were analyzed by SDS-PAGE and fluorography. The stability of the mutated proteins (K314L/R315L, W28S, S42K, S48D, and E59K) did not differ from that of the wt Munc18b. After the chase period no degradation or major loss of the labeled Munc18b was observed (Fig. 4).


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  		Fig. 4.   Stability of the mutant proteins. Myc-Munc18b or its mutant forms were expressed by transfection of constructs in pcDNA3.1 in CHO-K1 cells. The cells were [35S]methionine pulse-labeled for 1h and lysed directly (-) or after a 2 h chase (+) and subjected to immunoprecipitation with anti-myc mAb followed by SDS-PAGE and fluorography. The mutants are identified above the panels (wt, wild-type Munc18b; Mock, the plain vector plasmid).

For testing the Munc18b-syn2 and -syn3 interactions in vivo the CHO cells were transfected with wt or mutant myc-Munc18b constructs and immunoprecipitated with anti-myc antibody. The precipitates were analyzed with anti-Munc18b, anti-syn2, and anti-syn3 antibodies (Fig 5). The wt Munc18b was found to bind to both syn2 and syn3. In contrast, the S48D mutant bound only to syn2. Weak binding to syn2 was also observed with the K314L/R315L double mutant. The other mutants, W28S, S42K, and E59K, were found to bind neither of the syntaxins. The expression of the Munc18b variants was verified by Western blotting of the immunoprecipitates with anti-Munc18b antibody. All of the variants were present in similar amounts in the precipitated specimens. The results of these in vivo binding experiments were thus remarkably similar to the in vitro findings.


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  		Fig. 5.   Binding of Munc18b variants to syntaxin 2 and 3 in transfected CHO cells. CHO cells were transfected with myc-tagged Munc18b variants in pcDNA3.1 or the plain vector plasmid (Mock). After a 24-h transfection the cell lysates were immunoprecipitated with anti-myc mAb followed by SDS-PAGE and Western blotting with anti-Munc18b, anti-syntaxin 2, and anti-syntaxin 3 antibodies. The antibodies used are indicated on the right, and the mutants are identified above the panels. wt, wild-type Munc18b.

Dissection of the Functions of Syntaxin 2 and 3 Using the Munc18b S48D Mutant-- Because Munc18b S48D failed to bind syn3 but bound syn2 with normal efficiency it was regarded as highly interesting. The structural basis of the selectivity of S48D for syn2 is depicted in Fig. 6. S48D shows reduced affinity for syn3 because of repulsion of the like charges of Asp-48 in the mutant Munc18b and Asp-230 in syn3. Syn2 binding is still possible because of a favorite interaction between Asp-48 in Munc18b and Asn-232 in syn2, which corresponds to Asp-230 in syn3. Other potential interaction partners for Ser/Asp-48 are Lys-126, which is conserved between the different syntaxins, and Val-122, which is replaced by threonine in syn1A. The threonine could form a hydrogen bond with the newly introduced Asp-48 in the Munc18b mutant, partly compensating for the charge repulsion, which would explain that the mutant's affinity for syn1A is decreased only about 2-fold.


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  		Fig. 6.   The structural environment of Ser-48 in the Munc18b·syntaxin 3 complex. The residues around Ser-48 in the Munc18b-syn3 model are color coded according to their atom types, and the three interacting residues in syn3 are identified (Asp-230, Val-122, and Lys-126). Superimposed as thinner lines is the x-ray structure of nSec1-Munc18a (orange) and syntaxin 1A (green) (PDB code 1DN1). Distances (Å) to syntaxin residues that might interact with Ser-48 or the mutated residue Asp-48 are indicated by yellow dots.

Evidently, the S48D mutant could be used to dissect the functions of syntaxins 2 and 3 in the apical vesicle transport of epithelial cells. First, the expression levels of endogenous syn2 and 3 were investigated in three epithelial cell lines, Caco-2, LLC-PK1, and MDCK II. Total cell lysates were analyzed by SDS-PAGE and Western blotting with anti-syn2 and anti-syn3 antibodies. Both of the syntaxins were abundant in LLC-PK1 cells, whereas Caco-2 cells expressed syn3 abundantly but very little syn2. In MDCK II cells syn2 was abundant and syn3 present only in low amounts (Fig. 7). Although not seen in the figure, syn2 was clearly detectable in Caco-2 cells and syn3 in MDCK cells after longer exposure times. Based on this Western analysis we chose for further functional analysis the Caco-2 and MDCK II cell lines, which displayed highly different relative expression levels of syn2 and syn3. Syn3 has been reported to localize to the apical plasma membrane in Caco-2 and MDCK cells (25, 43-46), and also syn2 is reported to be predominantly apical in these cell lines (44). To make sure that this also holds true for the specific cells used in the present study, the two cell lines cultured on polycarbonate filters were transiently transfected with mouse syn2 or rat 3 cDNAs, and the distribution of the expressed proteins was studied by immunofluorescence microscopy. Both proteins were found to localize apically in both cell lines (data not shown).


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  		Fig. 7.   Endogenous syntaxin 2 and 3 expression in different epithelial cell lines. Caco-2, LLC-PK1, and MDCK II cells were lysed and analyzed by SDS-PAGE (15 µg of total protein was loaded) and Western blotting with anti-syntaxin 2 and anti-syntaxin 3 antibodies. The cell lines are identified above the panels, and the antibodies used are indicated on the right.

We next infected polarized Caco-2 and MDCK II cells grown on polycarbonate filters with recombinant SFVs expressing simultaneously wt Munc18b or the S48D mutant and influenza virus HA or with a control virus expressing a non-relevant cDNA insert together with the HA cDNA. After the infection period allowing expression of both proteins (Caco-2, 5 h; MDCK II, 7 h) (Production of the proteins at levels sufficient for quantitative confocal microscopy analysis required a longer infection time in MDCK cells.), cycloheximide was added, and incubation was continued to chase the HA to the cell surface. In MDCK cells a 2 h chase was sufficient for detection of a major portion of the HA at the apical surface, whereas in Caco-2 a longer 4 h chase was required. Confocal microscopy was then applied to determine the effects of Munc18b on the apical trafficking of HA. Distribution of the HA on the apical-basal axis of the infected cells was quantified (Fig. 8). The wt Munc18b inhibited the apical delivery of HA significantly in both cell lines as compared with the control. The extent of the inhibition was 33% in Caco-2 and 56% in MDCK II. Interestingly, the S48D mutant with a selective syn3 binding defect had no significant effect on the apical transport of HA in Caco-2 cells but induced a clear, 42% inhibition in the apical delivery of HA in MDCK II.


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  		Fig. 8.   Effect of Munc18b S48D over-expression on apical transport of HA in Caco-2 and MDCK cells. Polarized Caco-2 (A) or MDCK II (B) cells grown on polycarbonate filters were infected with recombinant SFVs expressing a non-relevant control protein (ctrl), wild-type Munc18b (wt), or the S48D mutant (S48D) together with influenza virus HA, as detailed under "Experimental Procedures." The cells were fixed and processed for immunofluorescence microscopy with HA antibodies. The distribution of the HA along the apical-basal axis was quantified using a confocal microscope. A percentage distribution of mean fluorescence intensity at four focal planes is presented. 1, apical surface; 2, a plane at 1/3 of cell thickness down; 3, a plane at 2/3 of cell thickness down; 4, basal surface. The results represent the mean ± S.E. from 20 cells analyzed from each infection. The statistical significance of the differences between intensity at the apical surface in wt Munc18b or S48D mutant expressing cells versus the control is indicated. *, p < 0.05; **, p < 0.01 (Student's t test).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study we created a molecular model of a complex between Munc18b and syn3. This model was used to design site-specific mutants of Munc18b with defects in syntaxin binding. The mutants thus generated were tested for binding to syn1A, 2, and 3 using both an in vitro binding assay and co-immunoprecipitation carried out with CHO cells expressing the Munc18b variants. The syntaxin-binding properties of the new mutants corresponded remarkably well to the phenotypes predicted by the model, suggesting that the model is of high quality and can be used as a basis for detailed interaction studies.

Although the previously reported K314L/R315L double mutant (34) still showed residual binding to all three syntaxins, the new mutants W28S, S42K, and E59K did not detectably bind any of the syntaxins tested. In K314L/R315L only a conserved hydrogen bond to Glu-170 of syn3 is removed, which weakens syntaxin binding, but no repulsive interactions are created as in some of the other mutants. The behavior of the S42K mutant, which was originally predicted to be defective only in binding to syn1A, was not as expected. Despite the possibility that lysine in position 42 forms favorable hydrogen bonds with Asn-233 in syn2 and the corresponding Asn-231 in syn3, a lysine side chain introduced in position 42 may be too bulky to enable complex formation between mutant Munc18b and these syntaxins. The new mutants all turned out to be as stable as the wt protein, in contrast to the previously used non-binding mutant D34N/M38V (34, 47). This makes the new single mutants excellent tools for studying a Munc18b that has no capability of syntaxin binding. Such mutants will be highly useful in studies aimed at exploring the functional significance of the other protein-protein interactions that Munc18 proteins participate in (37, 48-51).

With the modeling and mutagenesis approach we succeeded in creating a mutant Munc18b, S48D, with a significantly altered syntaxin specificity. Because over-expression of a given SM protein is known to inhibit the function of the syntaxins that the SM protein interacts with (13-15), we employed this mutant to dissect the function of syntaxins 2 and 3 in the apical membrane trafficking of epithelial cells. Syn3 has been suggested to control biosynthetic transport to the apical surface in MDCK (52, 53) and Caco-2 (54) cells as well as an apical recycling route in MDCK cells (52), but the function of syn2 at the apical plasma membrane is not well known. The two epithelial cell lines studied here express endogenous syntaxins 2 and 3 at different relative levels. We therefore anticipated that if syn3 in Caco-2 and syn2 in MDCK II fulfill a similar function in apical exocytosis, the apical delivery of HA should show differential sensitivity to over-expression of the Munc18b S48D mutant. This indeed turned out to be the case. S48D, which fails to bind syn3, had no significant inhibitory effect in Caco-2 cells (with abundant syn3 at the apical plasma membrane) but inhibited the HA delivery in MDCK II (with abundant syn2 expression). The results thus suggest that in MDCK II cells, in which the function of syn3 in exocytic transport to the apical cell surface is well established (52, 53), syn2 may also play an important role in this process. Furthermore, our results raise the possibility that the contribution of syn2 to the apical transport in this cell type may be quantitatively more important than that of syn3. The extent of apical transport inhibition induced in MDCK cells by Munc18b S48D was lower than that by the wt protein (42 versus 56%). The difference between these inhibitory effects may well represent the proportion of the transport accounted for by syn3. This interpretation is in apparent discrepancy with the results of Low et al. (52), who observed an inhibition of apical exocytosis upon over-expression of syn3, but not of syn2, in MDCK cells. However, as the authors of that study point out, the extent of syn2 over-expression achieved may not have been sufficient to disturb the transport step under study. One could argue that the inhibition observed here with the S48D mutant is due to the hardly detectable residual syn3-binding activity of the protein that might be sufficient to block the function of the endogenous syn3 present at low levels in MDCK. This, however, is highly unlikely because the K314L/R315L mutant (34), whose residual syn3-binding activity is clearly higher than that of S48D, failed to inhibit apical HA delivery in MDCK cells in the present assay (data not shown).

In specific cell types and tissues, such as intestinal and kidney proximal tubule epithelia, Munc18b expression correlates with that of syn3, and the two proteins form a physical complex (25, 27). However, this is not the case in all epithelial structures (27). The present results imply that even though syn3 is likely to be an important functional partner of Munc18b in certain tissues/cell types, interactions of Munc18b with syn2 (as well as syn1A) are likely to be of great functional importance in others. Of the non-neuronal syntaxins, the Munc18b S48D mutant binds only syn2. It will therefore be highly useful in dissecting the functional roles of syn2 and syn3 in cell types that express both proteins, and it is for this purpose superior to Munc18c, which binds both syn2 and syn4 (28).

The present study underlines the usefulness of a molecular modeling approach in analysis of the protein-protein interactions governing vesicle transport and provides new highly specific mutant tools for dissecting the functions of Munc18 proteins in association with different syntaxins and other interaction partners.

    ACKNOWLEDGEMENTS

We thank Seija Puomilahti and Pirjo Ranta for skilled technical assistance, Drs. Arja Band and Esa Kuismanen for the syn4-pGEX2T expression construct, and Dr. Sinikka Eskelinen for the LLC-PK1 cells.

    FOOTNOTES

* This study was supported by the Academy of Finland Grants 50641 and 54301 (to V. M. O.), the Finnish Cultural Foundation (to M. K. and V. M. O.), and the Sigrid Jusélius Foundation (to V. M. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A member of the Helsinki Graduate School of Biotechnology and Molecular Biology.

** Present address: Orion Pharma, P.O. Box 65, FIN-02101 Espoo, Finland.

|| To whom correspondence should be addressed: Dept. of Molecular Medicine, National Public Health Institute, Biomedicum, P.O. Box 104, FIN-00251 Helsinki, Finland. Tel.: 358-9-4744-8286; Fax: 358-9-4744-8960; E-mail: vesa.olkkonen@ktl.fi.

Published, JBC Papers in Press, August 26, 2002, DOI 10.1074/jbc.M208315200

    ABBREVIATIONS

The abbreviations used are: SNARE, soluble NSF (N-ethylmaleimide sensitive factor) attachment protein receptor; t-SNARE, target SNARE; syn, syntaxin; GST, glutathione-S-tranferase; HA, hemagglutinin; mAb, monoclonal antibody; PBS, phosphate buffered saline; SFV, Semliki Forest virus; SM protein, Sec1/Munc18 protein; CHO, Chinese hamster ovary cells; MDCK II, Madin-Darby canine kidney II cells; wt, wild-type.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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