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Originally published In Press as doi:10.1074/jbc.M006710200 on September 7, 2000

J. Biol. Chem., Vol. 275, Issue 47, 36691-36697, November 24, 2000
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A Syntaxin 7 Homologue Is Present in Dictyostelium discoideum Endosomes and Controls Their Homotypic Fusion*

Aleksandra BogdanovicDagger §, Franz BruckertDagger , Takahiro Morio, and Michel SatreDagger

From the From the Dagger  Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, Département de Biologie Moléculaire et Structurale, 38054 Grenoble Cedex 9, France and the  Institute of Biological Sciences, University of Tsukuba, Ibaraki 305-0006, Japan

Received for publication, July 27, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Endo-phagocytic activity is prominent in Dictyostelium discoideum and makes it a good model organism to study the molecular organization of membrane traffic in this pathway. We have identified a syntaxin 7 homologue (26% identity and 54% similarity to human syntaxin 7) in Dictyostelium cDNA and genomic data banks. In addition to the Habc and H3 helices and the C-terminal transmembrane domain characteristic of syntaxins, this protein contains a repetitive N-terminal extension of 68 amino acids. We first showed that Dictyostelium syntaxin 7 was able to form a complex with N-ethylmaleimide-sensitive fusion protein and alpha - and gamma -soluble N-ethylmaleimide-sensitive fusion protein attachment protein. Its intracellular localization was then studied by cell fractionation techniques and magnetic purification of the endocytic compartments. Most of D. discoideum syntaxin 7 is contained in endosomes. Finally, an in vitro endosome homotypic fusion assay (Laurent, O., Bruckert, F., Adessi, C., and Satre, M. (1998) J. Biol. Chem. 273, 793-799) was used to study a possible role for syntaxin 7 in this process. Purified anti-syntaxin 7 antibodies and a recombinant soluble fragment of syntaxin 7 both strongly inhibited fusion activity, indicating that this protein was necessary for endosome-endosome fusion. These results demonstrate the importance of this syntaxin 7 homologue in the early phases of Dictyostelium endo-phagocytic pathway.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Intracellular membrane fusion is a complex and multistage process that requires pairing of specialized membrane proteins called SNAREs,1 carried by both partners of the fusion reaction (1, 2). Three SNARE subfamilies, syntaxin, vesicle-associated membrane protein (VAMP)/synaptobrevin, and SNAP-25, are conserved from yeast to man (3). The structural basis of SNARE pairing is due to the characteristic presence in each SNARE of at least one alpha -helix able to adopt a coiled-coil conformation in association with other SNAREs (4, 5). In neurons, a tripartite SNARE complex is formed from four parallel alpha -helices, one contributed by the syntaxin, one by the synaptobrevin, and two by SNAP-25 (6, 7).

The most diverse and currently best known SNARE family is the syntaxin family. Specific syntaxins are present in almost every compartment undergoing membrane fusion, including endocytic compartments. Yeast syntaxins are organized in a sequential manner along the endocytic pathway, with Tlg1p and Tlg2p in early endosomes (8, 9), Pep12p in the pre-vacuolar compartment (10) and Vam3p in the vacuole (11, 12). A similar spatial organization is also found in Arabidopsis thaliana, where the yeast syntaxin homologues AtPep12p and AtVam3p are present on the prevacuolar and vacuolar compartments, respectively (13, 14). In mammalian cells that recycle plasma membrane receptors by endocytosis, syntaxin 7 is present in early (15, 16) and late endosomes, and its activity is required for efficient transport of internalized fluid-phase marker from early to late endosomes (17). In addition, syntaxin 7 is involved in the heterotypic fusion of late endosomes with lysosomes as well as in the homotypic fusion of late endosomes and lysosomes (18). Syntaxins 8 (19), 11 (20), and 13 (21, 22) are also located in endosomes. However, less is known about syntaxins of the endo-phagocytic pathway. In the macrophagic cell line J744, syntaxins 2, 3, and 4 are mainly found in the plasma membrane, yet they are also present on phagosomes, possibly as a consequence of a passive transport from the plasma membrane (23).

Dictyostelium discoideum is a highly phagocytic amoeba. In axenic strains the level of fluid-phase endocytosis activity is similar to that of professional phagocytic cells, such as activated macrophages (24). The endo-phagocytic pathway of D. discoideum has been well characterized (25, 26). Both fluid and particle internalization involve active actin cytoskeleton reorganization and formation of a phagocytic cup surrounded by coronin (27). Early recycling of the internalized fluid is almost undetectable. The internalized material, first contained in endosomes, is found in acidic lysosomes after 10 min. Undigested material then transits through a neutral post-lysosomal compartment and is released by exocytosis about 45 min after internalization (25, 26). All these endocytic compartments are therefore easily distinguished using different pulse-chase conditions. In addition, D. discoideum endocytic compartments can be purified after iron dextran internalization on a magnetic column (28-30). These advantages, combined with the available molecular tools, make D. discoideum a very attractive model organism to study the organization of the endo-phagocytic pathway at the molecular level.

In this work, we report the identification, localization, and functional study of a D. discoideum homologue of mammalian syntaxin 7. This protein is mainly localized in endosomes and is necessary for the in vitro reconstituted homotypic fusion of endosomes.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Cultures, Reagents, and General Procedures-- D. discoideum strain Ax-2 was cultivated at 21 °C in shaken suspensions (175 rpm) in an axenic medium (31). Exponential phase-growing amoebas (5 × 105 to 1 × 107 cells·ml-1) were harvested by centrifugation (1000 × g, 5 min, 4 °C).

Unless specified, biochemical reagents and chemicals were from Sigma or Roche Molecular Biochemicals. All plasmids and DNA constructions were sequenced on both strands. Protein concentration was determined by the BCA assay (Pierce) with bovine serum albumin as a standard. For Western blot analysis, polypeptides were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose for immunostaining (Mini Protean II, Bio-Rad). D. discoideum syntaxin 7 was detected by rabbit polyclonal antibodies, described in this study. Cathepsin D and vacuolar ATPase V40 subunit were detected by polyclonal and monoclonal antibodies, kindly provided by Dr. Jérôme Garin (Laboratoire de Chimie des Protéines, CEA-Grenoble, France) and Pr. Günther Gerisch (Max-Planck-Institute, Martinsried, Germany), respectively. HRP-conjugated secondary antibodies (Bio-Rad) were revealed by enhanced chemiluminescence reagents (ECL, Amersham Pharmacia Biotech) and Eastman Kodak Co. X-Omat AR film. When needed, the photographic image was digitized using an 8-bit Epson GT5000 linear densitometer and the ImageIn software. Comparing different time exposures ensured that the response was linear. Quantification of proteins of interest was performed after background subtraction with the public domain NIH Image program (developed at the U. S. National Institutes of Health and available on the Internet).

Preparation of His6-Syn782-331-- The His6-Syn782-331-expressing plasmid was build by inserting a polymerase chain reaction-generated fragment of the SLH661 clone in the pQE30 expression vector containing a 5'-polyhistidine-coding sequence (Qiagen). Primers were designed to allow in-frame ligation of the polymerase chain reaction product, so that the resulting recombinant protein is the portion 82-331 of D. discoideum syntaxin 7 fused with a MRGSHHHHHHHGI extension. Escherichia coli M15 cells (Qiagen) were transformed with the His6-Syn782-331-expressing plasmid and grown in Luria-Bertani medium supplemented with 100 µl·ml-1 ampicillin and 50 µg·ml-1 kanamycin up to a cell density of A600 = 0.8. Expression of the recombinant protein was then induced for 2 h at 37 °C with 1 mM isopropyl-thio-beta -D-galactoside (Appligene). Cells were harvested by centrifugation, suspended in lysis buffer (5 mM MgCl2, 50 mM KCl, 2 mM beta -mercaptoethanol, 10 µg·ml-1 leupeptin, 10 µg·ml-1 pepstatin, 25 mM HEPES-KOH, pH 7.5), and disrupted by a French press. The lysate was then clarified and purified on a Ni-nitrilotriacetic acid-agarose column (Qiagen), as described for D. discoideum His6-Rab7 (32). Further purification was performed on a 10-ml Q-Sepharose fast flow column (Amersham Pharmacia Biotech) equilibrated in 25 mM Tris/KOH, pH 8.0, 50 mM NaCl, 1 mM MgCl2, 5 mM beta -mercaptoethanol. The protein of interest was eluted in a salt gradient at 400-500 mM NaCl. In addition to the expected polypeptide, the purified His6-Syn782-331 pool contained two minor degradation products, as checked by MALDI-TOF analysis of their trypsin-digested peptides. After dialysis against phosphate-buffered saline, the recombinant protein was concentrated by centrifugation using the Ultrafree system (Millipore).

Preparation and Affinity Purification of Anti-syntaxin 7 Antibodies-- Two mg of purified His6-Syn782-331 protein were used to raise polyclonal antibodies in a rabbit (Elevage Scientifique des Dombes, Romans, France). An affinity column was made by coupling 1 mg of the same recombinant protein to glutaraldehyde-activated Affi-Gel-102 resin (Bio-Rad) and used to purify anti-syntaxin 7 antibodies from rabbit serum (33).

Preparation of D. discoideum Post-nuclear Supernatant and Membranes-- D. discoideum cells were washed three times in washing buffer (200 mM sucrose, 5 mM glycine-KOH, pH 8.5), suspended at 3.108 cells·ml-1 in breaking buffer (washing buffer supplemented with 1 mM dithiothreitol, 5 µg·ml-1 leupeptin, 5 µg·ml-1 pepstatin, and 2 µg·ml-1 aprotinin), and then broken by six strokes in a ball-bearing cell cracker (34). A post-nuclear supernatant (PNS) was prepared by centrifugation (1000 × g, 5 min, 4 °C). D. discoideum membranes were fractionated as "heavy" (P2) and "light membranes" (P3) by successive centrifugation in breaking buffer: 10,000 × g, 10 min, 4 °C, Beckman JA-20 rotor; 100,000 × g, 30 min, 4 °C, Beckman TL100.3 rotor. When total cell membranes were needed, the PNS was only subjected to the second step of centrifugation.

Sucrose Gradient Fractionation of D. discoideum Membranes-- To label endocytic compartments, D. discoideum cells (109 cells/batch) were pulsed for 5 min at 21 °C with 40 µM pyranine in axenic medium, washed three times in ice-cold washing buffer, then chased in fresh axenic medium for 0, 15, or 60 min at 21 °C. Under these conditions, the fluorescent fluid-phase marker reached endosomes, lysosomes, or post-lysosomes, respectively. For each pulse/chase conditions, total cell membranes were prepared, suspended in breaking buffer, and layered onto 10-ml linear (25-57%, w/v) sucrose gradients, prepared in the breaking buffer. After 3 h of centrifugation in a Beckman SW41 rotor at 100,000 × g, 4 °C, 1-ml fractions were collected from the bottom of the tube. Acid and alkaline phosphatase activities were determined using p-nitrophenyl phosphate. The pyranine concentration was measured by fluorometry (450-nm excitation, 510-nm emission wavelengths) after dilution in 100 mM Tris, pH 10, 0.5% Triton X-100. Proteins of interest were detected and quantified by Western blotting.

Magnetic Purification of Endocytic Compartments-- 109 amoebas were incubated for 90 min in 100 ml of axenic medium containing 1.2 mg·ml-1 superparamagnetic iron dextran (28) and 40 µM pyranine. A PNS was prepared and loaded on a magnetic column (30). The column was washed in the presence of the magnetic field with 150 ml of breaking buffer, releasing unbound material. The retained material was eluted in batch in the absence of the magnetic field by gently agitating the iron meshwork in 100 ml of breaking buffer. Membranous and soluble fractions in the retained- and non-retained materials were separated by centrifugation. P2 membranes were prepared by centrifugation at 10,000 × g, 20 min, 4 °C (Beckman JA-14 rotor), and P3 membranes by centrifugation at 100,000 × g, 30 min, 4 °C (Beckman Ti 70.1 rotor). Membranes were then analyzed for their iron (35), pyranine, and syntaxin 7 contents.

Endosome-Endosome Fusion Assay-- Avidin or biotin-HRP-loaded D. discoideum endosomes were prepared, and fusion assays were performed as described (32). Except where otherwise stated, the fusion reactions were conducted in the presence of 50 µM GTPgamma S, 0.8 mg·ml-1 D. discoideum cytosol, and an ATP-depleting system consisting of 10 units·ml-1 apyrase. To test syntaxin 7 involvement in endosome fusion, purified anti-syntaxin 7 antibodies and/or recombinant His6-Syn782-331 were added to the reaction mix. As a control, equal volumes of the corresponding buffers were added.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Molecular Identification of a Functional Syntaxin 7 Homologue in D. discoideum-- The D. discoideum EST data bank was searched for syntaxin homologues using human syntaxin 7 as a query. Six clones gave significant probability scores. This high number of clones was a clear indication of a well represented mRNA. All clones corresponded to the same cDNA but only their 5'- or 3'-end sequences were present in the data base. The longest member, clone SLH661 (AU039372), was fully sequenced. A putative starting codon was predicted according to the presence of Dictyostelium characteristic A residues at positions -2 and -3 relative to it. Another ATG located 354 base pairs downstream lacked the canonical environment. An AATAA polyadenylation consensus was present near an in-frame putative TAG stop codon, giving a potential coding sequence of 1071 base pairs. A subsequent BLAST search of the D. discoideum genomic data bases allowed the assembly of the corresponding 1302-base pair-long genomic DNA. The genomic sequence contained two short introns, typically AT-rich, the first one of 93 base pairs (nucleotides 375-467) and the second one of 138 base pairs (nucleotides 568-705).

The D. discoideum open reading frame encoded a 356-amino acid-long protein with a calculated molecular mass of 40.2 kDa showing 26% (54%) sequence identity (similarity) to human syntaxin 7. The characteristic syntaxin/epimorphin family signature was present at amino acids (aa) 265-304 (Fig. 1B). Using CLUSTAL W 1.7 software (36), the encoded protein was aligned together with known endocytic syntaxins: human syntaxins 7, 8, 11, and 13, yeast Vam3p, Pep12p, Tlg1p, and Tlg2p, and A. thaliana Vam3p and Pep12p. It showed a clear grouping of Dictyostelium protein with the syntaxin 7 subfamily in the resulting phylogenetic tree (Fig. 1A). A secondary structure was predicted using the Predict-Prot software (37), based on multiple alignment of six members of the syntaxin 7 subfamily (Fig. 1B). The largest part of the Dictyostelium protein (aa 69-356) conforms to a classical syntaxin pattern consisting of four alpha -helices followed by a C-terminal hydrophobic alpha -helical transmembrane domain (aa 334-354). The three alpha -helices (aa 89-111, 119-134, 159-186) correspond to the Habc complex (38). The fourth one (aa 260-291) is able to adopt a coiled-coil structure, as suggested by the presence of characteristic heptad repeats, and corresponds to the H3 domain taking part in the hydrophobic core of the four-helix rod in the SNARE complex (6, 7). In addition, the D. discoideum SLH661-encoded protein presents a 68-aa-long, Q, N-rich N-terminal extension that contains several repeats of the 4-aa motif GGYN. A 17-residue T, N-rich insertion (aa 202-225) is also present between the Hc helix and the core domain. Such insertions and extensions are common in D. discoideum proteins and may provide sites for regulation by threonine or tyrosine phosphorylation (39).


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  		Fig. 1.   Molecular analysis of the D. discoideum protein encoded by the clone SLH661. Human syntaxins 7, 8, 11, and 13 (Syn7, -8, -11, -13-Hs), Saccharomyces cerevisiae Pep12p (Pep12p-Sc), Vam3p (Vam3p-Sc), Tlg1p (Tlg1p-Sc), and Tlg2p (Tlg2p-Sc), A. thaliana Pep12p (Pep12p-At) and Vam3p (Vam3p-At) analogs and the polypeptide encoded by clone SLH661 were aligned using CLUSTAL W 1.7 software (36). A, phylogenetic relationship between endosome syntaxins. The numbers indicate the percentage of identical amino acids relative to SLH661. A tree was generated using the NJ-Plot software (57). B, alignment of D. discoideum SLH661-encoded protein and human syntaxin 7. The four domains of the protein are indicated: the N-terminal extension (aa 1-68; numbering refers to SLH661), the regulatory Habc domain (aa 69-201), the core domain (aa 226-333), and the transmembrane C-terminal domain (aa 334-354). The position of the Ha, Hb, Hc, and H3 helices was determined by the Predict-Prot software (37) using several syntaxin 7-like proteins (Syn7-Hs, Pep12p-Sc, Pep12p-At, Vam3p-Sc and Vam3p-At) and is indicated by light shading at positions where the output of the program was identical in four out of the six sequences. In the two proteins, hydrophobic residues along the heptad repeat of the potential coiled-coil structure are shown in bold, the syntaxin/epimorphin PROSITE signature (entry PS00914 in PROSITE data base obtained from the Internet) is underlined, and the unique glutamine residue conserved in all syntaxins is emphasized in black.

To develop molecular tools to study the intracellular localization and function of the putative D. discoideum syntaxin 7, its conserved cytoplasmic portion (residues 82-331) was expressed in bacteria as an His6 fusion protein (His6-Syn782-331) and was used to raise rabbit polyclonal antibodies (anti-syntaxin 7). Western blot analysis of the PNS, cytosol, and total membrane preparations revealed essentially the presence of a 43-kDa polypeptide enriched in the membrane fraction, but absent in the cytosol, as expected (Fig. 2). Detection of this polypeptide band was prevented by preincubating antibodies with an excess of His6-Syn782-331 recombinant protein (data not shown). These antibodies were affinity-purified on an His6-Syn782-331 column covalently coupled to protein A-agarose beads and used in immunoprecipitation experiments. A specific 43-kDa polypeptide was pulled down from a detergent-solubilized membrane extract2 whose peptide mass map, analyzed by MALDI-TOF, corresponds to the protein encoded by SLH661. These results establish the specificity of the purified antibodies. Furthermore, one of the peptides () comes from the sequence before the Met-119, confirming the position of the initial AUG codon and proving the existence of the N-terminal extension of the protein.


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  		Fig. 2.   Existence of a complex between D. discoideum syntaxin 7, alpha - and gamma -SNAP, and NSF proteins. Membrane (M) and cytosolic (C) extracts of NSF-myc-overexpressing (NSF-myc+) and control cells (WT) were prepared by centrifugation. Anti c-myc immunoprecipitation was carried out of detergent-solubilized extracts in the presence of ATP and EDTA as described previously (41). Stochiometric amounts of alpha - and gamma -SNAP co-immunoprecipitated (IP) with NSF-myc in the NSF-myc+ membrane extract (data not shown). The presence of syntaxin 7 in the starting material (corresponding to 5 × 106 cells) and in the immunoprecipitated material (corresponding to 2 × 108 cells) was revealed by Western blotting with anti-syntaxin 7 antibodies.

SNAREs are known to form stable complexes with NSF and SNAP proteins in the absence of ATP (40). A cell line expressing a c-myc-tagged form of D. discoideum NSF (NSF-myc+) was used to test for the presence of the putative syntaxin 7 in such complexes. Immunoprecipitation was performed on NSF-myc+ cell extract using 9E10 anti-myc antibody (Roche Molecular Biochemicals) in the same conditions under which alpha - and gamma -SNAP co-immunoprecipitate with NSF (41). The presence of the D. discoideum syntaxin 7 was revealed by Western blotting using the anti-syntaxin 7 antibodies described above. As shown in Fig. 2, this protein is pulled down in the membrane fractions originating from NSF-myc+ cells, which proves its specific interaction with NSF and alpha - and gamma -SNAP.

All of the above evidence therefore strongly indicate that the SLH661-encoded protein is a functional D. discoideum SNARE and a member of the syntaxin 7 subfamily. This protein will thus be designated as syntaxin 7.

D. discoideum Syntaxin 7 Is Associated with Membranes of Low Density-- To study syntaxin 7 intracellular localization, high and low density D. discoideum membranes were separated by differential centrifugation. A PNS was first centrifuged at 10,000 × g for 10 min to pellet high density membranes (P2), which were enriched in mitochondria, lysosomes, the Golgi apparatus, and remnants of the contractile vacuole apparatus meshwork known as acidosomes. A low density membrane pellet (P3) was obtained by further centrifugation of the P2 supernatant (S2) at 100,000 × g for 30 min. These membranes contained part of the endocytic apparatus, the contractile vacuole bladder, and the plasma membrane. Both P2 and P3 pellets were probed for the presence of syntaxin 7 by immunoblotting with anti-syntaxin 7 antibodies (Fig. 3). By quantitative densitometry, it was determined that about 70% of D. discoideum syntaxin 7 is recovered in the fraction of lower density.


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  		Fig. 3.   Differential centrifugation of D. discoideum membranes. A D. discoideum PNS was fractionated into high density (P2) and low density (P3) membrane pellets and cytosol by successive centrifugation. The amounts of syntaxin 7 in the protein material corresponding to 2.105 (PNS) or 8.106 (P2, P3) cells were quantified by Western blotting, ECL detection and densitometry scanning of the film. On this specific experiment, 63 and 37% of syntaxin 7 were found in the P3 and P2 pellets, respectively.

D. discoideum Syntaxin 7 Is Present in Endocytic Compartments-- To determine whether syntaxin 7 has an endocytic localization in D. discoideum, we used a magnetic purification technique that allows a single step preparation of endocytic compartments (28-30). D. discoideum cells were incubated in the presence of iron dextran and pyranine for 3 h to fill the endocytic space. A PNS was prepared and loaded onto an iron meshwork placed in an intense magnetic field. Nonspecifically bound material was washed out, then the magnetic field was removed, and the specifically retained material was recovered in batch. The purified vesicles were pelletized under conditions similar to that of a P2 pellet. Such low speed centrifugal forces are required to maximize vesicular integrity. Since we previously noted that most of syntaxin 7 was present in low density membranes, an additional high speed centrifugation step was performed on the S2 supernatants. Therefore, both retained and non-retained material were fractionated into P2 and P3 pellets and S3 supernatant as described above, and these fractions were analyzed for the presence of syntaxin 7, pyranine, acid phosphatase activity, and iron dextran (Fig. 4, A and B).


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  		Fig. 4.   Syntaxin 7 is associated to purified endocytic compartments. A. magnetic purification of D. discoideum endocytic pathway. Iron dextran (1.2 mg/ml) and pyranine (40 µM) were internalized by amoebae for 3 h, and a PNS was prepared and loaded on a magnetic column (30). The iron-containing vesicles were retained by the strong magnetic field, whereas the non-retained material was washed out in the flow-through. The retained material was eluted after the removal of the magnetic field. The membranous P2 and P3 fractions of the retained and non-retained material was recovered by two successive centrifugations (P2: 10,000 × g, 20 min; P3: 100,000 × g, 30 min) and analyzed for the presence of iron dextran, pyranine, total protein, acid phosphatase, and syntaxin 7. The results are normalized to the amount contained in the PNS. Note that 14 and 51% of pyranine were recovered in the S3 supernatants of the retained and non-retained material, respectively. B, the distribution of syntaxin 7 in the P2 and P3 fractions of the retained and non-retained material was detected by Western blotting using 1% of the starting material. The results of densitometry scanning are shown in A. C, syntaxin 7-containing compartments shift density upon iron dextran loading. The P2 and P3 membrane pellets and S3 cytosolic supernatant were prepared out of 109 cells preincubated for 1 h with (right lanes) or without (left lanes) 1.2 mg/ml iron dextran. 1% of each fraction was analyzed by Western blotting along with 0.2% of the PNS. Scanning densitometry of the blot shows that the P2 and P3 fractions from control cells contain 30 and 70% of the syntaxin 7, respectively, whereas those from iron dextran-loaded cells contain 85 and 15%, respectively.

The low speed pellet of the retained material contains purified endocytic compartments, as shown by the relative enrichment in iron dextran, pyranine, and acid phosphatase. More than 40% of total D. discoideum syntaxin 7 was also present in this fraction, whereas it contained only 5% of total proteins, corresponding to an 8-fold enrichment. The remaining syntaxin 7 was contained in the P2 and P3 pellets of non-retained material. The presence of small amounts of iron dextran, pyranine, and acid phosphatase activity in these fractions indicated that about 10% of endocytic compartments were not retained by the magnetic column. The majority of the syntaxin 7 not retained on the column is present in the P3 pellet. This pool likely corresponds to the endocytic compartments that have been broken, since 51% of the initial pyranine cell content were released in the S3 supernatant of the non-retained material.

Finally, the endocytic localization of syntaxin 7 was shown directly, taking advantage of the large increase in density that iron dextran internalization causes to the endocytic compartments (Fig. 4C). When the cells were not loaded with iron dextran, 70% of syntaxin 7 was present in the P3 pellet, whereas for cells loaded with iron dextran, only 15% of syntaxin 7 was in this fraction. This proves that at least 55% of D. discoideum syntaxin 7 is present in compartments whose density changes upon iron dextran loading.

Most of D. discoideum Syntaxin 7 Co-fractionates with Endosomes on Linear Sucrose Gradients-- To refine syntaxin 7 localization among the various endocytic compartments, D. discoideum membranes were further fractionated on a 25-57% linear sucrose density gradient. To distinguish the different endocytic compartments, three separate batches of D. discoideum cells were pulsed for 5 min with 40 µM pyranine, a fluorescent fluid-phase marker, and then chased for either 0, 15, or 60 min. Under these pulse-chase conditions, the fluid-phase marker is mainly contained in endosomes, lysosomes, or post-lysosomes, respectively (25, 26). Total cell membranes were then prepared in parallel and fractionated onto linear sucrose gradients.

The sharp pyranine peaks in the gradients obtained after a 15- or 60-min chase time indicated that the lysosomal and post-lysosomal compartments were concentrated at the bottom of the gradient (Fig. 5B, fractions 2-5). Both acid phosphatase and cathepsin D, two well known lysosomal marker enzymes (42, 43), were also found in the same portion of the gradient, confirming the presence of lysosomes in these high density fractions (Fig. 5, A and C). Endosomes labeled by a short pyranine pulse without chasing were contained in a broad peak encompassing fractions 5 to 9 of the gradient, well separated from lysosomes and post-lysosomes (Fig. 5B). Alkaline phosphatase activity was found in two peaks centered on fractions 5 and 9 (Fig. 5A). The distribution of this marker of the contractile vacuole apparatus and of the plasma membrane (44, 45) was quite different from that of fluid-phase markers. The sucrose density and the relative enrichment in the vacuolar ATPase C subunit around fraction 9 (Fig. 5C) confirmed that contractile vacuole bladder elements were indeed present in this peak (46). Finally, the presence of some acid phosphatase and cathepsin D in the top fractions of the gradient was due to the release of these soluble proteins from broken vesicles, as indicated by the presence of 25-30% of the fluorescent fluid-phase marker in the same fractions.


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  		Fig. 5.   Fractionation of D. discoideum membranes on linear sucrose gradient. Pyranine, a fluorescent fluid-phase marker, was internalized for 5 min and chased for 0, 15, and 60 min to label endosomes, lysosomes, and post-lysosomes, respectively. Total membranes were fractionated on a linear sucrose gradient. After 3 h of centrifugation at 100,000 × g, fractions were collected from the bottom of the tube, and an equal volume of each fraction was analyzed. A, acid () and alkaline (open circle ) phosphatase activities were determined using p-nitrophenyl phosphate. The sucrose concentrations are represented by the dotted line. B, the concentration of pyranine after 0 (black-triangle), 15 (black-square)-, and 60 ()-min chase was determined by fluorometry. C, fractions were analyzed by Western blotting for their content in syntaxin 7, cathepsin D, and vacuolar ATPase (v-ATPase) V40 subunit. The relative distribution of syntaxin 7 over the gradient was estimated by scanning densitometry of the film and is represented by asterisks in B.

Western blot analysis showed that syntaxin 7 was distributed over the upper half of the gradient, with 85% of the protein contained in fractions 5-9. There was therefore little overlap in the syntaxin 7, lysosome, and post-lysosome profiles. The distribution of syntaxin 7 shows a striking parallel to that of endosomes, except for fractions 9 to 11. The presence of pyranine in fractions 10 and 11 was due to vesicle breakage. This masked the endosome distribution, which was likely to go to zero, as observed for syntaxin 7. In fraction 9, the syntaxin 7 distribution departed significantly from that of endosomes, indicating the existence of a second minor (15%) overlapping peak. This could possibly correspond to broken endosomal membranes or arise from another cellular localization, as the density corresponds to that of the plasma membrane or contractile vacuole elements. In summary, these data show that the vast majority of the D. discoideum syntaxin 7 distribution correlates to that of endosomes.

Syntaxin 7 Is Required for D. discoideum Endosome Homotypic Fusion-- The presence of syntaxin 7 in endosomes prompted us to test whether this protein was involved in their homotypic fusion, a step of the endocytic traffic that can be reconstituted in vitro (32). In this test, avidin- and biotin-HRP-loaded endosomes were prepared and mixed in the presence of cytosol and GTPgamma S to allow fusion (50-µl volume). Upon membrane fusion, the resulting avidin-biotin-HRP complexes were immobilized in anti-avidin-coated enzyme-linked immunosorbent assay plates and quantified by the HRP enzymatic activity. First, purified anti-syntaxin 7 antibodies were added to the assay. As shown in Fig. 6A, the addition of 1 µg of antibodies to the fusion test resulted in an almost complete inhibition, bringing the fusion level to that of fusion performed at 0 °C. Addition of the cytoplasmic portion of the syntaxin 7 that was used for immunization (His6-Syn782-331) was also inhibitory (Fig. 6B). However, in the presence of anti-syntaxin 7, the addition of His6-Syn782-331 reversed the inhibition induced by antibodies (Fig. 6B). By normalizing the fusion activity obtained in the presence of both anti-syntaxin 7 and His6-Syn782-331 to that obtained in the presence of the syntaxin 7 fragment only, it can be shown that 0.1 µg of His6-Syn782-331 was able to neutralize 50% of the inhibition due to 0.1 µg of anti-syntaxin 7 (Fig. 6C). With a 10-fold molar excess in soluble fragment, the antibody inhibition was completely reversed. These data strongly indicate that syntaxin 7 takes part in the SNARE complex involved in the homotypic fusion process of endosomes.


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  		Fig. 6.   Syntaxin 7 is required for D. discoideum endosome-endosome fusion. A, anti-syntaxin 7 antibodies inhibit endosome homotypic fusion. Indicated amounts of affinity-purified anti-syntaxin 7 antibodies were added to in vitro reconstituted endosome fusion assays (50-µl volume) carried out in the absence of ATP (32). The reactions were preincubated for 15 min on ice before a 45-min incubation at 21 °C. Separate fusion reactions were also conducted in the absence of cytosol (a), or GTPgamma S (b), or at 0 °C (c). In d, 4 µg of anti-syntaxin 7 antibodies were added at the end of the 21 °C incubation, showing that these antibodies did not interfere with avidin- biotin-HRP detection. B, the addition of the soluble His6-Syn 782-331 fragment inhibits endosome fusion and reverses the effect of anti-syntaxin 7 antibodies. The fusion reaction was conducted in the presence of 0 () or 0.1 µg (black-square) of purified anti-syntaxin 7 antibodies and the indicated amount of purified recombinant His6-Syn782-331. Buffers alone were without effect. C, the relative inhibition of the antibodies at a given His6-Syn 782-331 concentration is calculated as the ratio of the specific activity (fusion activity at 21 °C minus fusion activity at 0 °C) obtained in the presence of the antibodies (Ab+) to the one obtained in their absence (Ab-). Data are from B. The solid line is hand-drawn. a.u., absorbance units.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Using D. discoideum as a model of phagocytic cells, we show evidence for the presence and activity of a SNARE homologous to mammalian syntaxin 7 in early endo-phagocytic compartments. D. discoideum syntaxin 7 bears 54% overall similarity with its human homologue and is able to form a complex with endogenous NSF and SNAP proteins. At this stage of the D. discoideum genome sequencing, this is the only member of the syntaxin 7/13 subfamily, in contrast with mammals, which at least contain two. Therefore, the syntaxin nomenclature might be refined as the complete set of D. discoideum syntaxins is identified.

Magnetically purified D. discoideum endocytic compartments contain about 50% of syntaxin 7. This endocytic localization is directly confirmed by a density shift of the compartments bearing syntaxin 7 upon loading of the endocytic pathway with iron dextran. As about half of the endocytic vesicles are broken during the purification procedure, it is likely that the proportion of syntaxin 7 contained in endocytic compartment is, in fact, higher. On linear sucrose gradients, the syntaxin 7 distribution extensively coincides with endosome-containing fractions. In D. discoideum, this compartment hosts fluid-phase markers during the first 10 min of internalization and is distinguishable from the remainder of the endocytic pathway by its low density, Rab7 enrichment and its ability to undergo homotypic fusion (32). Endosomes therefore constitute the main cellular localization for this protein in Dictyostelium.

The SNARE hypothesis states that proteins from separate compartments engage in a complex-catalyzing membrane fusion (1). This indirectly implies that in steady state conditions, some of the SNAREs should recycle back from the post-fusion to the pre-fusion compartment. Consequently, it is necessary to distinguish compartments where syntaxins are present from those where they are active. In vitro reconstitution of D. discoideum endosome homotypic fusion indicates that this process requires syntaxin 7, since fusion activity is specifically inhibited by the addition of anti-syntaxin 7 antibodies at nM concentrations. D. discoideum endosome homotypic fusion is also inhibited by larger concentrations of His6-Syn782-331. A similar inhibitory effect of the cytosolic portion of mammalian syntaxin 7 has been reported on the routing of internalized fluorescein isothiocyanate dextran (17) and on in vitro reconstituted lysosome fusion (18). This inhibition is likely to correspond to the substitution of the soluble syntaxin 7 fragment containing the H3 helix to endogenous syntaxin 7, resulting in unproductive SNARE complexes. These functional results prove that endosomes are therefore not only the main syntaxin 7 localization but also its activity zone.

The presence of syntaxin 7 or molecular analogues has been reported in the receptor-mediated endocytic pathway of various cells, from yeast to mammals. Evidence was given for its presence on late endosomes and its involvement in endo-lysosomal traffic (17, 18). In some cells, syntaxin 7 colocalizes with the transferrin receptor, the hallmark of early sorting endosomes (15, 16). Therefore, no consensus has yet emerged as to the exact syntaxin 7 localization in mammalian cells. In yeast, two syntaxin 7-like proteins organize the route from the early endosomes to the vacuole, with Pep12p present on prevacuolar compartments and Vam3p on the vacuole (3). The activity of both proteins partially overlaps, as shown by the rescue of a Pep12 null mutant phenotype, by the overexpression of Vam3p (47). Taking into account all these observations, a conserved role for syntaxin 7 can be proposed. Syntaxin 7 would be the target SNARE, allowing entry of internalized material into degradative compartments, and this in cooperation with Rab7. Thus, beside homotypic fusion, syntaxin 7 could also mediate fusion of D. discoideum endosomes with vesicles carrying components necessary for their maturation, such as proteases and vacuolar ATPase. The existence of such vesicles in D. discoideum has already been suggested by the study of Rab7 mutants (48, 49). Given the similarity between D. discoideum and professional phagocytes, it is likely that syntaxin 7 plays similar roles in the endo-phagosomes of mammalian phagocytes, organelles where homotypic as well as heterotypic fusions take place (50, 51).

It is noteworthy that the routes of syntaxin 7 and of the fluid-phase cargo separate early. Fluid-phase markers pass into lysosomes after 15 min, whereas this compartment contains less than 20% of cellular syntaxin 7. This suggests either the existence of a retention mechanism that prevents the majority of syntaxin 7 from reaching lysosomes or the presence of a recycling pathway that efficiently retrieves syntaxin 7. In this respect, incorporation of Vam3p into AP3-type coats through a di-leucine motif has already been suggested to explain the different locations of Vam3p and Pep12p in yeast (52). A similar motif is recognizable in Dictyostelium syntaxin 7 at the same position (EHQSLM: 235-240). The large N-terminal extension of D. discoideum syntaxin 7 might also constitute a support for a specific targeting motif.

The D. discoideum endosome fusion assay used in this study is performed in the presence of an ATP-depleting system, keeping the bulk ATP concentration below 1 nM (32). The sensitivity of this assay to anti-Rab7 antibodies (32) as well as to anti-syntaxin 7 antibodies and to the soluble cytoplasmic syntaxin 7 fragment confirms that the general docking and fusion mechanism involving Rabs and SNAREs applies under these special circumstances, where ATP exchange from the bulk solution is prevented. Accordingly, the group of J. E. Rothman (53-55) demonstrate that the presence of unpaired cognate SNAREs on separate membranes constitutes the minimum requirement for docking and fusion, without any need for NSF ATPase activity at this stage. Nevertheless, in the case of the fusion reaction reconstituted here, supplementary requirements are Rab7 activation by GTPgamma S and Mg2+ and the participation of soluble components brought up by the cytosol. Furthermore, the mechanisms of endosome fusion in D. discoideum and vacuolar fragment fusion in S. cerevisiae are strikingly parallel. Both processes are indeed activated by closely related GTPases, Rab7 and Ypt7, and involve target SNAREs of the syntaxin 7 subfamily, syntaxin 7 and Vam3p. The Dictyostelium endosomes, whose fusions are reconstituted in this assay, are therefore caught at the intermediate stage where SNARE proteins are "primed," skipping the ATP-dependent "priming" step (55, 56).

The syntaxin 7 localization coincides with the ability of endocytic compartments to fuse. It is therefore tempting to assume that the presence of syntaxin 7 controls the level of fusion activity, explaining why D. discoideum endosomes can undergo homotypic fusion, whereas lysosomes cannot (32). In this context, identifying protein partners that interact with D. discoideum syntaxin 7 could help to gain further insight into its function.

    ACKNOWLEDGEMENTS

We gratefully thank Drs. Günther Gerisch and Jérôme Garin for providing antibodies and hybridomas and Laurence Aubry for generous advice and many helpful discussions. We are deeply indebted to Dr. Jérôme Garin and Sylvie Kieffer for MALDI-TOF analysis of recombinant and immunoprecipitated syntaxin 7. The D. discoideum genomic sequence data used in this work were produced by the Dictyostelium Sequencing Group at the Sanger Center, the Dictyostelium Genome Sequencing Project at Jena, and at Baylor College of Medicine and are available on the Internet.

    FOOTNOTES

* This work was supported by the Université Joseph Fourier-Grenoble, the CNRS, and the Commissariat à l'Energie Atomique.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.

§ To whom correspondence should be addressed: DBMS/BBSI (UMR5092), CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 09, France. Tel.: 33 476 88 54 19; Fax: 33 476 88 44 99; E-mail: abogdanovic@cea.fr.

Published, JBC Papers in Press, September 7, 2000, DOI 10.1074/jbc.M006710200

2 A. Bogdanovic., F. Bruckert, S. Kieffer, J. Garin, T. Morio, and M. Satre, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: SNARE, soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptor; PNS, post-nuclear supernatant; SNAP-25, synaptosome-associated protein of 25 kDa; HRP, horseradish peroxidase; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight spectroscopy; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; aa, amino acids.

    REFERENCES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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