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1 Department of Anatomy and Neurobiology, Kyoto University Graduate School of Medicine, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
2 Laboratory for Cellular Morphogenesis, RIKEN Centre for Developmental Biology, Minatojima-minamimachi 2-2-3, Chu-o-ku, Kobe, 650-0047, Japan
3 Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan
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Introduction |
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Septins were first identified as the products of 4 cell division-related genes (CDC3, CDC10, CDC11 and CDC12) in budding yeast (Hartwell 1971; Longtine et al. 1996). Each of these gene products is present at the mother-bud neck in the wild-type strain, and temperature-sensitive mutations of any one of these four genes were found to cause a defect in bud-neck filament formation and to produce multinucleate cells with abnormal bud growth at the non-permissive temperature (Hartwell 1971; Byers & Goetsch 1976; Haarer & Pringle 1987; Kim et al. 1991; Ford & Pringle 1991). Immuno-isolated septin complexes filaments contained the above four proteins in an approximately 1:1:1:1 ratio and had a width of about 7 nm and lengths in multiples of 32 nm, suggesting that the bud-neck filaments may be composed of multiples of a unit complex (Frazier et al. 1998).
In Drosophila melanogaster, septins are also required for cytokinesis, at least in some cell types. (Neufeld & Rubin 1994; Adam et al. 2000). Like the yeast septin complex, immuno-isolated Pnut complexes contain at least two other septins, Sep1 and Sep2, which are in approximately 1:1 ratios with Pnut. Moreover, these complexes have a width of 7–9 nm and lengths in multiples of 26 nm (Field et al. 1996).
In mammals, more than 10 septin genes have been identified, many of which appear to undergo alternative splicing to produce multiple protein products (Trimble 1999; Field & Kellogg 1999; Kartmann & Roth 2001; Macara et al. 2002). Similar to those of yeast and Drosophila, the mammalian septins form hetero-oligomers (Hsu et al. 1998; Surka et al. 2002; Sheffield et al. 2003) and the mammalian septin Sept2/Nedd5 is necessary for cytokinesis (Kinoshita et al. 1997). However, mammalian septins might have functions distinct from their role in cytokinesis because many of them are expressed in postmitotic tissues such as the brain. Moreover, some septins, such as Sept5/CDCrel-1, are expressed almost exclusively in the brain (Caltagarone et al. 1998; Beites et al. 1999; Xue et al. 2000). Sept5 is involved in vesicle trafficking (Beites et al. 1999) and is a substrate of the E3 ubiquitin ligase Parkin, a causative agent in autosomal recessive forms of Parkinson's disease (Zhang et al. 2000).
Sept2 was originally isolated as a gene that was highly expressed in mouse neuronal precursor cells and down-regulated in the adult brain (Kumar et al. 1992). Sept2 and two other mammalian septins, Sept1/Diff6 and Sept4/H5, have been identified in neurofibrillary tangles in brains affected by Alzheimer's disease (Kinoshita et al. 1998). However, Sept2 expresses rather ubiquitously (Kinoshita et al. 1997). We found Sept2 is expressed mainly in astrocyte processes in normal adult mouse brain, especially in the cerebellar molecular layer. Bergmann glia is a characteristic astrocyte in the cerebellum. It is considered that Sept2 might be concerned with Bergmann glial function in molecular layer of the cerebellum. It is well known that GLAST, an astrocyte glutamate transporter, is expressed predominantly in Bergmann glial cell processes (Lehre et al. 1995; Chaudhry et al. 1995). In the present study, co-localization of Sept2 with GLAST and molecular interactions between Sept2 and GLAST were demonstrated for the first time.
Glutamate is the major excitatory neurotransmitter in the mammalian CNS (Fonnum 1984). Glutamatergic transmission is ultimately terminated by the binding of the released glutamate to its transporters and subsequent uptake into mainly glial cells and partially neurones. Glutamate uptake is an important role of glial cells. Glutamate uptake is accomplished mainly by a family of Na+-dependent, high-affinity glutamate transporters. Five subtypes of glutamate transporter have been identified: GLAST (excitatory amino acid transporter 1, EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5 (Storck et al. 1992; Pines et al. 1992; Kanai & Hediger 1992; Fairman et al. 1995; Arriza et al. 1997). GLAST and GLT-1 are localized in astrocytes (Danbolt et al. 1992; Rothstein et al. 1994), whereas EAAC1 (He et al. 2000), EAAT4 (Dehnes et al. 1998), and EAAT5 (Arriza et al. 1997) are localized in neurones. Although their distributions and enzymatic properties have been studied in detail (Danbolt 2001), little is known about the regulation of these enzymes or their interactions with other proteins.
The present study showed that Sept2 interacts with the astrocyte glutamate transporter GLAST in a GDP-dependent manner and that expression of constitutive GDP-form Sept2 mutant reduces glutamate uptake activity of GLAST. This finding suggests that Sept2 can be a regulator of glial glutamate uptake through interaction with GLAST, and that Sept2 may play an important role in providing an appropriate environment for efficient signal transmission in the cerebellum.
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Results |
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To explore the functions of septins in the mammalian brain, we first generated Sept2-specific antibody (Fig. 1A,B) and examined the localization of Sept2 in the mouse brain. Immunohistochemistry showed prominent staining of Sept2 in the molecular layer of the cerebellum (Fig. 1C). To identify the localization in detail, we next examined the subcellular localization of Sept2 in the cerebellar molecular layer by using immunogold electron microscopy. Gold particles were localized along the thin cell processes of astrocytes surrounding synapses and the parallel fibers near the axon terminal (Fig. 1E,F). As all astrocyte processes in the molecular layer of the cerebellum are known to belong to characteristic astrocytes called Bergmann glia, which are essential for the maturation and survival of Purkinje cells, Sept2 appeared to be localized predominantly in Bergmann glial processes facing synapses and axons.
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Due to the co-localization of Sept2 and GLAST indicated by immunofluorescence and immuno-electron microscopy, we next tested for possible protein interaction between Sept2 and GLAST. Immunoprecipitation assays using mouse brain membrane extracts showed complex formation between Sept2 and GLAST in vivo (Fig. 3A). Another mammalian septin, Sept4, was also co-immunoprecipitated with GLAST (Fig. 3A). In addition, the distribution of Sept2 was similar to that of Sept4 and Sept7 (other mammalian septins) reported from a previous study (Kinoshita et al. 2000). These facts suggest that septin hetero-complexes including Sept2 and Sept4 might interact with GLAST. All astrocytes are considered to express both GLAST and GLT-1, and we could also detected another astrocyte glutamate transporter, GLT-1, in Sept2-immunoprecipitates (data not shown). However, their regions of predominant expression in brain are different (Chaudhry et al. 1995). In the cerebellar cortex, GLAST expression is high, and that of GLT-1, low. Thus, the interaction of Sept2 with GLAST may be more significant physiologically than that with GLT-1. So we focused on the interaction between Sept2 and GLAST. Sept2–GLAST interaction was also detected by an immunoprecipitation assay with transfected COS7 cells (Fig. 3B) and by Sept2 affinity column chromatography with bacterial recombinant proteins (Fig. 3C). Furthermore, Sept2 affinity column chromatography showed that the carboxy-terminal region of GLAST (GLAST-CT; aa 341–544) bound to Sept2, but that the amino-terminal region containing six transmembrane domains (GLAST-NT; aa 1–340), did not (Fig. 3C). An immunoprecipitation assay with transfected COS7 cells also showed that GLAST-CT could interact with Sept2 (Fig. 3D). Purification of recombinant full length GLAST is very difficult because of its insolubility, but GST-GLAST-CT protein can be purified. The in vitro binding assay by using purified GLAST-CT and purified His6-tagged Sept2 also showed the Sept2–GLAST interaction (Fig. 3E). These results indicate that Sept2 binds directly to the carboxy-terminal region of GLAST.
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As septins are a family of GTPase proteins, we next focused on the guanine nucleotide dependency of the Sept2–GLAST interaction. As little is known about the septin guanine nucleotide reaction, we measured the guanine nucleotide-binding ability of Sept2 (Fig. 4A). Sept2-G47V is produced by the mutation within the G1 motif of the glycine at amino acid 47 to valine. The lack of GTP-binding ability and dominant inhibition of Sept2 septin filament formation of this mutant in HeLa and NIH-3T3 cells was reported earlier (Kinoshita et al. 1997). A similar mutant of another mammalian septin CDCrel-1 also behaved as a constitutive GDP-form or dominant-negative form (Beites et al. 1999). Indeed, Sept2-G47V had no GTP-binding ability and also showed no GDP-binding ability (Fig. 4A).
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S-bound GST-Sept2 showed significantly lower binding ability toward GLAST. Considering that the ratio of GTP-bound GST-Sept2 per total GST-Sept2 was at most about 0.4 (Fig. 4A), the difference in GLAST-binding ability between the GDP-form and GTP-form of Sept2 indicates that the GTP-form of Sept2 interacts very poorly with GLAST and the principal Sept2 interaction with GLAST is via the GDP-form. The results of an in vitro binding assay using radioisotope-labelled guanine nucleotide showed clearly this nucleotide dependency (Fig. 4C). [3H]GDP-bound Sept2 interacted with GLAST-CT, whereas [-35S]GTPS-bound Sept2 showed no binding ability. Sept2-G47V had no guanine nucleotide-binding ability; i.e. Sept2-G47V is a constitutive guanine nucleotide-unbound form of Sept2 (Fig. 4A). Nucleotide-unbound Sept2 and GDP-bound Sept2 showed equal GLAST-binding abilities (Fig. 4B). These results suggest that Sept2-G47V can be regarded as the constitutive GDP-form of Sept2 at least in terms of the interaction with GLAST. Immunoprecipitation assays with transfected COS7 cells showed that Sept2-G47V bound to GLAST more efficiently than did Sept2-WT in COS7 cells (Fig. 4D). These results suggest that the Sept2–GLAST interaction is also dependent on the guanine nucleotide binding state of Sept2 in vivo.
Sept2 modulates glutamate uptake activity of GLAST
To analyse the functional effects of the interaction between Sept2 and GLAST, we carried out glutamate uptake assays with COS7 cells co-expressing Sept2 and GLAST. In a time-course experiment, the amount of transported glutamate was modestly decreased by Sept2-WT and significantly decreased by Sept2-G47V at all time points tested (Fig. 5B). Expression of Sept2-WT or -G47V did not affect the expression of GLAST (Fig. 5A). Kinetic analysis of transfected COS7 cells showed that Sept2-G47V decreased the maximal velocity (Vmax) of glutamate uptake activity without a shift in glutamate affinity (Michaelis constant [Km]; Fig. 5C).
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Discussion |
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We showed in this study that Sept2 co-localizes with GLAST in thin Bergmann glial processes surrounding synapses and axons in the cerebellum. Other mammalian septins (Sept4 and Sept7) are reported to show a similar localization (Kinoshita et al. 2000). Also, both Sept2 and Sept4 were co-immunoprecipitated with GLAST from mouse brain membrane extracts. These facts suggest that septin hetero-complexes might interact with GLAST in vivo. The results of affinity column chromatography experiments and in vitro binding assays indicate that Sept2 can bind directly to the C-terminal of GLAST in a GDP form-dependent manner. Expression of constitutive GDP-form mutant of Sept2 (Sept2-G47V) decreased the maximal velocity of glutamate uptake activity of GLAST in COS7 cells and the primary culture of Bergmann glial cells. We also showed the internalization of GLAST from plasma membrane in the Sept2-G47V-infected Bergmann glial cells.
Expression level of glutamate transporters on the cell surface is a major regulatory factor of glutamate uptake activity (Danbolt 2001), and expression of GLAST at the cell surface is dependent on the cytoskeleton immediately beneath the plasma membrane (Duan et al. 1999). In another study, it was shown that GTRAP41 and GTRAP48, both EAAT4-interacting molecules, promoted EAAT4 activity by contributing to the stabilization of EAAT4 at the cell surface (Jackson et al. 2001). The authors of that study showed that GTRAP48 was a guanine nucleotide exchanging factor (GEF) for Rho and that GTRAP41 was a possible actin-binding protein, suggesting that stabilization of EAAT4 at the cell surface may be achieved by interaction with actin filaments and the small G-protein Rho. On the other hand, it has been proposed that glutamate up-regulates the maximal velocity (Vmax) of the astrocyte glutamate transporter without modulation of cell-surface expression or protein synthesis (Munir et al. 2000), although the mechanism is unclear.
It is known that mammalian septins form hetero-polymer filaments under normal culture conditions, which structures interact with the actin filaments immediately beneath the plasma membrane (Kinoshita et al. 1997). The polymerization and/or the stability of septin filaments are proposed to depend on their guanine nucleotide binding state. GTP-binding is required for polymerization of Sept2 (Mendoza et al. 2002), and expression of constitutive GDP-form mutant of Sept2 causes disruption of septin filaments (Kinoshita et al. 1997). These studies suggest that GTP hydrolysis of septin may lead to disassembly of septin filaments.
The present study suggests that the expression of Sept2-G47V (GDP-form Sep2) may disrupt septin filaments, causing destabilization of the membrane cytoskeleton. The binding of GLAST to GDP-form Sept2, together with the cytoskeletal instability might lead to internalization of GLAST from cell surface, resulting in the reduction of glutamate uptake by Bergmann glial cells.
Based on this consideration, we postulate as follows: The GDP-form Sept2 binds to GLAST and inactivates it. The binding of glutamate to GLAST, and/or some other signals linked with glutamate uptake, could elicit Sept2 guanine nucleotide exchange resulting in its conversion from the GDP-form to the GTP-form. GLAST can be released from the GTP-form of Sept2, and increase its activity. On the other hand, the GTP-form Sept2 promotes to polymerize septin filaments and stabilizes the juxtamembrane cytoskeletal matrix. GLAST released from GTP-form Sept2, together with the cytoskeletal stabilization might contribute to the cell surface expression and/or the conformational stabilization of GLAST.
However, another explanation would be possible as follows: Considering that immuno-isolated septin filaments contains more GDP-form than GTP-form septins (Field et al. 1996; Mitchison & Field 2002), co-localization of Sept2 and GLAST may reflect the binding of GLAST to GDP-form Sept2 in juxtamembrane septin filaments of Bergmann glial cell process. The possibility cannot be ruled out that GLAST bound to GDP-form Sept2 is stabilized on cell surface by anchoring to juxtamembrane septin filaments. Accordingly, the disruption of septin filaments by expression of Sept2-G47V could cause the instability of GLAST, leading to internalization of GLAST. However, the functional significance of GLAST–septin binding and assembly of septin filaments at different guanine nucleotide states of septin are to be evaluated in further study.
The demonstration and characterization of septin–glutamate transporter interaction reported here provides an important clue for exploring the functional significance of septins in the regulation of astrocyte glutamate uptake. Septins are not restricted to astrocytes in the cerebellum, but are distributed rather ubiquitously in the brain (Kinoshita et al. 2000). Septins are mainly localized next to the plasma membrane (Kinoshita et al. 2000), and various types of septin complexes have been reported (Hsu et al. 1998; Joberty et al. 2001; Sheffield et al. 2003). These facts suggest that septin may be involved in the regulation of various other membrane proteins and receptors. Little is known, however, about the effectors of guanine nucleotide exchange in septins. Identification of the regulatory proteins of, and the signal cues for, the septin guanine nucleotide exchange reaction is required for us to understand the physiological functions of septin. Elucidation of the role of septins in glutamate transporter regulation should provide important insights into basic brain functions.
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Experimental procedures |
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pBS SK (–)-Sept2-WT and -G47V were kindly donated by Dr M. Kinoshita (University of Kyoto, Japan). pEF-BOS-Myc and –HA (Mizushima & Nagata 1990) were kindly donated by Prof Kaibuchi (University of Nagoya, Japan). cDNAs encoding mouse Sept2 (wild-type and point-mutant) were obtained by PCR using BamHI site-linked appropriate primers. The cDNAs were inserted into the BamHI sites of the bacterial GST fusion protein expression vector pGEX-2T (Amersham Pharmacia, Piscataway, NJ, USA) or the mammalian Myc-tagged protein expression vector pEF-BOS-Myc. Sept2-WT was also inserted into BamHI site of a bacterial His6-tagged protein expression vector, pRSET (Invitrogen, Carlsbed, CA, USA). The mouse GLAST cDNA was obtained from a mouse brain Quick-Clone cDNA (Clontech, Palo Alto, CA) by the PCR method. GLAST-NT and -CT were also obtained by the PCR method. GLAST-CT were inserted into the BamHI site of pGEX-2T; and GLAST-full, -NT and -CT were inserted into the BamHI sites of pRSET and pEF-BOS-HA.
Antibodies
To produce rabbit anti-mouse Sept2 polyclonal antibody, we immunized a New Zealand White rabbit against GST-Sept2, and affinity-purified the antiserum. Rabbit and guinea pig anti-mouse GLAST polyclonal antibodies were generated as described (Shibata et al. 1997). Guinea-pig anti-mouse Sept4 polyclonal antibody was kindly donated by Dr M. Kinoshita (University of Kyoto, Japan). The anti-Myc 9E10 antibody was a generous gift from Prof K. Kaibuchi (University of Nagoya, Japan). Mouse anti-His antibody was purchased from Pharmacia. Guinea-pig anti-mouse GLT-1 polyclonal antibody and rabbit anti-rat NR1 subunit of NMDA receptor polyclonal antibody were obtained from Chemicon (Luton, UK), and mouse anti-GFAP monoclonal antibody from Sigma (St Louis, MO, USA). FITC-conjugated anti-rabbit Ig, HRP-conjugated anti-mouse Ig, HRP-conjugated anti-rabbit Ig, and HRP-conjugated streptavidin were purchased from Amersham Life Science (Buckinghamshire, UK). Biotinylated anti-guinea pig Ig was purchased from Vector Laboratories (Eurlingame, CA). Alexa Fluor 546-conjugated streptavidin came from Molecular Probes (Eugene, OR, USA). Colloidal gold (1.4-nm)-conjugated anti-rabbit Ig was purchased from Nanoprobe (Stony Brook, NY). Colloidal gold (10-nm)-conjugated anti-rabbit IgG and Colloidal gold (15-nm)-conjugated guinea pig IgG were purchased from British Bio Cell International (Cardiff, UK).
Recombinant adenoviruses
Recombinant adenoviruses encoding Sept2-WT and -G47V were generated by the use of the Adeno-X expression system (Clontech), according to the manufacturer's instructions.
Cell culture and transfection
COS7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 °C under an atmosphere of 5% CO2. For immunoprecipitation, the cells were plated in 10-cm dishes at 2 x 106 cells per dish, or for glutamate uptake assays, in 12-well dishes at 5 x 104 cells per well. The next day, the cells were transfected with each of the expression plasmids (10-cm dish: 5 µg/dish, 12-well: 1 µg/well) by using Lipofectamine Plus Reagent (Invitrogen). The transfected COS7 cells were cultured for 48 h before assays.
Bergmann glial cells were prepared as previously described with minor modifications (Ortega et al. 1991). Cerebella were dissected from postnatal day 1 mice and cut into small pieces. The pieces were incubated for 15 min at 37 °C into DMEM containing 0.125% trypsin, 0.1% DNase, penicillin and streptomycin, and subsequently triturated in DMEM containing 10% FBS, 0.1% DNase, penicillin, and streptomycin to inactivate the trypsin. Cells were pelleted by centrifugation at 1000 r.p.m. for 5 min and resuspended in DMEM containing 10% FBS, penicillin, and streptomycin. For the biotinylation assay, cells were cultured in 10-cm dishes and for glutamate uptake assay, in 24-well dishes until they had become confluent. The cultured Bergmann glia were infected with recombinant adenoviruses (10-cm dish: 5 x 106 pfu/mL x 8 mL, 24-well: 5 x 106 pfu/mL x 0.5 mL) for 5 h, then cultured for 48 h before assays.
Immunocytochemistry
Adult mice (LR White) were fixed by perfusion through the heart with 2% paraformaldehyde in 0.1 M phoshate buffer (PB) pH 7.4. For cryoprotection, brains were placed into 30% sucrose solution for 12 h. The brains were frozen in O.C.T. compound (Sakura, Tokyo, Japan) on dry ice, and cut into 7-µm-thick sections using a cryostat. The sections were washed in 0.1 M PB containing 0.01% saponin. Sections were incubated for 20 min in 20% Block Ace (Skim milk, Yukijirusi, Tokyo, Japan), then incubated with the primary antibody 4 h, washed, and incubated with the secondary antibody for 2 h. All antibodies were diluted to the final concentration of 1 µg/mL with 0.1 M PB containing 0.01% saponin and 5% Block Ace. For pre-embedding immuno-electron microscopy, after incubation with colloidal gold (1.4-nm)-conjugated secondary antibody for 2 h, the sections were fixed with 1% glutaraldehyde in 0.1 M PB and the gold particles were intensified by use of a HQ silver kit (Nanoprobe). The sections were postfixed in 1% osmium tetroxide for 90 min at 4 °C, dehydrated through graded concentrations of ethanol, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate before observation with an electron microscope (JEM 1200EX, JEOL, Tokyo, Japan). For post-embedding immuno-gold analysis, adult mice were perfused transcardially with 4% paraformaldehyde/0.1% glutaraldehyde in 0.1 M PB. Cerebellar sections (400 µm in thickness) were prepared on a microslicer, cryoprotected with 30% glycerol in 0.1 M PB, and frozen rapidly with liquid propane in a Leica EM CPC unit (Vienna, Austria). Frozen sections were immersed in 0.5% uranyl acetate in methanol at –90 °C in a Leica AFS freeze-substitution unit, infiltrated at –45 °C with Lowicryl HM-20 resin (Lowi, Waldkraiburg, Germany), and polymerized with UV light. Ultrathin sections were mounted on nickel grids precoated with neoprene W (Nisshin EM, Tokyo, Japan). After having been etched with saturated sodium-ethanolate solution for 3 s, the sections were treated successively with 1% human serum albumin/0.1% Tween 20 in TBS (HTBST, pH 7.5) for 1 h, rabbit anti-Sept2 antibody in HTBST for 3 h, colloidal gold (10-nm)-conjugated anti-rabbit IgG in HTBST for 1 h, guinea pig anti-GLAST IgG antibody in HTBST for 3 h, and colloidal gold (15-nm)-conjugated guinea-pig IgG in HTBST for 1 h. Finally, grids were stained with uranyl acetate for 15 min and then with mixed lead solution for 1 min, and examined using an H-7100 electron microscope (Hitachi, Tokyo, Japan).
Bergmann glial cells cultured on poly L-lysine coated coverslips were fixed with 4% paraformaldehyde and permeated with 0.2% Triton X-100/0.1 M PB for 10 min. The samples were treated with 20% Block Ace/0.1 M PB for 20 min, then incubated with the primary antibody for 1 h, washed, and incubated with the secondary antibody for 30 min. All antibodies were diluted to the final concentration of 1 µg/mL with 0.1 M PB containing 5% Block Ace.
Recombinant proteins
Production of recombinant proteins was induced in BL21 (DE3) pLysS cells (Stratagene, La Jolla, CA, USA) by incubation with 0.1 mM IPTG for 4 h at 30 °C.
GST-tagged Sept2 constructs and GLAST-CT construct were purified with glutathione-Sepharose 4B (Pharmacia), according to the manufacturer's instructions. His6-tagged Sept2 was purified with His-Bind Resin (Novagen, Madison, WI, USA), according to the manufacturer's instructions.
The bacterial cell lysates for affinity column chromatography were prepared as follows: The cell pellets expressing His6-tagged GLAST-full, -NT and -CT were suspended in 3 volumes of homogenizing buffer (20 mM Tris-HCl [pH 7.5], 1 mM EDTA, 1 mM DTT, 0.3% Triton X-100, 10 µg/mL leupeptin, 10 µg/mL aprotinin), and homogenized by sonication. The homogenates were centrifuged (1 h, 100 000 g), and the supernatants were collected. All procedures were carried out at 4 °C.
Preparation of mouse membrane extract
Mouse brain was homogenized in 3 volumes of homogenizing buffer (20 mM Tris-HCl [pH 8.0], 2 mM EDTA, 5 mM MgCl2, 1 mM DTT, 10% sucrose, 10 µg/mL leupeptin, 10 µg/mL aprotinin) by five up-and-down strokes with a Teflon/glass homogenizer and centrifuged (10 min, 1000 g). The supernatant was centrifuged again (1 h, 27 000 g), and the pellet was suspended in 3 volumes of extraction buffer (homogenizing buffer containing 2% CHAPS and 150 mM NaCl). The mixture was incubated for 1 h with rotation, and centrifuged (1 h, 27 000 g). The supernatant contained about 90% of the membrane protein at a concentration of 20–30 mg/mL. All procedures were carried out at 4 °C.
Immunoprecipitation
Transfected COS7 cells cultured in 10-cm dish were solubilized in 1 mL of IP buffer (20 mM Tris-HCl; pH 8.0, 1 mM EDTA, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 10 µg/mL leupeptin, 10 µg/mL aprotinin) at 4 °C for 2 h with rotation and then centrifuged (10 min, 20 000 g) to remove the cellular debris. The mouse brain membrane extract was precleared by incubation with Protein-G-Sepharose (Pharmacia).
The COS7 cell lysate or mouse brain extract containing 10 mg protein was mixed with 5 µg of antibody and incubation was done for 2 h. 30 µL of Protein-G beads were then added, and incubation was continued for 2 h. The beads were washed with 500 µL of wash buffer (1.5% Triton X-100 IP buffer) four times, and the antibody was eluted with 100 µL of 0.1 M glycine adjusted with HCl to pH 2.5. The eluates were subjected to immunoblot.
Guanine nucleotide-binding assay
The guanine nucleotide-bound forms of GST-Sept2 were made by incubating GST-Sept2 for 30 min at 30 °C with 100 µM GDP or GTPS (containing [3H]GDP (Pharmacia) or [-35S]GTPS (Pharmacia)) in reaction buffer (20 mM Tris-HCl [pH 7.5], 1 mM EDTA, 1 mM DTT, 5 mM MgCl2). The radioactivities were measured with a liquid scintillation counter, LS 6000SC (Beckman, Fullerton, CA).
Affinity column chromatography
Purified proteins of GST-Sept2 constructs (0.5 mg/column) were immobilized on glutathione-Sepharose 4B (500 µL/column), and the beads were packed into columns. The mouse brain membrane extracts (50 mg protein/column) or lysates of His6-tagged GLAST construct-expressing bacteria (250 mL culture/column) were loaded on to the columns. After the columns had been washed at 4 °C with 10 bed volumes of buffer A (20 mM Tris-HCl; pH 7.5, 1 mM EDTA, 1 mM DTT, 5 mM MgCl2) containing 200 mM NaCl and 0.5% Triton X-100, the bound proteins were eluted with 3 bed volumes of buffer A containing 30 mM glutathione. The eluates were subjected to immunoblot.
Affinity column assays using guanine nucleotide-bound GST-Sept2, which was prepared as described under Guanine nucleotide binding assay, were carried out under high Mg2+ conditions (20 mM MgCl2) after guanine nucleotide binding reactions to prevent guanine nucleotide release from Sept2.
In vitro binding assay
Purified 5 nmol GST or GST-GLAST-CT was mixed with purified 1 nmol His6-Sept2, and the mixture was incubated for 2 h at 4 °C. 100 µL glutathione-Sepharose 4B were then added and incubation was continued for 1 h at 4 °C. The beads were washed with wash buffer A six times, and the bound proteins were eluted with 100 µL of buffer A containing 30 mM glutathione. The eluates were subjected to immunoblot.
The in vitro binding assay to reveal the guanine nucleotide dependency of the Sept2–GLAST-CT interaction was performed as follows: Radioisotope-labelled guanine nucleotides bound to purified His6-Sept2 were prepared as described under Guanine nucleotide binding assay. All of the procedures after guanine nucleotide binding reactions were carried out as described in the preceding paragraph, except that the high Mg2+ buffers were used. The radioactivities of the eluates were measured with a liquid scintillation counter.
Uptake assay
Glutamate uptake assays were performed as previously described (Shimada et al. 1999). Two days after transfection, the medium in the dish was replaced by Krebs-Ringer-HEPES (KRH) solution (12-well; 0.5 mL, 24-well; 0.25 mL) consisting of 120 mM NaCl, 1.2 mM MgSO4, 4.7 mM KCl, 2.2 mM KH2PO4, 2.2 mM CaCl2, 10.0 mM glucose, 10.0 mM HEPES adjusted with KOH to pH 7.4, and the cells were incubated for 30 min. At time zero, KRH solution containing L-[3H]glutamate (Pharmacia) was added (12-well; 0.5 mL, 24-well; 0.25 mL). The assay was terminated by washing the cells 3 times with 1 mL of ice-cold KRH solution. The cells were then solubilized with 1% SDS (12-well; 250 µL, 24-well; 125 µL). The radioactivities of 25 µL aliquots were measured with a liquid scintillation counter.
Biotinylation of cell-surface proteins
Biotinylation of cell-surface proteins was performed as previously described (Munir et al. 2000). Bergmann glial cells cultured in 10-cm dish were rinsed with ice-cold PBS containing 0.1 mM CaCl2 and 1.0 mM MgCl2 (PBS-Ca/Mg). The plates were then incubated in 3 mL of PBS-Ca/Mg containing 1 mg/mL EZ-Link Sulfo-NHS-LC-Biotin (PIERCE, Rockford, IL, USA) for 20 min at 4 °C. Next they were rinsed with 5 mL of PBS-Ca/Mg containing 100 mM glycine 3 times. The cells were lysed by incubation in 1 mL of lysis buffer (100 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 µg/mL leupeptin, 10 µg/mL aprotinin) for 1 h at 4 °C. The cell lysates were centrifuged (10 min, 20 000 g), and the supernatants were collected. 100 µL of Streptavidin Sepharose High Performance beads (Pharmacia) were added to them, and incubation was done for 1 h at 4 °C. By centrifugation (10 min, 10 000 g), biotinylated proteins bound to the beads were separated from non-biotinylated proteins in the supernatant. The beads were washed with 500 µL of lysis buffer 3 times, and then the biotinylated proteins were eluted from the beads by boiling with 1 mL of SDS sample buffer. Samples were finally subjected to immunoblotting.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: n-kinoshita{at}cdb.riken.jp
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References |
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Received: 16 July 2003
Accepted: 14 October 2003
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