Human Molecular Genetics, 2000, Vol. 9, No. 2 303-310
© 2000 Oxford University Press
Alzheimer’s presenilin 1 is a putative membrane receptor for rab GDP dissociation inhibitor
Laboratory Neurozintuigen and Department of Neurology, Academic Medical Center, PO Box 22700, 1105 AZ Amsterdam, The Netherlands, 1Department of Cell Biology, Utrecht University School of Medicine, 3584 KX Utrecht, The Netherlands and 2Neuronal Cell Biology and Gene Transfer Laboratory, Flanders Interuniversitary Institute for Biotechnology, Center for Human Genetics, KULeuven, B-3000 Leuven, Belgium
Received 30 September 1999; Revised and Accepted 16 November 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in the presenilin 1 (PS-1) gene cause Alzheimer’s disease (AD). These mutations alter the processing of the amyloid precursor protein (APP) by increasing the production of the fibrillogenic amyloid fragment, Aß1–42/43. Since the secretase activities that process APP are localized in different intracellular compartments, it is likely that membrane transport is a key factor in the pathogenesis of AD. In this report we provide evidence for a direct connection between PS-1 and membrane transport. We show that the N-terminus of PS-1 binds to rab GDP dissociation inhibitor (rabGDI), a regulatory factor in vesicle transport. In PS-1-deficient neurons we found a 2-fold decrease in the amount of rabGDI associated with membranes. Our findings are compatible with PS-1 being a membrane receptor for rabGDI. This is in line with a role of PS-1 in the regulation of protein trafficking in the ER/Golgi, which can modulate the production of Aß.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Alzheimer’s disease (AD) is a common, devastating disorder of the brain, ultimately leading to death. It is characterized by progressive memory loss and other cognitive and behavioral deficits. Neuropathological characteristics include the presence of intracellular neurofibrillary tangles and extracellular ß-amyloid deposits in plaques and around cerebral blood vessels. The sporadic form of the disease is the most common, but rare familial cases exist. The first mutations identified in pedigrees with familial Alzheimer’s disease (FAD) were found in the gene encoding the amyloid precursor protein (APP) (1). These mutations, however, account for only ~2% of the FAD cases and genetic linkage analysis eventually led to the identification of two other FAD genes, presenilin 1 (PS-1) and PS-2 (2,3).
Presenilins (PSs) are integral membrane proteins with eight predicted transmembrane domains. They are predominantly localized to the endoplasmic reticulum (ER) and Golgi apparatus (4,5). All PS mutations identified to date are missense mutations [except one (6)] and give rise to autosomal-dominant forms of AD. The FAD mutations are randomly distributed throughout the protein; many of them are located in or adjacent to transmembrane regions. Since the PS-1 knock-out phenotype is lethal and can be rescued by both wild-type and mutant PS-1 (7,8), the PS FAD mutations most likely result in a gain-of-(deleterious) function phenotype. In spite of the strong homology between PS-1 and PS-2 (~65% amino acid identity) they are not functionally redundant. Various reports suggest a role of PSs in Notch signaling, apoptosis or protein trafficking and sorting, but how these putative functions relate to the pathology of AD remains to be established (reviewed in ref. 9).
The most direct connection between AD and presenilins is the observation that all FAD mutations affect the distinct APP processing pathways, by an, as yet, unknown mechanism (10). The constitutive 
-secretase pathway generates soluble sAPP fragments. The -secretase activity is located at or very close to the plasma membrane (11–13) and cleaves APP within the amyloid region, thereby precluding the formation of ß-amyloid (Aß). A second APP processing pathway involves a combined action of ß- and 
-secretase and leads to the formation of Aß fragments varying in length from 39 to 43 residues. The subcellular localization of this amyloidogenic APP processing pathway appears to be cell-type specific. In neuronal cells, cleavage by both ß- and -secretases occurs predominantly in the early secretory pathway (11,12). In these cells, Aß1–42/43 is produced primarily in the ER and Aß1–40 in or beyond the Golgi apparatus (14,15). The compartimentalization of the APP processing activities implies that correct trafficking of either APP or the secretases is pivotal for proper processing of APP, and that mistrafficking can lead to increased Aß production. PS FAD mutations cause an increase in the formation of Aß, in particular of the more amyloidogenic 1–42/43 form (16,17). In addition, -secretase cleavage of APP is strongly inhibited in PS-1–/– mice, whereas - and ß-secretase cleavages are unaffected (18). The localization of PS-1 in the ER/Golgi and the effect of PS FAD mutations and PS-1 deficiency on APP processing, are compatible with a role for PS-1 in protein sorting or trafficking in the cell.
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RESULTS |
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The N-terminus of PS-1 binds to rab GDP dissociation inhibitor (rabGDI)
To further define the functions of PS-1, we employed the yeast two-hybrid system in order to identify PS-1 interacting proteins. The N-terminal cytoplasmic domain of PS-1 (amino acids 1–80; Fig. 1A) was fused to the Gal4 DNA-binding domain in vector pAS-2. This fusion construct was used to screen a human adult brain cDNA library cloned downstream of the Gal4 activation domain of the two-hybrid prey plasmid pACT2. One of the putative PS-1 interacting proteins we identified in this screen encoded the C-terminal part of rabGDI
(amino acids 205–447), which includes the conserved regions 3A and B (Fig. 1B). Figure 1C shows the result of a two-hybrid assay with PS-1 and this C-terminal part of rabGDI. RabGDI interacts with the N-terminus of PS-1, not with the pAS-2 vector without insert and also not with another part of PS-1, HL-VI, a large hydrophilic loop that, like the N-terminus, is oriented towards the cytoplasm (Fig. 1A).
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RabGDI binds to members of the rab family of monomeric GTPases (19). GTP-bound rab proteins are associated with the cytoplasmic surface of transport vesicles and are thought to regulate membrane fusion through the interaction of their effector proteins with components of the conserved SNARE membrane fusion complex. Upon GTP hydrolysis, rabGDI binds to rab proteins in their GDP-bound form, extracts them into the cytoplasm and subsequently serves to deliver rab GDP to a donor organelle. This cyclical mechanism allows rab proteins to engage in multiple rounds of vesicular transport between two compartments and it is thought that rabGDI improves the fidelity of transport (20,21).
RabGDIs constitute a small family of highly conserved proteins. In yeast a single essential rabGDI is present (22), but in higher species two or more isoforms have been identified. RabGDIs bind rab proteins with similar selectivity (23,24). The intracellular localization of rabGDI isoforms, however, appears to be different, providing yet another level of specificity in trafficking (23,25–27). Two human rabGDI isoforms have been cloned thus far: the ubiquitously expressed rabGDIß and the brain-specific rabGDI that was identified in our screen with the PS-1 N-terminus (28,29). The two rabGDIs are highly homologous, even the least conserved C-terminal region that we found to bind PS-1 displays 80% identity at the amino acid level, suggesting that they have similar functions. In support of this notion, and shown in Figure 1C, the rabGDIß isoform can also bind PS-1.
Endogenous full-length rabGDI binds the PS-1 N-terminus
To confirm and extend the two-hybrid result, we next investigated whether endogenous rabGDI can also bind PS-1. Therefore, we expressed the PS-1 N-terminus as a glutathione S-transferase (GST) fusion protein in Escherichia coli and incubated this tagged protein with mouse brain homogenate. In the absence of the non-hydrolyzable GTP analog, GTPS, we did not observe binding of rabGDI to the PS-1 fusion protein, as shown in Figure 2. In contrast, GTPS pre-incubation of the brain extract at 37°C for 30 min resulted in formation of a complex between rabGDI and the PS-1 fusion protein (Fig. 2) as detected with a rabGDI antibody cross-reacting with the ~55 kDa rabGDI mouse homolog, rabGDI1. Possibly, stabilization of rab proteins in their GTP-bound conformation enhances the interaction between GDI and PS-1. Some rabGDI binding to the GST protein alone was seen, but this was only a minor fraction compared with the GDI bound to the PS-1–GST fusion protein. Thus, the presence of a PS-1 N-terminus on a GST fusion protein strongly increases binding of endogenous rabGDI to the fusion protein. The increased association in the presence of GTPS suggests that binding is facilitated when rabGDI is not complexed with rab proteins.
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RabGDI binds to endogenous PS-1
We next wanted to investigate whether rabGDI can also interact with endogenous PS-1. We had to circumvent the use of a secondary antibody for detection of co-precipitated proteins, because the GDI and PS-1 bands co-migrate with the IgG bands, which interferes with the detection. We therefore used transfected 293 cells with an HA-tagged rabGDI
construct, which allows detection with an HRP-conjugated anti-HA antibody. We preferred not to use overexpression of PS-1, since this may introduce artefacts (30). When we analyzed proteins co-precipitating with the endogenous PS-1 from the 293 cells, we could identify the HA-rabGDI; however, the signal was very low (data not shown). To increase the amount of PS-1 in the immunoprecipitation we incubated a detergent lysate from mouse brain membranes with a lysate from rabGDI transfected cells. After pre-incubation with GTPS, PS-1 protein complexes were immunoprecipitated with an antibody directed to the PS-1 N-terminus, and analyzed with the HRP-conjugated anti-HA antibody. This approach did indeed increase the amount of HA-rabGDI that is precipitated with the PS-1 antibody, indicating that the amount of precipitated HA-rabGDI is proportional to the level of PS-1. The PS-1 antibody co-precipitates HA-rabGDI from the transfected 293 lysate, but not from the mock-transfected 293 lysate (Fig. 3). This is not due to a non-specific interaction of rabGDI with the PS-1 antibody or the protein A beads, since no HA-rabGDI was immunoprecipitated if a 14-3-3 polyclonal antibody was used for the immunoprecipitation (data not shown). This result confirms that rabGDI binds endogenous PS-1.
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RabGDI and PS-1 localize to the same compartment
In order to interact in the context of an intact cell, rabGDI and PS-1 have to be present at the same location in the cell. We therefore analyzed the subcellular localization of rabGDI in primary mouse hippocampal neurons. In biochemical analysis, the great majority of rabGDI is found in the cytosolic fraction (25). However, studies on the localization of rabGDI in situ by immunofluoresence showed that in MDCK cells more rabGDI is either associated with or located in close proximity to membranous structures in the cell than the fractionation studies suggest, as may be expected for a membrane-associated protein (31). We observed that rabGDI in neurons is localized in a reticular, perinuclear pattern with some punctate staining in the neurites as well, suggesting that rabGDI is primarily localized in the vicinity of the ER (Fig. 4A). To confirm this we performed double labelings with the rabGDI antibody and the ER-resident enzyme protein disulfide isomerase (PDI). Although the staining of PDI (Fig. 4B) is somewhat more confined to the perinuclear region than rabGDI, there is strong overlap between PDI and rabGDI localization (Fig. 4C). The abundance of rabGDI in the vicinity of the ER, where PS-1 is localized, makes an in vivo interaction between the proteins feasible.
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RabGDI membrane association is impaired in PS-1-deficient neurons
In contrast to the well established role of rabGDI in the rab cycle, little is known about the proteins involved in recruiting the rabGDI/rabGDP to a membrane and how GDI is dissociated when the rab protein is inserted in a membrane. Since PS-1 is an integral membrane protein that interacts in a cytosolic domain with rabGDI, we tested whether PS-1 acts as a membrane receptor for rabGDI. To this end, we prepared a total membrane fraction from neuronal cultures derived from wild-type or PS-1 knock-out mouse embryos and assayed the amount of rabGDI associated with these membranes. To control for the amount of membranes loaded, the rabGDI values were corrected for the amount of calnexin, which is, like PS-1, an integral membrane protein in the ER. As shown in Figure 5, a 2-fold decrease in membrane-associated rabGDI (51.3 ± 7.7% compared with wild-type) was observed in the PS-1-deficient cells. This is not due to a difference in expression of rabGDI in these brain cultures, since the total amount of rabGDI is the same for wild-type and knock-out cells (data not shown). We also did not observe a difference in the cytosolic fractions of wild-type or knock-out cells. It is, however, not surprising that the difference in the membrane fraction is not reflected in the cytosolic fraction. Given the fact that the majority of rabGDI ends up in the cytosolic fraction after fractionation, this pool is too large to show this. This result suggests that PS-1 is involved in the membrane association of rabGDI in mouse brain.
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DISCUSSION |
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In this paper we report that PS-1 binds to a regulator of vesicle transport, rabGDI. As PS-1 is localized to ER/Golgi compartments, it is tempting to postulate a role for PS-1 in protein trafficking. This is supported by studies in which deletion of PS-1 or its Caenorhabditis elegans homolog affect sorting/maturation of proteins such as the TrkB receptor (32) and the Notch receptor (33). In addition, the transport of ß-catenin to the nucleus is disturbed in PS-1 FAD mutants (34). Recent findings indicate that cleavage of the Notch receptor is inhibited in the absence of PS-1 and that mutation of conserved membrane aspartates in PS-1 inhibits
-secretase cleavage (35–38). These data link PS-1 to -secretase cleavage; however, they do not provide conclusive evidence since the collective data can also be explained by protein mis-targeting (39). This is an attractive explanation, since PS-1 is localized in the ER/Golgi, whereas Notch cleavage occurs at the plasma membrane. Alterations in protein trafficking have been hypothesized to be important in the pathogenesis of AD. Since Aß is produced in the ER/Golgi, increased trafficking of APP or the -secretases to these compartments may increase Aß formation. Our data show that PS-1 binds rabGDI and we propose that this interaction is required for normal vesicular transport through the biosynthetic pathway. The observation that rabGDI, like PS-1, localizes to the ER makes it likely that this interaction can occur in the context of an intact cell. In agreement with this, APP processing is also affected by perturbation of rab1B and rab6 function (40,41).
Membrane receptors for rabGDI have been postulated on endocytic compartments (42–45). Our finding that the membrane association of rabGDI is decreased in the absence of PS-1 (Fig. 4) leads us to propose that PS-1 might be such a receptor for rabGDI on ER/Golgi membranes. Although we cannot exclude that the reduced amount of rabGDI at the membranes is a secondary effect caused by the developmental defects of the PS-1 defeciency, the finding that PS-1 and rabGDI can physically interact supports the hypothesis that the PS-1 deficiency directly affects the amount of rabGDI on the membranes. Since there will be several receptors for GDI in the cell, it is not surprising that, although a significant decrease of GDI in the membrane fraction is observed in the absence of PS-1, there is still GDI associated with membranes. Possibly, PS-1 sequesters rabGDI in the ER/Golgi complex, thereby allowing GDI to function between or in these compartments. The binding of rabGDI to PS-1 is increased in the presence of GTPS, suggesting that GDI binds when it is not complexed with rab proteins (Fig. 2). This could mean that the binding of a rabGDI/rab complex to PS-1 allows dissociation of this complex, thereby delivering the rab protein to the membrane. Alternatively, PS-1 may sequester rabGDI to the membrane, so that it can bind rab proteins that have hydrolyzed their GTP. The binding of the rab protein may then release rabGDI from PS-1. Interestingly, a recent paper reports the binding of the trans-Golgi network rab11 to another part of the PS-1 protein, HLVI, which suggests that PS-1 may also be in direct contact with rab proteins (46).
How could an FAD mutation in PS-1 affect the function of rabGDI? The FAD mutations show a dominant gain-of-function phenotype. This could imply that rabGDI binds with higher affinity to mutant PS-1 or that it is not properly released. Either way this would affect the normal function of rabGDI in the ER/Golgi complex, where PS-1 is localized. In this respect it is interesting that the amount of rab8 associated with membranes is increased in the brain of AD patients, suggesting either increased delivery or decreased release of rab proteins from membranes (47). Preliminary data from our laboratory that the membrane association of rab6 is decreased in PS-1–/– cells would be in agreement with this (W. Scheper, unpublished data). In vitro, addition of rabGDI to ER/Golgi membranes releases rab proteins from membranes, thereby inhibiting ER/Golgi transport (48). Recently, very potent inhibitory rabGDI mutants were described with limited ability to extract rab1A from membranes (49). Since these mutant rabGDI proteins efficiently bind to membranes, it was suggested that the inhibition of transport was caused by altered affinity of rabGDI for a membrane receptor, thereby blocking normal rabGDI activity. Since the PS-1 FAD mutations only increase the production of Aß 1.5- to 2-fold, any effect of an FAD mutation on the interaction with rabGDI will be very subtle. It can be expected that mutations in the N-terminus of PS-1 would directly interfere with rabGDI binding. Whereas FAD mutations occur throughout the PS-1 protein, to our knowledge, no FAD mutations were described in the N-terminus, except one immediately adjacent to the first transmembrane domain (9). This is consistent with our model since mutations that prevent binding of rabGDI would lead to loss of function of PS-1 and thus not to an FAD phenotype. The level of rabGDI in neuronal cells is several times higher than in non-neuronal cells (28,29). This may explain a neuron-specific effect of the putative rabGDI–PS-1 interaction. A role for rabGDI in maintenance of neuronal function is supported by the recent identification of mutations in rabGDI in X-linked non-specific mental retardation (50). Our data suggest that rabGDI may play a fundamental role in the pathogenesis of AD as well.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Materials
Restriction endonucleases, a High Fidelity PCR kit, T4 ligase and HRP-conjugated anti-HA monoclonal antibody were obtained from Boehringer Mannheim (Mannheim, Germany). Yeast strain Y190, cloning vector pAS2-1 and pACT2 human adult brain cDNA library were obtained from Clontech (Palo Alto, CA), protein A–Sepharose beads from Pharmacia Biotech (Uppsala, Sweden), cycloheximide, 3-amino-1,2,4-triazole (3-AT), glutathione agarose beads, GTP
S, FITC-conjugated goat anti-rabbit antibodies and Cy3-conjugated sheep anti-mouse antibodies from Sigma (St Louis, MO). Cell culture reagents were purchased from Gibco BRL (Gaithersburg, MD), HRP-conjugated rabbit anti-mouse IgGs from Dako (Glostrup, Denmark), DAC from Eurogentec (Seraing, Belgium), Vectashield mounting medium from Vector Laboratories (Burlingame, CA) and an ECL kit from Amersham (Amersham, UK).
Construction of plasmids
The fragments encoding the N-terminus (amino acids 1–80) and HL-VI (amino acids 263–407) of PS-1 were obtained by PCR with a forward primer containing a 5' EcoRI site and a reverse primer containing a 5' SalI site and cloned in the EcoRI/SalI sites of pAS2-1, in frame with the Gal4DNA-BD. The fragment encoding amino acids 205–445 from rabGDI was amplified by PCR with a forward primer containing a 5' EcoRI site and a reverse primer containing a 5' XhoI site, and cloned into the EcoRI/XhoI sites of pACT2, in frame with the Gal4-AD. The N-terminal PS-1 fragment (amino acids 1–80) was cloned using a forward primer containing a 5' BamHI site and a reverse primer containing a 5' SalI site in the BamHI/SalI sites of prp269, in frame with the GST protein. The fragment encoding amino acids 1–447 from rabGDIß was amplified by PCR with a forward primer containing a 5' SalI site and a reverse primer containing a 5' XbaI site and cloned in the SalI/XbaI sites of pMT-HA, in frame with the HA tag, rendering construct pMT-HA rabGDI. Cloning was performed according to standard protocols. All constructs were sequenced on an ABI 377 sequencer.
Yeast two-hybrid analysis
A human adult brain cDNA/GAL4 AD fusion library was screened with the pAS2-1/PS-1 N-terminal construct according to the manufacturer’s instructions. Activation of the His4 and ß-galactosidase reporters present in yeast strain Y190 by this construct alone was excluded. A total of 4 x 105 independent clones were analyzed in the presence of 35 mM 3-AT. Clones that remained positive after restreaking were separated from the bait by selection on cycloheximide. This allowed direct assay for activation of the resulting cDNAs to activate reporters. After isolation of plasmid DNA and retransformation into bacteria, the clones were sequenced on an ABI 377 sequencer. Clones were designated as putative PS-1-interacting proteins if they did not activate the reporter genes by themselves or in cotransformation with the pAS-2 vector lacking an insert. Triplicate clones from three separate experiments were analyzed for binding to the PS-1 N-terminus.
Preparation of brain homogenate and membrane fractions
Total mouse brain homogenate was prepared in homogenization buffer [5 mM Tris–HCl pH 7.4, 250 mM sucrose, 1 mM EGTA supplemented with protease inhibitors (0.24 U/ml aprotinin, 5 mM EDTA, 1 µg/ml pepstatin)] by dounce homogenization, and centrifuged at 800 g for 10 min to obtain a post-nuclear supernatant. Mixed brain cultures were obtained from 14-day-old wild-type or PS-1 knock-out mouse embryos as described (22) and grown for 4 days prior to harvesting. Wild-type and PS-1 knock-out mouse mixed brain cultures were scraped in homogenization buffer and homogenized by passaging 10 times through a cell-cracker (EMBL, Heidelberg, Germany). Post-nuclear supernatants were obtained after centrifuging for 10 min at 800 g. The post-nuclear supernatants were centrifuged for 60 min at 150 000 g in a TL-100 centrifuge (Beckman) to pellet the membranes. Membranes were resuspended in PBS and the protein concentration was determined using Bio-Rad (Hercules, CA) Protein Reagent. Aliquots of 25 µg of each brain-culture-derived membrane fraction were analyzed on a precast 4–20% Tris–glycine SDS gel (Novex) and blotted onto nitrocellulose membranes using a semi-dry electroblotting apparatus.
GST pull-down assay
The prp269 GST plasmid or the prp269 GST–PS-1 plasmid was transformed into E.coli DH5 and expression of the fusion proteins was induced in the presence of 0.1 mM IPTG for 4 h. The proteins were purified over glutathione agarose beads. Samples of every batch were analyzed on Coomassie-stained SDS–polyacrylamide gels, to check the size and purity of the fusion proteins. The relative amounts of protein present in each sample were estimated and adjustments were made to ensure similar input of the different proteins in the pull-down assay. Equal amounts of GST beads and GST–PS-1 beads were incubated with total mouse brain homogenate solubilized in 0.5% NP-40 and preincubated for 30 min at 37°C in the presence or absence of 0.5 mM GTPS (Sigma). After overnight incubation at 4°C with homogenate in lysis buffer [100 mM KCl, 50 mM Tris–HCl pH 7.5, 0.5% NP-40, 2 mM MgCl2 supplemented with protease inhibitors (100 µg/ml leupeptin, 0.24 U ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml soy bean trypsin inhibitor)], the beads were washed in lysis buffer supplemented with 10 µM GTPS four times, bound proteins were released in SDS sample buffer, analyzed by SDS–PAGE and blotted onto PVDF membrane using a semi-dry electroblotting apparatus.
Cell culture and transfections
HEK 293 were maintained in DMEM supplemented with 10% fetal calf serum (FCS), 100 IU/ml penicillin, 100 µg/ml streptomycin and 300 µg/ml glutamine. The cells were transiently transfected in 9 cm dishes using 15 µg DAC and 10 µg plasmid or sonicated herring sperm DNA (mock transfections) in each transfection. Five hours after addition of the DNA–DAC complex, medium was replaced. Cells were grown for ~24 h after transfection and harvested in lysis buffer.
Co-immunoprecipitations
Lysates from transfected 293 cells were pre-incubated at 37°C for 30 min in the presence of 0.5 mM GTPS and subsequently incubated overnight at 4°C with membranes (~500 µg) isolated from mouse brain solubilized in 0.5% NP-40. The formed complexes were immunoprecipitated using an anti-PS-1 N-terminus antibody precoupled to protein A–Sepharose beads. The immunocomplexes were washed three times in lysis buffer supplemented with 10 µM GTPS. The precipitated proteins were released in SDS sample buffer and loaded on a 10% SDS–polyacrylamide gel. Gels were blotted onto PVDF membrane using a semi-dry electroblotting apparatus.
Western blotting
A bovine GDI cDNA was kindly donated by Yoshima Takai (Osaka University Medical School, Osaka, Japan) and cloned in the BamHI site of pGEX2T. A GDI polyclonal antibody was raised by injecting rabbits with GST–GDI, and affinity purified by sequential passage of the GDI antisera over DH5 total protein extract and GST columns, respectively. Blots were pre-incubated with Blotto [5% non-fat dried milk in TBST (0.05% Tween, 10 mM Tris–HCl pH 8, 150 mM NaCl)] and incubated with anti-GDI antibody or anti-calnexin antibody (kindly provided by Dr A. Helenius) for 2 h at room temperature in Blotto. The blots were washed four times in TBST and incubated for 90 min with HRP-conjugated goat anti-rabbit IgGs. Alternatively, blots were incubated with HRP-conjugated HA antibody for 2 h at room temperature in Blotto. Blots were washed five times in TBST and once in TBS before analysis by ECL and exposure on film.
Immunofluorescence microscopy
Coverslips of primary mouse hippocampal neurons were prepared from 17-day-old mouse embryos as described (36). The coverslips were blocked in PBS with 5% FCS for 1 h. Incubations with the primary antibodies were in PBS/5% FCS for 90 min; subsequently, the coverslips were washed in PBS and incubated with either FITC-conjugated anti-rabbit or Cy3-conjugated anti-mouse antibody for 1 h. After washing in PBS the coverslips were mounted in Vectashield. Microscopy was performed on an Olympus AH3 Vanox fluorescence microscope and the images were photographed with Extrachrome 200 ASA film (Kodak).
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ACKNOWLEDGEMENTS |
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We thank the members of the Neurozintuigen laboratory for their support, Dr B. De Strooper for hospitality, materials and advice, Dr P. Saftig for materials, K. Craessaerts for expert technical assistance, Dr A. Helenius for the calnexin antibody, Dr P. Borst for comments on the manuscript, and Dr R.M.F. Wolthuis for advice on protein assays and stimulating discussions. This research was supported by the Dutch Organisation for Scientific Research (NWO 903-51-106 to W.A.v.G. and NWO 902-23-214 to P.v.d.S.). W.A. was supported by the NWO-Vlaanderen, the VIB and the KULeuven (VIS project).
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FOOTNOTES |
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+ Present address: University of Cambridge, Cambridge Institute for Medical Research, Hills Road, Cambridge CB2 2XY, UK
§ To whom correspondence should be addressed. Tel: +31 20 5665998; Fax: +31 20 5664440; Email: f.baas@amc.uva.nl
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