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Address correspondence to Kai Simons, Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany. Tel.: 49-351-2102800. Fax: 49-351-2102900. E-mail: simons{at}mpi-cbg.de
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Abstract |
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Key Words: lipid rafts; ß-amyloid; BACE; Alzheimer's disease; endocytosis
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Introduction |
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There is growing evidence that cholesterol is of particular importance in regulating - and ß-cleavage (Simons et al., 2001). The E4 allele of apolipoprotein E has been shown to be a major risk factor for AD (Corder et al., 1993; Strittmatter et al., 1993), levels of total cholesterol and LDL in serum were reported to correlate with the amount of Aß in AD brains (Kuo et al., 1998), and there is epidemiological evidence that elevated cholesterol levels during mid-life increase the risk of developing AD (Kivipelto et al., 2001). Elevated dietary cholesterol uptake increased amyloid plaque formation in rabbits and transgenic mice (Sparks et al., 1994; Refolo et al., 2000), and cholesterol loading and depletion affected Aß generation in cultured cells and in an animal model (Simons et al., 1998; Fassbender et al., 2001). There is also a correlation between cellular cholesteryl-ester levels and Aß production (Puglielli et al., 2001), and it was demonstrated that aggregated Aß binds cholesterol in vitro (Avdulov et al., 1997). Interestingly, two independent retrospective studies reported a strong decrease in the incidence of AD and dementia in patients treated with 3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitors (Jick et al., 2000; Wolozin et al., 2000).
All of these studies point out that cholesterol is critically involved in Aß generation. However, little is known about the mechanisms by which cholesterol affects this process. We previously hypothesized that the association of APP with lipid rafts determines Aß production (Simons et al., 1998). Rafts are lateral assemblies of sphingolipids and cholesterol within the membrane (for review see Simons and Toomre, 2000). They are thought to form ordered platforms, which float around in the liquid-disordered matrix of the cellular membrane and represent versatile devices to compartmentalize membrane processes. Biochemically, the components of lipid rafts are characterized by their insolubility in detergents such as Triton X-100 or CHAPS at 4°C (Fiedler et al., 1993; Brown and London, 1997). A fraction of APP and BACE1 were shown to be associated with detergent-resistant membranes (DRMs) in a cholesterol-dependent manner (Bouillot et al., 1996; Simons et al., 1998; Riddell et al., 2001). -Secretase cleavage, on the other hand, was elevated after inhibition of ß-secretase activity by cholesterol depletion, and ADAM 10, a putative
-secretase, was soluble after detergent extraction (Kojro et al., 2001). The most straightforward interpretation of these data is that APP is present in two cellular pools, one associated with lipid rafts where Aß is generated and another outside of rafts where
-cleavage takes place. This model of membrane compartmentalization would explain how the same protein could be processed in two different mutually exclusive ways.
If cleavage of APP by BACE1 occurred in rafts, it would be important to know how and where this interaction is regulated. Therefore, in this paper we have studied these relationships and provide evidence that Aß generation critically depends on lipid rafts for enzyme activation to occur.
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Results |
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To easily monitor the influence of cholesterol depletion on APP processing, N2a cells were infected with adenoviruses to express either human wild-type APP (wtAPP) or the Swedish mutant of APP (swAPP). swAPP is dominantly ß cleaved, resulting in a several-fold higher production of Aß than for wtAPP. After MßCD extraction, the cells were metabolically labeled with [35S]methionine and chased for up to 2 h. Immunoprecipitations from conditioned medium revealed that Aß production was dependent on cellular cholesterol levels (Fig. 1 A). Decreasing cellular cholesterol by 85% totally abolished Aß secretion. Remarkably, already relatively small changes in total cellular cholesterol levels were found to have strong effects. A 2030% decrease showed a 5060% reduction in Aß secretion. ßCTF, which is generated by ß-cleavage, was also clearly reduced. On the other hand, the production of CTF by
-cleavage was increased (Fig. 1 B). As expected from results of Kojro et al. (2001), the soluble ectodomain generated by
-cleavage was also strongly increased in cholesterol-depleted cells. In general, processing of wtAPP and swAPP were similarly affected by cholesterol depletion.
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APP and BACE copatch with placental alkaline phosphatase but segregate from transferrin receptor
Lipid rafts are most abundant in the plasma membrane. In fibroblasts, individual rafts have a size of 50 nm, corresponding to
3,500 sphingolipid molecules and probably not more than 1030 proteins (Pralle et al., 2000). This means that two different species of raft proteins would rarely be in the same raft. However, it was shown previously that raft and nonraft markers could be cross-linked with antibodies into distinct patches (Harder et al., 1998; Janes et al., 1999; Prior et al., 2001). Raft markers copatch and segregate away from nonraft markers. Cross-linking with antibodies can thus be used as an assay for raft association. We tested whether antibody cross-linking induced copatching of APP and BACE1 with a raft marker, the glycosyl phosphatidylinositol (GPI)-anchored protein placental alkaline phosphatase (PLAP). As a nonraft marker, we used a mutant human transferrin receptor (TfR), where the cytosolic aa 541 (TfR del 541) have been removed. This mutant is defective in endocytosis due to the deletion of the sorting signal. Patches of TfR del 541 were shown to be segregated from components found in lipid rafts (Harder et al., 1998).
Our experiments were performed with BACE1A-CFP and YFP-wtAPP. CFP and YFP are the cyan and yellow color variants of the green fluorescent protein, respectively. Control experiments demonstrated that these fluorescent protein (FP)-containing constructs showed the same proteolytic processing and immunofluorescence behavior as the corresponding untagged proteins (unpublished data). BACE1A-CFP was cross-linked with the polyclonal antibody 7523 recognizing the NH2-terminal part of BACE1 (Capell et al., 2002). YFP-wtAPP was cross-linked with antiserum KG77 or mouse monoclonal antibody 3E6, both directed against the FP. Control experiments with wtAPP or BACE1A-VSVG and anti-APP antibody 5313 or anti-BACE1 antibody 7523 showed essentially the same patching (unpublished data). Also, we did not see significant differences in staining of swAPP and wtAPP.
Both BACE1 and wtAPP colocalized with PLAP at the plasma membrane in the majority of cells, but they clearly segregated from TfR del 541 (Fig. 2). BACE1 and wtAPP could also be localized to the same patches upon cross-linking (Fig. 3). For quantitative analyses of the extent of copatching, images of 10 randomly selected cells on one coverslip were taken and assigned into four categories: (1) coclustering (>80% overlap); (2) partial coclustering (clearly overlapping spots 3080%); (3) random distribution, and (4) segregation. The data from five independent experiments (Fig. 4) indicate that cross-linked wtAPP and BACE1 copatched with the raft protein PLAP and segregated from the nonraft protein TfR del 541.
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Initial experiments revealed that in N2a cells only a minor amount (<5%) of both APP and BACE1 were resistant to extraction with 1% Triton X-100. However, when the cells were extracted with 20 mM CHAPS a significantly higher amount of BACE1 and APP floated to the low density membrane fraction in a cholesterol-dependent manner (unpublished data). Therefore, we used CHAPS-extracted membranes to examine the effect of antibody-induced patching on DRM association. N2a cells were infected with adenoviruses to express YFP-swAPP or BACE1A-CFP, metabolically labeled for 2 h with [35S]methionine, and chased for 2 h in the absence of antibody or in the presence of anti-FP (KG77) or anti-BACE1 (7523) antibodies, respectively. Cells were then extracted with 20 mM CHAPS, and the detergent extracts were subjected to OptiPrep step gradient centrifugation. A significantly higher fraction of APP and BACE1 floated with DRMs after antibody-induced patching (Fig. 5). Quantification revealed that without cross-linking 18.0 ± 2.6% of APP (n = 3) and 24.6 ± 2.3% of BACE1 (n = 4) were found in the upper two fractions (DRMs). Antibody cross-linking increased the DRM-associated fraction to 25.1 ± 1.2% (n = 3) and 32.3 ± 0.7% (n = 4) of APP and BACE1, respectively. Thus, both APP and BACE1 increased their detergent resistance upon cross-linking, probably reflecting increased raft affinity caused by oligomerization. Similar results have been obtained for other raft proteins, which increase their raft association by forming oligomers (Simons and Toomre, 2000; Cheng et al., 2001).
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Endocytosis is essential for ß-cleavage
Antibody cross-linking might not only lead to copatching of raft components, it could also alter endocytosis of cross-linked proteins. Previous studies suggest that endocytosis is required for Aß generation (Koo and Squazzo, 1994; Perez et al., 1999; Huse et al., 2000; Daugherty and Green, 2001). Therefore, we decided to inhibit endocytosis by transiently expressing RN-tre or the dynamin II mutant K44A in N2a cells. RN-tre is a Rab5-specific GTPase-activating protein and inhibits clathrin-dependent endocytosis (Lanzetti et al., 2000). Dynamin is involved in fission of vesicles from the plasma membrane. It was shown that expression of the mutant K44A inhibits both clathrin-dependent and some clathrin-independent endocytotic pathways (Damke et al., 1994; Henley et al., 1998).
N2a cells were transiently transfected with equal amounts of plasmids encoding for swAPP, RN-tre, or dynamin K44A and labeled for 1 h with [35S]methionine. Immunoprecipitations from cell lysates with antibody IP60 (raised against the COOH terminus of APP) and from media with antibody 70JE (Aß) were performed (Fig. 7 A). APP biosynthesis was unchanged after expression of RN-tre or dynamin K44A; however, the COOH-terminal fragment generated by ß-cleavage (ßCTF) and secretion of Aß were significantly reduced. Expression of dynamin K44A inhibited Aß secretion by 8090% (Fig. 7 A). Remarkably, the membrane-bound fragment generated by -cleavage (
CTF) was only slightly increased (correlated to total APP). Thus, under our experimental conditions endocytosis was essential for ß-cleavage to occur, whereas
-cleavage was not appreciably stimulated by inhibiting endocytosis.
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Discussion |
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Another important result in support of raft association was that after antibody-induced cross-linking both APP and BACE1 copatched on the surface of living cells with each other and with PLAP, a GPI-anchored raft-associated protein. All three proteins segregated from patches containing cross-linked transferrin receptor, which served as a nonraft marker. We and others have previously used this assay to monitor how proteins associate with rafts at the cell surface (Harder et al., 1998; Janes et al., 1999; Prior et al., 2001). Antibody cross-linking of raft surface antigens leads to the formation of large clusters, which are easily observable in the light microscope. Copatching is dependent on cholesterol, since patching is inhibited by cholesterol removal.
Importantly, antibody cross-linking increased the DRM association of both APP and BACE1, as was previously shown for other raft-associated proteins (Harder et al., 1998). Because only a small fraction of APP and BACE1 associated with DRMs, these proteins are probably found (at steady state) in two membrane pools, one raft associated and another localized outside of rafts. How partitioning between these two pools is regulated is not clear. It has been reported that both APP and BACE1 can dimerize and that homodimerization of APP increases Aß production (unpublished data; Scheuermann et al., 2001). Oligomerization of raft components can lead to increased raft affinity. Many surface receptors, such as Fc() receptors and T and B cell receptors, dimerize or oligomerize after ligand binding, and this has been shown to increase association with DRMs (Janes et al., 2000; Langlet et al., 2000; Cheng et al., 2001). Therefore, dimerization might be important for regulating raft association of APP and BACE1.
Partitioning of APP and BACE1 into rafts alone seems not to be sufficient to induce ß-cleavage. Cleavage normally also depends on endocytosis. This was demonstrated by our experiments designed to inhibit endocytosis. In two approaches, we either expressed RN-tre, a Rab5 GTPase-activating protein (Lanzetti et al., 2000), or the dynamin mutant K44A (Damke et al., 1994). The former perturbs clathrin-dependent endocytosis (Lanzetti et al., 2000) and the latter both clathrin-dependent and some clathrin-independent endocytic pathways (Damke et al., 1994; Henley et al., 1998). The results were clear cut: Aß generation was strongly inhibited, whereas APP was still cleaved. The latter was expected from reports demonstrating that
-cleavage occurs at the cell surface (Haass et al., 1992; Parvathy et al., 1999). Previous work has reported that ß-cleavage may happen already late in the secretory pathway, or after delivery to the cell surface, and during endocytosis (Koo and Squazzo, 1994; Perez et al., 1999; Huse et al., 2000; Daugherty and Green, 2001; Kamal et al., 2001). Inhibition of endocytosis by our approaches suggests that, at least in N2a cells, most of this cleavage occurs after internalization. Since cholesterol depletion is also known to decrease the rate of endocytosis (Rodal et al., 1999), this is also likely to contribute to the decreased ß-cleavage. However, the inhibitory effect on Aß production by inhibition of endocytosis could be overcome by cross-linking surface APP and/or BACE1 with antibodies.
To account for these results, we envisage that ß-cleavage would normally not take place at the cell surface because surface APP and BACE1 are most likely present in separate rafts (Fig. 8). Rafts are small and highly dispersed at the cell surface and are suggested to contain only a subset of 1030 protein molecules (Pralle et al., 2000). Therefore, the likelihood is low that APP and BACE1 are in the same individual raft. For ß-cleavage to occur rafts would have to be clustered to get APP and BACE1 into the same raft platform. Thus, we hypothesize that APP and BACE1 meet after endocytosis by clustering and coalescence of APP- or BACE1-containing rafts within endosomes (Fig. 8 A). How and where clustering is accomplished during internalization from the plasma membrane is not known. However, raft clustering can be artificially induced at the cell surface by cross-linking with antibodies (Fig. 8 B). This could lead to the increased ß-cleavage in clusters/patches containing both APP and BACE1 that we had observed. Remarkably, we did not detect a dramatic increase in
-secretase processing of APP after inhibition of endocytosis. We assume that this is due to a continued raft association of a fraction of cell surface APP, which would not be accessible to
-cleavage. On the other hand, cholesterol depletion would shift the partitioning of APP from lipid rafts to the surrounding lipid bilayer and lead to the observed increase of
-cleavage.
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Also the -secretase complex was shown to be raft associated (Li et al., 2000; Wahrle et al., 2002). Moreover, the ß-cleaved COOH-terminal fragment, the substrate for
-cleavage, is found in DRMs and so is the product Aß (Lee et al., 1998; Riddell et al., 2001). How and where
-secretase acts to cleave out Aß is not known. Interestingly, Yanagisawa and coworkers have in a series of publications demonstrated that cholesterol-dependent sequestration of Aß promotes fibrillogenesis of soluble Aß and suggested that Aß associated with rafts undergoes a conformational change, which promotes amyloid plaque formation (Yanagisawa et al., 1995; Mizuno et al., 1999; Kakio et al., 2001). Thus, the stage is set for a molecular dissection of how cholesterol and lipid rafts contribute to amyloid plaque formation in the pathogenesis of AD.
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Materials and methods |
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Reagents and antibodies
MßCD, mevalonate, and cycloheximide were from Sigma-Aldrich, and lovastatin was from Calbiochem. The following antibodies were used against APP: rabbit polyclonal antibody IP60 (Ehehalt et al., 2002) directed against the very COOH terminus of APP, mouse monoclonal antibody 6E10 (Senetek) that detects APP and Aß, rabbit polyclonal antibody 70JE (Ehehalt et al., 2002) against aa 111 of Aß, and the rabbit polyclonal antibody 5313 (Steiner et al., 1999) directed against the NH2-terminal part of the APP ectodomain. Antibodies against FP were rabbit polyclonal antibody KG77 raised against recombinant GFP expressed in bacteria and mouse monoclonal antibody 3E6 (Molecular Probes). Rabbit polyclonal antibody 7523 (Capell et al., 2002) was against the NH2-terminal end of BACE1. The antihuman transferrin receptor monoclonal antibody was from Roche. Mouse monoclonal and rabbit polyclonal anti-PLAP antibodies were from Dako.
Constructs and generation of recombinant adenoviruses
The BACE1A-YFP/CFP constructs were described previously (Ehehalt et al., 2002); BACE1A containing a VSVG tag (BACE1A-VSVG) was constructed as follows. BACE1A-YFP in pGEMT (Promega) was digested with AflII and NotI to release the YFP moiety. The VSVG epitope (MYTDIEMNRLGK) was then added using two complementary oligonucleotides to reconstitute the AflII and NotI restriction sites. The oligonucleotides used were 5'-TTAAGGGTATGTATACTGATATCGAAATGAATCGATTGGGTAAGTGAGC-3' and 5'-GGCCGCTCACTTACCCAATCGATTCATTTCGATATCAGTATACATACCC-3'. BACE1A-VSVG was subsequently transferred as a SalI-NotI fragment into the mammalian expression vector pShuttlecytomegalovirus (CMV) (He et al., 1998).
The YFP-APP construct was obtained as follows. APP was tagged in the ectodomain by replacing the naturally occurring Kunitz-type protease inhibitor domain with the FP. This was necessary because insertion of the FP at the very NH2 terminus of APP resulted in a misfolded chimeric protein, incapable of leaving the ER. YFP was amplified by PCR from pEYFP-N1 (CLONTECH Laboratories, Inc.) to add an XcmI site at the 5' end, an XhoI site at the 3' end, and spacers on either side of YFP. The oligonucleotides used were 5'-ACCACAGAGTCTGTGGAAGAGGTGGTTCGAGGCGGCGGATCTACCGTGGGCAGCGCACCGGTCGCCACCATG-3' (the XcmI site is underlined and the bolded sequence matches plasmid pEYFP-N1) and 5'-TCTCGAGATACTTGTCAACGGCATCAGGGGTACTGGCTGCTGTTGTAFGAACTCCGCCGCCGGTAGATGCGGTCACGCTGCCGGTG-CCCTTGTACAGCTCGTCCATG-3' (the XhoI site is underlined, and the bolded sequence matches plasmid pEYFP-N1). This PCR product was ligated as an XcmI-XhoI fragment into pGEMT-APP695 digested with XcmI and XhoI. YFP-APP was subsequently transferred as a SalI-NotI fragment into the mammalian expression vector pShuttle-CMV. Adenoviruses were prepared as described (He et al., 1998).
PLAP under control of the Rous sarcoma virus promoter and the TfR del 541 expression construct in pCMV5 were described previously (Harder et al., 1998). Dynamin II K44A in pCMV5 was provided by S. Schmid (Scripps Research Institute, La Jolla, CA) (Damke et al., 1994; Fish et al., 2000), and the cDNA of RN-tre (Lanzetti et al., 2000) was provided by M. Zerial (Max Planck Institute of Molecular Cell Biology and Genetics).
Transfection, viral infection, and cholesterol depletion
24 h after seeding of N2a cells into 3.5-cm dishes, they were infected with recombinant adenoviruses for 0.5 h at 37°C in complete medium. After a change of medium, the cells were incubated for 1016 h at 37°C and then used for biochemical assays.
Transient transfections using calcium phosphate precipitation were performed with 13 µg of each expression plasmid as described by Chen and Okayama (1988). For cholesterol depletion, the cells were grown for 1 d in normal medium and then for 1 d in either DME supplemented with 2 mM L-glutamine, 10% lipid-deficient FCS, 2 µM lovastatin, and 0.25 mM mevalonate, or in complete medium. They were then infected and grown for a further 1216 h in the same medium. The cells were treated for 530 min with 10 mM MßCD in methionine-free medium (labeling medium) and thereafter metabolically labeled with 100 µCi/dish of [35S]methionine (NEN). Depending on the experiment, the cells were chased for 0.52 h in labeling medium containing an excess of methionine (150 µg/ml) and 20 µg/ml cycloheximide to inhibit protein synthesis.
Cholesterol determinations were done with the Amplex Red Cholesterol Assay kit (Molecular Probes), which revealed a depletion of up to 80% of total cellular cholesterol after treatment with a combination of lovastatin and MßCD.
Immunoprecipitation and quantification
After metabolic labeling, the cell culture medium was collected and cell extracts were prepared using PBS containing 2% NP-40, 0.2% SDS, and 25 µg/ml each of chymostatin, leupeptin, antipain, and pepstatin A. Immunoprecipitates were recovered on protein ASepharose CL4B beads (Amersham Biosciences) and analyzed either on 10% polyacrylamide (Laemmli, 1970) or 1020% Tris-Tricine (Invitrogen) gels. Individual bands were quantified using the Fujifilm BAS 1800II image plate reader and Science Lab 99 Image Gauge v3.3 software (Raytest Isotopenmessgeraete).
Immunofluorescence and antibody-induced patching
For immunofluorescence microscopy, the cells were fixed for 4 min at 8°C with 4% paraformaldehyde in PBS followed by an incubation in methanol for 4 min at 20°C. Fixed cells were incubated for 1 h at RT with a proper dilution of antibodies in PBS/0.2% gelatin. After three washes with PBS/0.2% gelatine, they were incubated with the respective secondary antibodies in PBS/0.2% gelatin for 1 h at RT.
To cluster raft proteins, the respective antibodies were diluted in CO2-independent medium (Invitrogen) containing 20 mg/ml BSA. Antibodies against PLAP were diluted 1:35, the antihuman transferrin receptor monoclonal antibody 1:100, the polyclonal anti-FP (KG77) and polyclonal anti-BACE1 (7523) antibodies 1:100, and the monoclonal anti-FP (3E6) 1:50. The cells were incubated for 45 min with the respective combination of antibodies at 10°C, briefly washed, and further incubated for 45 min at 10°C with mixed fluorescently labeled secondary antibodies. Cy3-labeled secondary antibodies were diluted 1:500, and the Cy5-labeled secondary antibodies were diluted 1:100. The cells were fixed as described above. Fluorescent images were acquired on an Olympus BX61 microscope.
Quantification of copatching
Because of variation in expression levels of transiently expressed proteins and differences in cell shape, quantification of copatching was done as described before (Harder et al., 1998). Briefly, images of 10 randomly selected cells expressing the two marker proteins were taken from one coverslip. An individual not involved in recording scored the images in a blind fashion into four categories: (1) coclustering (>80% overlap), (2) partial coclustering (clearly overlapping patches; 3080%), (3) random distribution, and (4) segregation. The percentage of cells in each class from five independent experiments were expressed as mean ± SD.
Preparation of DRMs
Detergent extraction with CHAPS was performed as described (Fiedler et al., 1993). N2a cells were grown in 3.5-cm dishes, infected with adenoviruses to express BACE1A-YFP or YFP-APP, labeled for 2 h with [35S]methionine, and chased for 2 h in labeling medium containing an excess of methionine (150 µg/ml), 20 µg/ml cycloheximide, and for some samples antibodies 7523 or KG77 (1:100). The cells were washed once with PBS and scraped on ice into 300 µl 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 3 mM EDTA (TNE) buffer containing 25 µg/ml each of chymostatin, leupeptin, antipain, and pepstatin A. The cells were homogenized through a 25 G needle and centrifuged for 5 min at 3,000 rpm. The postnuclear supernatant was subjected to extraction for 30 min at 4°C in 20 mM CHAPS/TNE. The extracts were adjusted to 40% OptiPrep (Nycomed) and overlaid in a TLS 55 centrifugation tube with 1.4 ml of 30% OptiPrep/TNE and 200 µl TNE. After centrifugation for 2 h at 55,000 rpm, five fractions were collected from the top, and BACE1A-YFP or YFP-APP were recovered by immunoprecipitation with antibody KG77.
Uptake of biotin transferrin
N2a cells seeded on coverslips were transfected with either human transferrin receptor (TfR) and dynK44A-GFP or with TfR and pEGFP-N1 (CLONTECH Laboratories, Inc.). On the next day, the coverslips were washed by dipping in ice-cold PBS and then incubated for 20 min on ice with human biotin transferrin (50 µg/ml; Sigma-Aldrich) in 30 µl of low carbonate MEM supplemented with 2 mg/ml BSA (medium/BSA). After another wash with ice-cold PBS, the coverslips were incubated for 20 min on ice with a 1:40 dilution of the monoclonal antihuman TfR antibody (Roche) in 30 µl of medium/BSA or with medium/BSA alone. The cells were washed again in ice-cold PBS and subsequently incubated for 10 min at 37°C in 50 µl of medium/BSA to allow internalization of biotin transferrin. Biotin transferrin that remained on the surface was removed by three washes (2 min each) with ice-cold 0.5 M acetic acid and 0.5 M NaCl. Control cells used to determine the total amount of biotin transferrin present were incubated with ice-cold PBS for the same time.
The cells were lysed in 200 µl of PBS, 2% NP-40, and 0.2% SDS. Part of the lysate was incubated with streptavidin-coated magnetic beads (Dynal), a sheep antitransferrin antibody (Scottish Antibody Production Unit), and a rabbit antisheep secondary antibody (Dianova) coupled to a ruthenium trisbipyridine chelate (IGEN International, Inc.). These reagents have been described previously by Horiuchi et al. (1997). Biotin transferrin was then quantified on an Origen M8 analyzer (IGEN International, Inc.).
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Footnotes |
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Acknowledgments |
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R. Ehehalt was supported by grant EH196/1-1 from the Deutsche Forschungsgemeinschaft (DFG), P. Keller was supported by a grant from the Max Planck Gesellschaft, and K. Simons and P. Keller were supported by the DFG Schwerpunktprogramm SPP 1085 "Zellulaere Mechanismen der Alzheimer Erkrankung."
Submitted: 22 July 2002
Revised: 20 November 2002
Accepted: 22 November 2002
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References |
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