Membrane Biology Section, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland
Submitted 30 December 2004 ; accepted in final form 13 April 2005
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ABSTRACT |
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Alzheimers disease; -secretase;
-amyloid; intramembranous protease; transmembrane topology
PS1 is a 50-kDa integral membrane protein. In cells the PS1 holoprotein is rapidly endoproteolyzed by cleavage near Met292. The resulting
30 kDa NH2 terminal fragment (NTF) and
20 kDa COOH terminal fragment (CTF) are found in high molecular weight complexes with the integral membrane proteins nicastrin, PEN-2, and APH-1, which together are thought to constitute the
-secretase (27). Recent evidence suggests that the components of these complexes not only stabilize PS1 but are also involved in its maturation by endoproteolysis (20, 26).
An understanding of the transmembrane topology of PS1 is clearly essential to the interpretation of its role in -secretase activity. Hydropathy analysis shows that PS1 contains 10 hydrophobic regions (HRs) sufficiently long to form
-helical membrane spanning segments (MSSs; Fig. 1A). Essentially two types of studies have been performed by various groups to study the PS1 topology: 1) experiments examining antibody epitope accessibility before and after plasma membrane permeabilization (58), and 2) experiments examining the membrane integration of truncated forms of PS1 (17, 21) or SEL-12 (18, 19), a PS1 homologue from Caenorhabditis elegans. In these latter experiments, reporter peptides were fused to PS1 or SEL-12 truncated after each HR and these chimeric proteins were expressed in isolated microsomes or intact cells. Assays were then carried out to determine the location of the reporter peptide in the cytosolic or extracellular compartment, allowing one to infer the ability of each successive HR to integrate into the membrane.
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Recently, however, Annaert et al. (2) have found evidence that the COOH terminal 39 amino acids of PS1 bind specifically to the MSSs of both APP and telencephalin, another -secretase substrate. This 39 amino acid stretch begins at the COOH terminal end of HR 9 and thus includes HR 10 (Fig. 1B). On the basis of their results, Annaert et al. (2) have proposed that the binding site in PS1 for type I membrane protein substrates actually lies within the membrane and that these 39 amino acids form a part of this binding pocket. This hypothesis is clearly difficult to reconcile with the commonly accepted view that the sequence downstream of HR 9 lies in the cytosol. In the experiments presented here, we reexamine the location of the COOH terminus of PS1 and the association of HR 10 with the membrane. We present the results of experiments carried out using three independent methodologies, which provide strong evidence that HR 10 spans the membrane and that the COOH terminus of PS1 lies in the extracytosolic compartment.
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MATERIALS AND METHODS |
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Cell culture and transfection. Human embryonic kidney (HEK)-293 and HEK-293T cells were cultured and transfected as previously described (9). The PS1/PS2 double-knockout cell line BD1 (11), a generous gift from Dr. Alan Bernstein (Mt. Sinai Hospital, Toronto, Ontario, Canada), was cultured as described (30) and transfected with the use of FuGENE (Roche).
Preparation of ER vesicles and proteinase K digestion. The procedure for the proteinase K experiments was based on that of Feramisco et al. (12) with some modifications. Briefly, HEK-293 cells were collected in buffer A, composed of (in mM) 10 HEPES-KOH, pH 7.4, 10 KCl, 1.5 MgCl2, 5 Na-EDTA, 5 Na-EGTA, and 250 sucrose (all steps at 4°C), homogenized by being passed through a 22-gauge needle 15 times, and centrifuged at 3,000 g for 10 min. The supernatant was centrifuged at 20,000 g for 15 min, and the resulting pellet, containing sealed cytosolic side out ER vesicles (see RESULTS), was resuspended in buffer A containing 100 mM NaCl. Aliquots of this preparation (24 µg of protein) were treated with 2 mg/ml proteinase K (Sigma, P5568) in the presence or absence of 1% Triton X-100 in a total volume of 12 µl for 30 min on ice. The reaction was stopped by the addition of PMSF at a final concentration of 2030 mM. Samples were then subjected to SDS-PAGE and immunoblot analysis.
DNA constructs.
The segments of the human PS1 sequence indicated were cloned into the mammalian expression vector pEGFP- (10). This vector drives the expression of a fusion protein consisting of the enhanced GFP (EGFP), followed by BglII and HindIII restriction sites for the insertion of (PS1) sequence and a COOH terminal glycosylation tag.
Site-directed mutagenesis was carried out using the Quikchange and Quikchange II XL kits (Stratagene) used according to the manufacturers instructions.
Preparation and deglycosylation of membrane fractions.
Particulate fractions were prepared from HEK-293T cells transiently transfected with pEGFP- constructs, as previously described (10). A membrane fraction was prepared from this particulate fraction by alkaline floatation essentially as previously described (10), except that all sucrose solutions were buffered with 100 mM Na2CO3 (pH 11.5). The membrane fractions were deglycosylated with the use of peptide:N-glycosidase F (PNGase F; New England Biolabs) (10).
SDS-PAGE, Western blot, and data analysis. SDS-PAGE and Western blot analysis were carried out as previously described (10). Quantitation of Western blots was done with the use of ImageQuant 5.2 software (Molecular Dynamics).
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RESULTS AND DISCUSSION |
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In contrast to our results for PS1-N and PS1-loop, we find that 100.2 ± 7.6% (n = 9) of the immunoreactive signal of the antibody PS1-C, raised against the extreme COOH terminus of PS1, remains after proteinase K treatment. This signal is present in two bands, one at or near the molecular weight of the undigested CTF and the other at 10.0 ± 0.3 kDa, representing 26.0 ± 4.3% and 74.2 ± 8.3% of the starting signal, respectively (Fig. 2B). We have not attempted to determine the reason why only 5% of the full-length CTF signal is resistant to proteinase K when assayed by the PS1-loop antibody vs. 26% when assayed by the PS1-C antibody. However, it is possible that the epitope recognized by PS1-loop (a monoclonal antibody) lies near the NH2 terminal end of the CTF and may be digested from some of the molecules detected by PS1-C near the molecular weight of the full-length CTF. In this regard, in our preliminary experiments (not shown) we noted that PS1 was considerably more resistant to digestion by proteinase K than calnexin, possibly because, as a part of a large membrane-bound protein complex, PS1 is relatively less accessible from the surrounding medium. Nevertheless, what is strikingly clear from Fig. 2B is that the accessibility of the extreme COOH terminus of PS1 to proteinase K is quite different from that of the NH2 terminus and the loop region, strongly suggesting that it does not lie in the cytosolic compartment.
Reporter gene fusion.
To further examine the location of the COOH terminus of PS1, we have used a gene fusion approach, where portions of the PS1 sequence were fused between EGFP and a COOH terminal glycosylation tag (see MATERIALS AND METHODS). This latter sequence contains five consensus sites for N-linked glycosylation (10); when translocated into the interior of the ER it acquires 14 kDa of apparent molecular weight due to glycosylation, an increase that is easily discerned on SDS-PAGE. The utility of this experimental system for membrane topology and biogenesis studies has been previously documented (9, 10).
In Fig. 3 we illustrate the results of experiments where PS1 fragments encoding HRs 79 (cHR 79), HRs 710 (cHR 710), HRs 19 (cHR 19), and HRs 110 (full-length PS1; cHR 110) were expressed as EGFP fusion proteins in HEK-293T cells. In Fig. 3A, we show the results of a typical experiment where the membrane fraction from HEK-293T cells, transiently transfected with the construct indicated, was treated with (+) or without () PNGase F (see MATERIALS AND METHODS). These membrane fractions were run on SDS-PAGE and probed by Western blot analysis using an antibody against EGFP to determine their extent of glycosylation and thus the location of the glycosylation tag inside or outside the ER lumen (Fig. 3B). Little glycosylation is seen for either cHR 79 or cHR 19, where the PS1 sequence ends after HR 9, consistent with the general consensus that the COOH terminal end of HR 9 faces the cytoplasm. However, both fusion proteins that incorporate HR 10 (cHR 710 and cHR 110) are highly glycosylated, indicating that HR 10 spans the membrane in these constructs. To confirm this interpretation, in the fusion protein cHR 110 (RD), we have mutated amino acids Leu443 and Val444 near the middle of HR 10 (Fig. 1B) in cHR 110 to Arg and Glu, respectively, dramatically reducing the hydrophobicity of HR 10. As illustrated in Fig. 3B, the glycosylation of this mutated construct is markedly reduced relative to cHR 110, confirming that the glycosylation of cHR 110 is dependent on the hydrophobicity of HR 10, consistent with its role as a MSS.
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To test whether PS1-NGT could produce functional -secretase activity, we transiently expressed it in BD-1 cells [which have no endogenous
-secretase activity because they lack both PS1 and PS2 (11)], and measured the production of APP cleavage products (Total A
) in the extracellular solution. As illustrated in Fig. 4C, this glycosylation mutant yielded similar activity to wild-type PS1. In Western blots of membranes from transfected BD-1 cells, we were unable to detect any significant component of unglycosylated PS1-NGT (Fig. 4D).
Relationship to previous topology studies of PS1. As already discussed, our data from endogenously expressed PS1 indicating that the NH2 terminus and loop region between HRs 6 and 8 are cytosolic (Fig. 2) are in agreement with the conclusions of most other groups that have studied the PS1 topology. Dewji and Singer (6, 7) have presented evidence that the epitopes of antibodies directed against the NH2 terminus and loop regions are accessible on the surface of unpermeabilized cells. Similar results were found by Schwarzman et al. (23) using an NH2 terminal antibody. While we cannot exclude the possibility that there is a pool of PS1 on the cell surface with an opposite orientation, our results are consistent with the conclusion that the vast majority of PS1 molecules that are synthesized in the ER have their NH2 terminus and loop regions in the cytoplasm.
Our conclusion that the COOH terminus of HR 9 is cytosolic (Fig. 3) is in agreement with all groups that have studied the PS1 topology. In addition, our results provide strong evidence that HR 10 is a MSS and that the COOH terminal tail of PS1 is located in the ER lumen where it is inaccessible to proteinase K applied from the cytosolic compartment and accessible to luminal oligosaccharyl transferase (Figs. 3 and 4). As discussed previously, these findings are in agreement with the results of Nakai et al. (21) obtained using a reporter gene fusion approach and are consistent with the proposal of Annaert et al. (2) that the PS1 binding site for type I membrane protein includes HR 10 and lies within the membrane. Although the topology model of Li and Greenwald (18, 19) for the PS1 homologue SEL-12 places HR 10 in the cytosol of C. elegans, our results provide strong evidence that this is not the case for PS1 in the mammalian cell lines we have tested.
Two groups that have examined the accessibility of PS1 COOH terminal antibody epitopes also concluded that the COOH terminus of PS1 is cytosolic (68), i.e., that COOH terminal epitopes are not accessible in cells without treatment with membrane permeabilizing agents. However, experiments of this type are complicated by the fact that that the majority of PS1 is localized to intracellular membranes (8), and that permeabilizing agents may also alter epitope affinity and/or accessibility independent of their permeabilizing effects. To distinguish between cytosolic and luminal epitopes these experiments depend on agents that selectively permeabilize the plasma membrane without permeabilizing intracellular compartments. Because of the large amount of intracellular PS1, even a small permeabilization of intracellular membranes could potentially confound their interpretation. Also, the COOH terminal antibody used by one of these groups (6, 7) was raised against a 9-amino-acid peptide from human PS2 (STDNLVRPF), which is homologous to amino acids 448456 of human PS1 (ATDYLVQPF); but the first 6 amino acids of this peptide actually lie within HR 10 of PS1 (Fig. 1B). Because we place HR 10 in the bilayer we would not have expected this epitope to be accessible from either side of the membrane in the absence of detergent.
Finally, we note that in a recent publication Friedmann et al. (13) have studied the topology of five homologues of the presenilins found in the human genome. These include signal peptide peptidase and four signal peptide peptidase-like (putative) proteases. Interestingly, they propose that all five of these proteins have a nine MSS topology with extracellular NH2 termini and intracellular COOH termini, i.e., with termini in the reverse orientation to our findings for PS1. Because of their reverse orientation in the membrane these proteins are thought to be intramembranous proteases acting on type II membrane proteins as substrates (13).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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