Mutation of Conserved Aspartates Affects Maturation of Both Aspartate Mutant and Endogenous Presenilin 1 and Presenilin 2 Complexes
2000; Elsevier BV; Volume: 275; Issue: 35 Linguagem: Inglês
10.1016/s0021-9258(19)61517-6
ISSN1083-351X
AutoresGang Yu, Fusheng Chen, Masaki Nishimura, Harald Steiner, Anurag Tandon, Toshitaka Kawarai, Shigeki Arawaka, Agnes Supala, You‐Qiang Song, Ekaterina Rogaeva, Erin Holmes, Dong Mei Zhang, Paul Milman, Paul E. Fraser, Christian Haass, Peter St George‐Hyslop,
Tópico(s)Signaling Pathways in Disease
ResumoPresenilin (PS1 and PS2) holoproteins are transiently incorporated into low molecular weight (MW) complexes. During subsequent incorporation into a higher MW complex, they undergo endoproteolysis to generate stable N- and C-terminal fragments. Mutation of either of two conserved aspartate residues in transmembrane domains inhibits both presenilin-endoproteolysis and the proteolytic processing of β-amyloid precursor protein and Notch. We show that although PS1/PS2 endoproteolysis is not required for inclusion into the higher MW N- and C-terminal fragment-containing complex, aspartate mutant holoprotein presenilins are not incorporated into the high MW complexes. Aspartate mutant presenilin holoproteins also preclude entry of endogenous wild type PS1/PS2 into the high MW complexes but do not affect the incorporation of wild type holoproteins into lower MW holoprotein complexes. These data suggest that the loss of function effects of the aspartate mutants result in altered PS complex maturation and argue that the functional presenilin moieties are contained in the high molecular weight complexes. Presenilin (PS1 and PS2) holoproteins are transiently incorporated into low molecular weight (MW) complexes. During subsequent incorporation into a higher MW complex, they undergo endoproteolysis to generate stable N- and C-terminal fragments. Mutation of either of two conserved aspartate residues in transmembrane domains inhibits both presenilin-endoproteolysis and the proteolytic processing of β-amyloid precursor protein and Notch. We show that although PS1/PS2 endoproteolysis is not required for inclusion into the higher MW N- and C-terminal fragment-containing complex, aspartate mutant holoprotein presenilins are not incorporated into the high MW complexes. Aspartate mutant presenilin holoproteins also preclude entry of endogenous wild type PS1/PS2 into the high MW complexes but do not affect the incorporation of wild type holoproteins into lower MW holoprotein complexes. These data suggest that the loss of function effects of the aspartate mutants result in altered PS complex maturation and argue that the functional presenilin moieties are contained in the high molecular weight complexes. presenilin 1 presenilin 2 N-terminal fragment C-terminal fragment β-amyloid precursor protein murine embryonic fibroblast wild type Presenilin 1 (PS1)1 (1Sherrington R. Rogaev E. Liang Y. Rogaeva E. Levesque G. Ikeda M. Chi H. Lin C. Holman K. Tsuda T. Mar L. Fraser P. Rommens J.M. St. George-Hyslop P. Nature. 1995; 375: 754-760Crossref PubMed Scopus (3579) Google Scholar) and presenilin 2 (PS2) (2Rogaev E.I. Sherrington R. Rogaeva E.A. Levesque G. Ikeda M. Liang Y. Chi H. Lin C. Holman K. Tsuda T. Mar L. Sorbi S. Nacmias B. Piacentini S. Amaducci L. 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Cell. 1999; 99: 691-702Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). The precise role of PS1 in this unusual form of proteolytic processing is unknown. Mutating either of the two conserved aspartate residues within transmembrane domains of PS1 (Asp257 and Asp385) or PS2 (Asp366) inhibits the endoproteolytic cleavage of the mutant presenilin and reduces the intramembranous cleavage of βAPP and Notch (23Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.S. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1685) Google Scholar, 24Steiner H. Duff K. Capell A. Romig H. Grim M.G. Lincoln S. Hardy J., Yu, X. Picciano M. Fechteler K. Citron M. Kopan R. Pesold B. Keck S. Baader M. Tomita T. Iwatsubo T. Baumeister R. Haass C. J. Biol. Chem. 1999; 274: 28669-28673Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 25Ray W.J. Yao M. Mumm J. Schoeter E.H. Saftig P. Wolfe M. Selkoe D.J. Kopan R. Goate A.M. J. Biol. Chem. 1999; 274: 36801-36807Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). It has been proposed that mutation of these aspartate residues has a dominant negative effect on a catalytic site and that the presenilins are aspartyl proteases (23Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.S. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1685) Google Scholar). One observation possibly inconsistent with this hypothesis is that whereas PS1−/−, PS1 aspartate mutant, and PS2 aspartate mutant cells all have impaired γ-secretase activity, PS2−/− cells have normal γ-secretase activity (24Steiner H. Duff K. Capell A. Romig H. Grim M.G. Lincoln S. Hardy J., Yu, X. Picciano M. Fechteler K. Citron M. Kopan R. Pesold B. Keck S. Baader M. Tomita T. Iwatsubo T. Baumeister R. Haass C. J. Biol. Chem. 1999; 274: 28669-28673Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 26Herreman A. Hartmann D. Annaert W. Saftig P. Craessaerts K. 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Taken together, these structural effects on presenilin complex formation explain all previous observations without the need to postulate a novel catalytic activity for these residues. These results also argue that the low MW holoprotein complexes, regardless of whether they have wild type PS1/PS2 or aspartate mutant PS1/PS2, are nonfunctional intermediaries between PS monomers and PS-NTF·CTF-containing higher MW functional complexes. We generated HEK293 cells (using LipofectAMINE transfection) and murine embryonic fibroblasts (MEF) (using theCLONTECH replication-defective recombinant retroviral system) stably or transiently overexpressing cDNA constructs corresponding to: wild type human PS1 (PS1wt), a FAD-associated clinical mutation causing missplicing of residues 290–319 encoded by exon 10 (also termed exon 9) (PS1ΔE10), aspartate mutant human PS1 (PS1D257A and PS1D385A), an exon 10 splicing mutant cDNA containing the PS1D385A mutation incis (PS1ΔE10D385A), PS2wt, or the PS2D366A mutant (16Citron M. Westaway D. Xia W. Carlson G. Diehl T.S. Levesque G. Johnson-Wood K. Lee M. Seubert P. Davis A. Kholodenko D. Motter R. Sherrington R. Perry B. Yao H. Strome R. Lieberburg I. Rommens J. Kim S. Schenk D. Fraser P. St. George-Hyslop P. Selkoe D.J. Nature Med. 1997; 3: 67-72Crossref PubMed Scopus (1161) Google Scholar, 23Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.S. 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We elected to investigate the Asp → Ala mutants because the change in both the size and charge of the amino acid would maximize our ability to detect structural effects, which we would predict to be subtle because the aspartate mutant PS1 molecules are not destabilized and degraded. Expression of PS1, PS2, or one of their mutants was investigated by standard Western blots as described (16Citron M. Westaway D. Xia W. Carlson G. Diehl T.S. Levesque G. Johnson-Wood K. Lee M. Seubert P. Davis A. Kholodenko D. Motter R. Sherrington R. Perry B. Yao H. Strome R. Lieberburg I. Rommens J. Kim S. Schenk D. Fraser P. St. George-Hyslop P. Selkoe D.J. Nature Med. 1997; 3: 67-72Crossref PubMed Scopus (1161) Google Scholar, 24Steiner H. Duff K. Capell A. Romig H. Grim M.G. Lincoln S. Hardy J., Yu, X. Picciano M. Fechteler K. Citron M. Kopan R. Pesold B. Keck S. Baader M. Tomita T. Iwatsubo T. Baumeister R. Haass C. J. Biol. Chem. 1999; 274: 28669-28673Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 27Nishimura M., Yu, G. Levesque G. Zhang D.M. Ruel L. Chen F. Milman P. Holmes E. Liang Y. Kawarai T. Jo E. Supala A. Rogaeva E. Xu D.M. Janus C. Levesque L. Bi Q. Duthie M. Rozmahel R. Mattila K. Lannfelt L. Westaway D. Mount H.T. Woodgett J. Fraser P. St. George-Hyslop P. Nature Med. 1999; 5: 164-169Crossref PubMed Scopus (205) Google Scholar). Biochemical subcellular fractionation was performed on a step gradient consisting of 1 ml each of 30, 25, 20, 15, 12.5, 10, 7.5, 5, and 2.5% (v/v) iodixanol (Accurate, NY) in homogenization buffer (130 mmKCl, 25 mm NaCl, 25 mm Tris, 1 mmEGTA, pH 7.4) as described previously (20Chen, F., Yang, D.-S., Tandon, A., Rozmahel, R., Yu, G., Nishimura, M., Kawarai, T., Westaway, D., Gandy, S. E., Fraser, P. E., St. George-Hyslop, P. (2000) J. Biol. Chem., in pressGoogle Scholar, 28Majoul I.V. Bastiaens P.I.H. Soling H.D. J. Cell Biol. 1996; 133: 777-789Crossref PubMed Scopus (136) Google Scholar). Discontinuous sucrose gradient fractionation was performed as described previously (4Yu G. Chen F. Levesque G. Nishimura M. Zhang D.-M. Levesque L. Rogaeva E. Xu D. Liang Y. Duthie M. St. George-Hyslop P. Fraser P.E. J. Biol. Chem. 1998; 273: 16470-16475Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). Glycerol velocity gradient centrifugation was performed on cells lysed in 1.0% digitonin buffer as described previously (4Yu G. Chen F. Levesque G. Nishimura M. Zhang D.-M. Levesque L. Rogaeva E. Xu D. Liang Y. Duthie M. St. George-Hyslop P. Fraser P.E. J. Biol. Chem. 1998; 273: 16470-16475Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). Fractions were collected, and the proteins in each fraction were separated on Tris-glycine polyacrylamide gels (Novex), transferred to polyvinylidene difluoride (Millipore), and the Western blots were probed with appropriate antibodies and detected using ECL (Amersham Pharmacia Biotech). Each fractionation experiment was performed multiple times on PS1D257A, PS1D385A, PS1wt, PS1ΔE10, PS1ΔE10D385A, PS2wt, or PS2D366A cells in parallel experiments. Molecular mass marker proteins (cytochrome c, 12 kDa; carbonic anhydrase, 29 kDa; bovine serum albumin, 69 kDa; β-amylase, 200 kDa; apoferritin, 443 kDa; thyroglobulin, 669 kDa) were included in each gradient fractionation to allow direct comparison of fraction run in parallel as described previously (4Yu G. Chen F. Levesque G. Nishimura M. Zhang D.-M. Levesque L. Rogaeva E. Xu D. Liang Y. Duthie M. St. George-Hyslop P. Fraser P.E. J. Biol. Chem. 1998; 273: 16470-16475Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). In studies using transiently transfected cells, the location of the endogenous presenilin complexes in the untransfected cells within the culture served as an additional internal control. Antibody Ab14 (rabbit polyclonal to PS1-NTF), antibody NT1 (mouse anti-human PS1-NTF monoclonal), rabbit polyclonal anti-PS1-CTF antibody, DT-2 (mouse anti-human monoclonal to PS2), and antibody 369 (to α- and β-C-terminal stubs of βAPP) have been reported elsewhere (9Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovisky T. Davenport F. Nordstedt C. Seeger M. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar, 29Levesque L. Annaert W. Craessaerts K. Mathews P.M. Seeger M. Nixon R. van Leuven F. Gandy S. Westaway D. St. George-Hyslop P. De Strooper B. Fraser P.E. Mol. Med. 1999; 5: 542-554Crossref PubMed Google Scholar). Initial characterization of our cell lines overexpressing wild type human PS1 (PS1wt), wild type PS2 (PS2wt), aspartate mutant PS1 (PS1D257A and PS1D385A), or aspartate mutant PS2D366A confirmed previous reports of the effects of overexpression of aspartate mutant PS1/PS2 proteins (23Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.S. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1685) Google Scholar,24Steiner H. Duff K. Capell A. Romig H. Grim M.G. Lincoln S. Hardy J., Yu, X. Picciano M. Fechteler K. Citron M. Kopan R. Pesold B. Keck S. Baader M. Tomita T. Iwatsubo T. Baumeister R. Haass C. J. Biol. Chem. 1999; 274: 28669-28673Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Thus, overexpression of human PS1D257A or human PS1D385A in MEF lines resulted in the expected “displacement/replacement” of the endogenous mouse PS1, inhibition of the endoproteolysis of the aspartate mutant human PS1, and inhibition of γ-secretase cleavage of endogenous mouse βAPP to levels comparable to those in fibroblasts from homozygous PS1-null (PS1−/−) mice (Fig. 2) (23Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.S. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1685) Google Scholar). However, note that the human specific PS1 antibody NT1 (29Levesque L. Annaert W. Craessaerts K. Mathews P.M. Seeger M. Nixon R. van Leuven F. Gandy S. Westaway D. St. George-Hyslop P. De Strooper B. Fraser P.E. Mol. Med. 1999; 5: 542-554Crossref PubMed Google Scholar) did detect small amounts of the PS1D257A and PS1D385A proteins as processed fragments (Fig. 2). Aβ levels were too low to measure in MEF cells. However, expression of PS1D257A and PS1D385A in HEK293 cells overexpressing βAPPSwedish drastically reduced both Aβ40 and Aβ42 (not shown). Similar effects were detected when the PS2D366A mutant was overexpressed in HEK293 cells (not shown) and have already been published (24Steiner H. Duff K. Capell A. Romig H. Grim M.G. Lincoln S. Hardy J., Yu, X. Picciano M. Fechteler K. Citron M. Kopan R. Pesold B. Keck S. Baader M. Tomita T. Iwatsubo T. Baumeister R. Haass C. J. Biol. Chem. 1999; 274: 28669-28673Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Glycerol velocity gradient analysis revealed that both the PS1wt N- and C-terminal fragments (Fig.3 A), and the PS2wtN- and C-terminal fragments (Fig. 3 F) were predominantly present in ∼250-kDa complexes. The holoproteins of PS1wt(Fig. 3 A) and PS2wt (Fig. 3 F) were observed within lower molecular weight complexes. These results are in agreement with previously published data (4Yu G. Chen F. Levesque G. Nishimura M. Zhang D.-M. Levesque L. Rogaeva E. Xu D. Liang Y. Duthie M. St. George-Hyslop P. Fraser P.E. J. Biol. Chem. 1998; 273: 16470-16475Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 5Capell A. Grunberg J. Pesold B. Diehlmann A. Citron M. Nixon R. Beyreuther K. Selkoe D.J. Haass C. J. Biol. Chem. 1998; 273: 3205-3211Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). In contrast, the aspartate mutant presenilins were predominantly present as holoproteins located in the lower molecular weight fractions (PS1D257A, Fig. 3 B; PS1D385A, not shown; PS2D366A, Fig. 3 E). Longer exposures also revealed trace amounts of the aspartate mutant holoproteins distributed into fractions of much higher molecular mass (≫250 kDa) than the mature wild type presenilin NTF·CTF complexes (not shown). Quantitative image analyses (Fig. 3, G and I) confirm these results. The presenilins interact with β-catenin and, in brain, with neuronal plakophilin-related armadillo protein (NPRAP)/δ-catenin, which are components of both the immature holoprotein PS1 complex and the mature NTF·CTF PS1 complex (4Yu G. Chen F. Levesque G. Nishimura M. Zhang D.-M. Levesque L. Rogaeva E. Xu D. Liang Y. Duthie M. St. George-Hyslop P. Fraser P.E. J. Biol. Chem. 1998; 273: 16470-16475Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 30Zhou J. Liyanage U. Medina M. Ho C. Simmons A.D. Lovett M. Kosik K.S Neuroreport. 1997; 8: 2085-2090Crossref PubMed Scopus (200) Google Scholar, 31Levesque G., Yu, G. Nishimura M. Zhiang D.M. Levesque L., Yu, H. Xu D. Liang Y. Ikeda M. Rommens J. Fraser P.E. St. George-Hyslop P. J. Neurochem. 1999; 72: 999-1008Crossref PubMed Scopus (98) Google Scholar). Immunoprecipitation studies showed no obvious alteration in the affinity of PS1 aspartate mutants for β-catenin (Fig. 4). This result is not unexpected because the presenilin aspartate residues are in transmembrane domains, whereas β-catenin interacts with the cytoplasmic hydrophilic domain between transmembranes 6 and 7 (31Levesque G., Yu, G. Nishimura M. Zhiang D.M. Levesque L., Yu, H. Xu D. Liang Y. Ikeda M. Rommens J. Fraser P.E. St. George-Hyslop P. J. Neurochem. 1999; 72: 999-1008Crossref PubMed Scopus (98) Google Scholar). The identity of proteins uniquely present in the high molecular weight complex has not yet been established. Consequently, we cannot directly test the possibility that the aspartate mutations might impede interactions with components putatively unique to the high MW complex. Biochemical fractionation studies using either discontinuous sucrose gradients or iodixanol gradients revealed that the aspartate mutant presenilin holoproteins were distributed in the rough endoplasmic reticulum, smooth endoplasmic reticulum/endoplasmic reticulum-like, and Golgi fractions (data not shown). Although this is slightly different from the distribution of wild type presenilin holoproteins (predominantly rough endoplasmic reticulum and smooth endoplasmic reticulum/endoplasmic reticulum-like fractions) (4Yu G. Chen F. Levesque G. Nishimura M. Zhang D.-M. Levesque L. Rogaeva E. Xu D. Liang Y. Duthie M. St. George-Hyslop P. Fraser P.E. J. Biol. Chem. 1998; 273: 16470-16475Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 32Kim S.H. Lah J.J. Thinakaran G. Levey A. Sisodia S.S. Neurobiol. Dis. 2000; 7: 99-117Crossref PubMed Scopus (49) Google Scholar), it overlaps that seen for the Alzheimer-associated PS1ΔE10 mutant (5Capell A. Grunberg J. Pesold B. Diehlmann A. Citron M. Nixon R. Beyreuther K. Selkoe D.J. Haass C. J. Biol. Chem. 1998; 273: 3205-3211Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 33Zhang J. Kang D.E. Xia W. Okochi M. Mori H. Selkoe D. Koo E.H. J. Biol. Chem. 1998; 273: 12436-12442Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) (which supports increased γ-secretase activity but which does not undergo endoproteolysis (9Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovisky T. Davenport F. Nordstedt C. Seeger M. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar)). The aberrant glycerol velocity gradient fractionation profile of aspartate mutant presenilins might be because of either of two distinct mechanisms. The aspartate mutant presenilins might be excluded from the mature, larger complexes (e.g. possibly because of failure to interact with components selectively present only in the mature complexes). Alternatively, it could be argued that it is their failure to undergo endoproteolysis, which inhibits their inclusion in the mature fragment containing complex. To discriminate between these possibilities, we compared the fractionation profiles of the Alzheimer-associated PS1ΔE10 mutant with that of the same construct containing a PS1D385A mutation incis (PS1ΔE10D385A) (which has already been shown to inhibit γ-secretase (23Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.S. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1685) Google Scholar)). PS1ΔE10D385A and PS1ΔE10 were transiently transfected into HEK293 cells, and their distributions on glycerol gradients were analyzed using the endogenous PS1 complexes in untransfected cells within these cultures as internal controls. In agreement with previous reports (5Capell A. Grunberg J. Pesold B. Diehlmann A. Citron M. Nixon R. Beyreuther K. Selkoe D.J. Haass C. J. Biol. Chem. 1998; 273: 3205-3211Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar), the uncleaved PS1ΔE10 was detected in gradient fractions that overlapped that of the endogenous mature PS1·NTF·CTF high molecular weight complex (Fig.3 C), indicating that the PS1ΔE10holoprotein is appropriately integrated into the mature higher molecular weight complex. In contrast, the PS1ΔE10D385A holoprotein predominantly exists in the lower MW fractions (Fig. 3 D). As with the PS1D385A mutation, longer exposures detected trace amounts of the PS1ΔE10D385A holoprotein in fractions of much higher molecular mass (≫250 kDa) than those containing the mature PS1wt complex. Quantitative image analyses again confirm the distinct but overlapping distributions of holoprotein- and NTF·CTF-containing complexes (compare Fig. 3,G, H, and I). Cumulatively, the present results suggest that the effect of the PS1D385Amutation on complex formation is independent of endoproteolysis. Finally, to address the question of how aspartate mutants (and especially PS2 aspartate mutants) might suppress normal γ-secretase activity, we examined endogenous PS1 levels in cells overexpressing the PS2D366A mutation (Fig.5 A). Conversely, we also analyzed endogenous PS2 levels in cells overexpressing the PS1D385A mutation (Fig. 5 A). In agreement with prior studies on overexpression of wild type presenilins (10Thinakaran G. Harris C.L. Ratovitski T. Davenport F. Slunt H.H. Price D.L. Borchelt D.R. Sisodia S.S. J. Biol. Chem. 1997; 272: 28415-28422Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar), these experiments showed that overexpression of either wild type or aspartate mutant PS2 displaced and destabilized endogenous PS1 and vice versa. However, the displacement phenomenon induced by overexpression of either wild type or aspartate mutant presenilin proteins mainly affects the formation of NTF·CTF fragment complexes. Thus the amounts of endogenous PS1 holoprotein detected upon overexpression of aspartate mutant PS2 is relatively unchanged despite nearly complete suppression of endogenous PS1 fragment formation (Fig.5 A). Similarly, overexpression of aspartate mutant PS1 shows persistence of endogenous PS2 holoprotein expression but suppression of endogenous PS2 NTF·CTF formation (Fig. 5 B). The persistent endogenous presenilin holoproteins detected in aspartate mutant expressing cells could exist either as monomers or be incorporated into the low MW holoprotein complexes. To resolve this, we examined the abundance and size distribution of endogenous PS1 holoprotein epitopes in PS2D366A cells and in control cells expressing endogenous PS1. These studies revealed that the abundance and size distribution of endogenous PS1 holoprotein epitopes were identical in PS2D366A cells and in wild type control cells (Fig. 6). In contrast, the abundance of PS1-NTF in the higher MW fractions was dramatically reduced compared with wild-type control cells (Fig. 6). This observation supports the argument that the aspartate mutant holoprotein blocks maturation of endogenous presenilin holoprotein complexes and that it is the loss of the higher molecular weight endogenous NTF·CTF-containing complex that likely causes the loss of function effect. Two lines of evidence from our data support the notion that mutation of conserved intramembrane aspartate residues of either PS1 or PS2 has structural effects on the normal presenilin protein complex formation. First, these mutations are associated with failed maturation of aspartate mutant presenilin protein complexes such that the majority of aspartate mutant presenilins remain in the smaller (immature) holoprotein-containing complex. This failure of aspartate mutant PS complexes to mature into the higher molecular weight forms cannot be ascribed simply to failure of presenilin endoproteolysis. Even when the PS1D385A mutant is placed within the PS1ΔE10 protein, which does not require endoproteolysis for biological activity, the chimeric PS1ΔE10D385A protein does not mature and has impaired activity. The most parsimonious interpretation of these results is that the aspartate residues are important in the formation of intra- or intermolecular interactions in the mature presenilin complexes. For instance, the aspartate residues might be involved in the interaction of presenilins with as yet unidentified membrane-bound proteins specific to the higher molecular weight mature complex.
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