Title: The Proteolytic Fragments of the Alzheimer's Disease-associated Presenilin-1 Form Heterodimers and Occur as a 100–150-kDa Molecular Mass Complex
Abstract: Mutations in the presenilin (PS) genes are linked to early onset familial Alzheimer's disease (FAD). PS-1 proteins are proteolytically processed by an unknown protease to two stable fragments of ∼30 kDa (N-terminal fragment (NTF)) and ∼20 kDa (C-terminal fragment (CTF)) (Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. H., Spitzer, L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M., Hardy, J., Levey, A. I., Gandy, S. E., Jenkins, N. A., Copeland, N. G., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 181–190). Here we show that the CTF and NTF of PS-1 bind to each other. Fractionating proteins from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-extracted membrane preparations by velocity sedimentation reveal a high molecular mass SDS and Triton X-100-sensitive complex of approximately 100–150 kDa. To prove if both proteolytic fragments of PS-1 are bound to the same complex, we performed co-immunoprecipitations using multiple antibodies specific to the CTF and NTF of PS-1. These experiments revealed that both fragments of PS-1 occur as a tightly bound non-covalent complex. Upon overexpression, unclipped wild type PS-1 sediments at a lower molecular weight in glycerol velocity gradients than the endogenous fragments. In contrast, the non-cleavable, FAD-associated PS-1 Δexon 9 sediments at a molecular weight similar to that observed for the endogenous proteolytic fragments. This result may indicate that the Δexon 9 mutation generates a mutant protein that exhibits biophysical properties similar to the naturally occurring PS-1 fragments. This could explain the surprising finding that the Δexon 9 mutation is functionally active, although it cannot be proteolytically processed (Baumeister, R., Leimer, U., Zweckbronner, I., Jakubek, C., Grünberg, J., and Haass, C. (1997) Genes & Function1, 149–159; Levitan, D., Doyle, T., Brousseau, D., Lee, M., Thinakaran, G., Slunt, H., Sisodia, S., and Greenwald, I. (1996)Proc. Natl. Acad. Sci. U. S. A. 93, 14940–14944). Formation of a high molecular weight complex of PS-1 composed of both endogenous PS-1 fragments may also explain the recent finding that FAD-associated mutations within the N-terminal portion of PS-1 result in the hyperaccumulation not only of the NTF but also of the CTF (Lee, M. K., Borchelt, D. R., Kim, G., Thinakaran, G., Slunt, H. H., Ratovitski, T., Martin, L. J., Kittur, A., Gandy, S., Levey, A. I., Jenkins, N., Copeland, N., Price, D. L., and Sisodia, S. S. (1997) Nat. Med. 3, 756–760). Moreover, these results provide a model to understand the highly regulated expression and processing of PS proteins. Mutations in the presenilin (PS) genes are linked to early onset familial Alzheimer's disease (FAD). PS-1 proteins are proteolytically processed by an unknown protease to two stable fragments of ∼30 kDa (N-terminal fragment (NTF)) and ∼20 kDa (C-terminal fragment (CTF)) (Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. H., Spitzer, L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M., Hardy, J., Levey, A. I., Gandy, S. E., Jenkins, N. A., Copeland, N. G., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 181–190). Here we show that the CTF and NTF of PS-1 bind to each other. Fractionating proteins from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-extracted membrane preparations by velocity sedimentation reveal a high molecular mass SDS and Triton X-100-sensitive complex of approximately 100–150 kDa. To prove if both proteolytic fragments of PS-1 are bound to the same complex, we performed co-immunoprecipitations using multiple antibodies specific to the CTF and NTF of PS-1. These experiments revealed that both fragments of PS-1 occur as a tightly bound non-covalent complex. Upon overexpression, unclipped wild type PS-1 sediments at a lower molecular weight in glycerol velocity gradients than the endogenous fragments. In contrast, the non-cleavable, FAD-associated PS-1 Δexon 9 sediments at a molecular weight similar to that observed for the endogenous proteolytic fragments. This result may indicate that the Δexon 9 mutation generates a mutant protein that exhibits biophysical properties similar to the naturally occurring PS-1 fragments. This could explain the surprising finding that the Δexon 9 mutation is functionally active, although it cannot be proteolytically processed (Baumeister, R., Leimer, U., Zweckbronner, I., Jakubek, C., Grünberg, J., and Haass, C. (1997) Genes & Function1, 149–159; Levitan, D., Doyle, T., Brousseau, D., Lee, M., Thinakaran, G., Slunt, H., Sisodia, S., and Greenwald, I. (1996)Proc. Natl. Acad. Sci. U. S. A. 93, 14940–14944). Formation of a high molecular weight complex of PS-1 composed of both endogenous PS-1 fragments may also explain the recent finding that FAD-associated mutations within the N-terminal portion of PS-1 result in the hyperaccumulation not only of the NTF but also of the CTF (Lee, M. K., Borchelt, D. R., Kim, G., Thinakaran, G., Slunt, H. H., Ratovitski, T., Martin, L. J., Kittur, A., Gandy, S., Levey, A. I., Jenkins, N., Copeland, N., Price, D. L., and Sisodia, S. S. (1997) Nat. Med. 3, 756–760). Moreover, these results provide a model to understand the highly regulated expression and processing of PS proteins. Most cases of Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid β-peptide; βAPP, β-amyloid precursor protein; CTF, C-terminal fragment; FAD, familial Alzheimer's disease; NTF, N-terminal fragment; PS, presenilin; TM, trans-membrane domain; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; wt, wild type. occur sporadically, with a strong increase in risk during aging (for review see Ref. 5Selkoe D.J. J. Biol. Chem. 1996; 271: 18295-18298Abstract Full Text Full Text PDF PubMed Scopus (761) Google Scholar). However, in at least 10–15% of cases, autosomal dominant mutations have been found to cause early onset familial AD (FAD). Mutations in three genes are known so far to cause FAD. Mutations within the gene encoding the β-amyloid precursor protein (βAPP) all cause the enhanced production of the 42-amino acid version of the amyloid β-peptide (Aβ42; see Ref. 6Suzuki N. Cheung T.T. Cai X.D. Odaka A. Otvos Jr., L. Eckman C. Golde T.E. Younkin S.G. Science. 1994; 264: 1336-1340Crossref PubMed Scopus (1358) Google Scholar; for review, see Ref. 5Selkoe D.J. J. Biol. Chem. 1996; 271: 18295-18298Abstract Full Text Full Text PDF PubMed Scopus (761) Google Scholar). Aβ42 is a major component of amyloid plaques (7Lemere C.A. Lopera F. Kosik K.S. Lendon C.L. Ossa J. Saido T. Yamaguchi H. Ruiz A. Martinez A. Madrigal L. Hincapie L. Arango J. Anthony D.C. Koo E.H. Goate A.M. Selkoe D.J. Carlos Arango J. Nat. Med. 1996; 2: 1146-1150Crossref PubMed Scopus (436) Google Scholar), which are the pathological hallmark of the disease (5Selkoe D.J. J. Biol. Chem. 1996; 271: 18295-18298Abstract Full Text Full Text PDF PubMed Scopus (761) Google Scholar). Aβ42 exhibits enhanced neurotoxicity, which might be due to its increased ability to form insoluble fibers (8Burdick D. Soreghan B. Kwon M. Kosomoski J. Knauer M. Henschen A. Yates J. Cotman C. Glabe C. J. Biol. Chem. 1992; 267: 546-554Abstract Full Text PDF PubMed Google Scholar, 9Jarrett J.T. Berger E.P. Lansbury P.T. Biochemistry. 1993; 32: 4693-4697Crossref PubMed Scopus (1768) Google Scholar). Increased production of Aβ42 (10Scheuner D. Eckman C. Jensen M. Song X. Citron M. Suzuki N. Bird T.D. Hardy J. Hutton M. Kukull W. Larson E. Levy-Lahad E. Viitanen M. Peskind E. Poorkaj P. Schellenberg G. Tanzi R. Wasco W. Lannfelt L. Selkoe D. Younkin S. Nat. Med. 1996; 2: 864-870Crossref PubMed Scopus (2282) Google Scholar, 11Borchelt D. Thinakaran G. Eckman C. Lee M. Davenport F. Ratovitsky T. Prada C.-M. Kim G. Seekins S. Yager D. Slunt H. Wang R. Seeger M. Levey A. Gandy S. Copeland N. Jenkins N. Price D. Younkin S. Sisodia S.S. Neuron. 1996; 17: 1005-10013Abstract Full Text Full Text PDF PubMed Scopus (1348) Google Scholar, 12Duff K. Eckman C. Zehr C. Yu X. Prada C.M. Perez-Tur J. Hutton M. Buee L. Harigaya Y. Yager D. Morgan D. Gordon M.N. Holcomb L. Refolo L. Zenk B. Hardy J. Younkin S. Nature. 1996; 383: 710-713Crossref PubMed Scopus (1325) Google Scholar, 13Citron 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. Nat. Med. 1997; 3: 67-72Crossref PubMed Scopus (1169) Google Scholar, 14Xia W. Zhang J. Kholodenko D. Citron M. Podlisny M.B. Teplow D.B. Haass C. Seubert P. Koo E.H. Selkoe D.J. J. Biol. Chem. 1997; 272: 7977-7982Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 15Tomita T. Maruyama K. Saido T.C. Kume H. Shinozaki K. Tokuhiro S. Capell A. Walter J. Grünberg J. Haass C. Iwatsubo T. Obata K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2025-2030Crossref PubMed Scopus (355) Google Scholar) was also found to result from the much more common mutations within the presenilin (PS) genes (16Sherrington R. Rogaev E.I. Liang Y. Rogaeva E.A. Levesque G. Ikeda M. Chi H. Lin C. Li G. Holman K. Tsuda T. Mar L. Foncin J.-F. Bruni A.C. Montesi M.P. Sorbi S. Rainero I. Pinessi L. Nee L. Chumakov I. Pollen D. Brookes A. Sanseau P. Polinsky R.J. Wasco W. da Silva H.A.R. Haines J.L. Pericak-Vance M.A. Tanzi R.E. Roses A.D. Fraser P.E. Rommens J.M. St. George-Hyslop P.H. Nature. 1995; 375: 754-760Crossref PubMed Scopus (3599) Google Scholar, 17Levy-Lahad E. Wasco W. Poorkaj P. Romano D.M. Oshima J. Pettingell W.H. Yu C. Jondro P.D. Schmidt S.D. Wang K. Crowley A.C. Fu Y.-H. Guenette S.Y. Galas D. Nemens E. Wijsman E.M. Bird T.D. Schellenberg G.D. Tanzi R.E. Science. 1995; 269: 973-977Crossref PubMed Scopus (2241) Google Scholar, 18Rogaev E.I. Sherrington R. Rogaeva E.A. Levesque G. Ikeda M. Liang Y. Chi H. Lin C. Holamn K. Tsuda T. Mar L. Sorbi S. Nacmias B. Piacentini S. Amaducci L. Chumakov I. Cohen D. Lannfelt L. Fraser P.E. Rommens J.M. St. George-Hyslop P.H. Nature. 1995; 376: 775-778Crossref PubMed Scopus (1801) Google Scholar). Since mutations within the PS genes are responsible for many FAD cases, the analysis of the cellular biology of these proteins will undoubtedly lead to a better understanding of the molecular mechanisms involved in AD (19Hardy J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2095-2097Crossref PubMed Scopus (203) Google Scholar). Two highly homologous PS proteins (PS-1 and PS-2) are known (Refs.16Sherrington R. Rogaev E.I. Liang Y. Rogaeva E.A. Levesque G. Ikeda M. Chi H. Lin C. Li G. Holman K. Tsuda T. Mar L. Foncin J.-F. Bruni A.C. Montesi M.P. Sorbi S. Rainero I. Pinessi L. Nee L. Chumakov I. Pollen D. Brookes A. Sanseau P. Polinsky R.J. Wasco W. da Silva H.A.R. Haines J.L. Pericak-Vance M.A. Tanzi R.E. Roses A.D. Fraser P.E. Rommens J.M. St. George-Hyslop P.H. Nature. 1995; 375: 754-760Crossref PubMed Scopus (3599) Google Scholar, 17Levy-Lahad E. Wasco W. Poorkaj P. Romano D.M. Oshima J. Pettingell W.H. Yu C. Jondro P.D. Schmidt S.D. Wang K. Crowley A.C. Fu Y.-H. Guenette S.Y. Galas D. Nemens E. Wijsman E.M. Bird T.D. Schellenberg G.D. Tanzi R.E. Science. 1995; 269: 973-977Crossref PubMed Scopus (2241) Google Scholar, 18Rogaev E.I. Sherrington R. Rogaeva E.A. Levesque G. Ikeda M. Liang Y. Chi H. Lin C. Holamn K. Tsuda T. Mar L. Sorbi S. Nacmias B. Piacentini S. Amaducci L. Chumakov I. Cohen D. Lannfelt L. Fraser P.E. Rommens J.M. St. George-Hyslop P.H. Nature. 1995; 376: 775-778Crossref PubMed Scopus (1801) Google Scholar; for review see Refs. 20Tanzi R.E. Kovacs D.M. Kim T.-W. Moir R. Guenette S.Y. Wasco W. Neurobiol. Dis. 1996; 3: 159-168Crossref PubMed Scopus (240) Google Scholar and 21Haass C. Neuron. 1997; 18: 687-690Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). PS-1 and PS-2 are membrane-bound proteins with 7 or 8 transmembrane (TM) domains (22Doan A. Thinakaran G. Borchelt D.R. Slunt H.H. Ratovitsky T. Podlisny M. Selkoe D.J. Seeger M. Gandy S.E. Price D.L. Sisodia S.S. Neuron. 1996; 17: 1023-1030Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). Mutations accumulate within the TM domains but are also frequently found within the hydrophilic domains, specifically the large cytoplasmic loop between TM6 and putative TM7 (22; for review see Refs.19Hardy J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2095-2097Crossref PubMed Scopus (203) Google Scholar, 20Tanzi R.E. Kovacs D.M. Kim T.-W. Moir R. Guenette S.Y. Wasco W. Neurobiol. Dis. 1996; 3: 159-168Crossref PubMed Scopus (240) Google Scholar, 21Haass C. Neuron. 1997; 18: 687-690Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Based on the results from genetic rescue experiments of the mutant PS homologue (the sel-12 gene (23Levitan D. Greenwald I. Nature. 1995; 377: 351-354Crossref PubMed Scopus (631) Google Scholar)) in Caenorhabditis elegans and gene deletions in mice, PS-1 is most likely involved in cell fate decisions via the Notch signaling pathway (2Baumeister R. Leimer U. Zweckbronner I. Jakubek C. Grünberg J. Haass C. Genes & Function. 1997; 1: 149-159Crossref PubMed Scopus (185) Google Scholar,3Levitan D. Doyle T. Brousseau D. Lee M. Thinakaran G. Slunt H. Sisodia S. Greenwald I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14940-14944Crossref PubMed Scopus (344) Google Scholar, 24Shen J. Bronson R.T. Chen D.F. Xia W. Selkoe D.J. Tonegawa S. Cell. 1997; 89: 629-639Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar, 25Wong P.C. Zheng H. Chen H. Becher M.W. Sirinathsinghji D.J.S. Trumbauer M.E. Chen H.Y. Price D.L. Van der Ploeg L.H.T. Sisodia S.S. Nature. 1997; 387: 288-292Crossref PubMed Scopus (647) Google Scholar). In C. elegans, wild type human PS-1 and PS-2 were found to rescue all aspects of the sel-12 mutant phenotype (2Baumeister R. Leimer U. Zweckbronner I. Jakubek C. Grünberg J. Haass C. Genes & Function. 1997; 1: 149-159Crossref PubMed Scopus (185) Google Scholar, 3Levitan D. Doyle T. Brousseau D. Lee M. Thinakaran G. Slunt H. Sisodia S. Greenwald I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14940-14944Crossref PubMed Scopus (344) Google Scholar). The sel-12 gene is known to facilitateNotch signaling (23Levitan D. Greenwald I. Nature. 1995; 377: 351-354Crossref PubMed Scopus (631) Google Scholar); therefore, the results from the rescue experiments strongly suggest that human PS proteins play an important role in the Notch signaling cascade of vertebrates as well. This conclusion was further supported when knock-outs of the PS-1 gene were generated in mice, since the loss of PS-1 expression resulted in a phenotype reminiscent of Notch knock-outs (24Shen J. Bronson R.T. Chen D.F. Xia W. Selkoe D.J. Tonegawa S. Cell. 1997; 89: 629-639Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar, 25Wong P.C. Zheng H. Chen H. Becher M.W. Sirinathsinghji D.J.S. Trumbauer M.E. Chen H.Y. Price D.L. Van der Ploeg L.H.T. Sisodia S.S. Nature. 1997; 387: 288-292Crossref PubMed Scopus (647) Google Scholar). Surprisingly, all FAD-associated point mutations of PS-1 tested so far exhibited a strongly reduced ability to rescue the sel-12mutant phenotype in C. elegans (2Baumeister R. Leimer U. Zweckbronner I. Jakubek C. Grünberg J. Haass C. Genes & Function. 1997; 1: 149-159Crossref PubMed Scopus (185) Google Scholar, 3Levitan D. Doyle T. Brousseau D. Lee M. Thinakaran G. Slunt H. Sisodia S. Greenwald I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14940-14944Crossref PubMed Scopus (344) Google Scholar), indicating that theses mutations might change functionally important amino acids (23Levitan D. Greenwald I. Nature. 1995; 377: 351-354Crossref PubMed Scopus (631) Google Scholar). This notion is supported by the finding that FAD-associated mutations occur at positions that are highly conserved during evolution in all PS genes analyzed so far (for review, see Ref. 21Haass C. Neuron. 1997; 18: 687-690Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). In contrast, expression of PS-1 lacking exon 9 due to a naturally occurring FAD causing splicing mutation (26Perez-Tur J. Froehlich S. Prihar G. Crook R. Baker M. Duff K. Wragg M. Busfield F. Lendon C. Clark R.F. Roques P. Fuldner R.A. Johnston J. Cowburn R. Forsell C. Axelman K. Lilius L. Houlden H. Karran E. Roberts G.W. Rossor M. Adams M.D. Hardy J. Goate A. Lannfelt L. Hutton M. Neuroreport. 1995; 7: 297-301Crossref PubMed Scopus (238) Google Scholar) rescued the sel-12 mutant phenotype surprisingly well (Refs. 2Baumeister R. Leimer U. Zweckbronner I. Jakubek C. Grünberg J. Haass C. Genes & Function. 1997; 1: 149-159Crossref PubMed Scopus (185) Google Scholar and 3Levitan D. Doyle T. Brousseau D. Lee M. Thinakaran G. Slunt H. Sisodia S. Greenwald I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14940-14944Crossref PubMed Scopus (344) Google Scholar; also see below). Interestingly, PS proteins have been found to occur predominantly as stable C-terminal and N-terminal fragments (CTF and NTF; see Fig. 1), whereas only low levels of unclipped PS holoprotein can be detected within all cell lines and tissues analyzed to date (1Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovitsky T. Davenport F. Nordstedt C. Seeger M. Hardy J. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar, 27Mercken M. Takahashi H. Honda T. Sato K. Murayama M. Nakazato Y. Noguchi K. Imahori K. Takashima A. FEBS Lett. 1996; 389: 297-303Crossref PubMed Scopus (120) Google Scholar, 28Podlisny M. Citron M. Amarante P. Sherrington R. Xia W. Zhang J. Diehl T. Levesque G. Fraser P. Haass C. Koo E. Seubert P. St. George-Hyslop P. Teplow D. Selkoe D. Neurobiol. Dis. 1997; 3: 325-337Crossref PubMed Scopus (274) Google Scholar). Surprisingly, mutations within the TM domains of the PS-1 NTF appear to result not only in the hyperaccumulation of the NTF by itself but also in the accumulation of the complementary CTF (4Lee M.K. Borchelt D.R. Kim G. Thinakaran G. Slunt H.H. Ratovitski T. Martin L.J. Kittur A. Gandy S. Levey A.I. Jenkins N. Copeland N. Price D.L. Sisodia S.S. Nat. Med. 1997; 3: 756-760Crossref PubMed Scopus (130) Google Scholar). Because it is highly unlikely that mutations that occur far away from the cleavage site (28Podlisny M. Citron M. Amarante P. Sherrington R. Xia W. Zhang J. Diehl T. Levesque G. Fraser P. Haass C. Koo E. Seubert P. St. George-Hyslop P. Teplow D. Selkoe D. Neurobiol. Dis. 1997; 3: 325-337Crossref PubMed Scopus (274) Google Scholar) of PS-1 directly influence the rate of cleavage, other mechanisms allowing the accumulation of both fragments in a stoichiometrically regulated manner must be considered. In this regard, it is interesting to note that overexpression of PS proteins does not result in a linear increase of fragment formation (1Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovitsky T. Davenport F. Nordstedt C. Seeger M. Hardy J. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar, 28Podlisny M. Citron M. Amarante P. Sherrington R. Xia W. Zhang J. Diehl T. Levesque G. Fraser P. Haass C. Koo E. Seubert P. St. George-Hyslop P. Teplow D. Selkoe D. Neurobiol. Dis. 1997; 3: 325-337Crossref PubMed Scopus (274) Google Scholar). Moreover, expression of the Δexon 9 mutation (26Perez-Tur J. Froehlich S. Prihar G. Crook R. Baker M. Duff K. Wragg M. Busfield F. Lendon C. Clark R.F. Roques P. Fuldner R.A. Johnston J. Cowburn R. Forsell C. Axelman K. Lilius L. Houlden H. Karran E. Roberts G.W. Rossor M. Adams M.D. Hardy J. Goate A. Lannfelt L. Hutton M. Neuroreport. 1995; 7: 297-301Crossref PubMed Scopus (238) Google Scholar), which is known to inhibit conventional proteolytic processing of PS-1, also markedly decreases the formation of the endogenous PS fragments (1Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovitsky T. Davenport F. Nordstedt C. Seeger M. Hardy J. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar, 4Lee M.K. Borchelt D.R. Kim G. Thinakaran G. Slunt H.H. Ratovitski T. Martin L.J. Kittur A. Gandy S. Levey A.I. Jenkins N. Copeland N. Price D.L. Sisodia S.S. Nat. Med. 1997; 3: 756-760Crossref PubMed Scopus (130) Google Scholar, 29Walter J. Grünberg J. Capell A. Pesold B. Schindzielorz A. Citron M. Mendla K. St. George-Hyslop P. Multhaup G. Selkoe D.J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5349-5354Crossref PubMed Scopus (101) Google Scholar). These results indicate a highly regulated mechanism that allows the accumulation of only certain levels of both fragments. Any disturbance in this regulation, such as hyperaccumulation of the PS fragments or of the unclipped PS-1 Δexon 9 protein (4Lee M.K. Borchelt D.R. Kim G. Thinakaran G. Slunt H.H. Ratovitski T. Martin L.J. Kittur A. Gandy S. Levey A.I. Jenkins N. Copeland N. Price D.L. Sisodia S.S. Nat. Med. 1997; 3: 756-760Crossref PubMed Scopus (130) Google Scholar), appears to be associated with early onset FAD, probably due to the enhanced production of Aβ42. However, nothing is known about the nature of the regulation mechanism. We have therefore analyzed whether PS-1 fragments interact with each other. We find that the NTF of PS-1 co-immunoprecipitates with the CTF. Moreover, both fragments form a 100–150-kDa complex in untransfected cells. Binding of PS fragments might therefore explain their concomitant accumulation in transgenic animals expressing mutations within the N-terminal portion of the PS protein. These results might also suggest that the highly regulated fragment formation could be due to the formation of a stoichiometric high molecular weight complex. K293 cells were cultured in Dulbecco's minimal essential medium (Glutamax1; Life Technologies, Inc.) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin (30Haass C. Schlossmacher M. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B. Lieberburg I. Koo E. Schenk D. Teplow D. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1765) Google Scholar). K293 cells stably transfected with wt PS-1 or the Δexon 9 mutation were described previously (13Citron 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. Nat. Med. 1997; 3: 67-72Crossref PubMed Scopus (1169) Google Scholar). Kidney 293 cells were grown to confluence. Cells of 10 10-cm dishes were scraped in phosphate-buffered saline and pelleted. The cell pellet was washed three times in phosphate-buffered saline. Cells were then resuspended in 5 ml of RSB buffer (10 mm Tris, pH 7.5, 20 mm KCl, 1.5 mm MgAc2) containing protease inhibitors as described (30Haass C. Schlossmacher M. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B. Lieberburg I. Koo E. Schenk D. Teplow D. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1765) Google Scholar) and homogenized with 30 strokes in a glass Dounce homogenizer. To prepare a postnuclear supernatant, the homogenate was centrifuged at 1000 × g for 15 min at 4 °C. Membranes from the postnuclear supernatant were then pelleted by centrifugation for 1 h at 100,000 × gat 4 °C. Membranes were washed in a high salt HEPES buffer (1m KCl, 20 mm HEPES, pH 7.2, 2 mmEGTA, 2 mm EDTA, 2 mm DTT) containing protease inhibitors (30Haass C. Schlossmacher M. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B. Lieberburg I. Koo E. Schenk D. Teplow D. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1765) Google Scholar). The purified membranes were extracted with 2% CHAPS in HEPES buffer (100 mm KCl, 20 mm HEPES pH 7.2, 2 mm EGTA, 2 mm EDTA, 2 mmDTT, containing protease inhibitors as described (30Haass C. Schlossmacher M. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B. Lieberburg I. Koo E. Schenk D. Teplow D. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1765) Google Scholar)) for 1 h on ice. Alternatively, proteins were also extracted with 1% Triton X-100 or 0.5% SDS in HEPES buffer. For SDS extraction, K+ was substituted by Na+ in the HEPES buffer. Membrane extracts were cleared by ultracentrifugation for 1 h at 100,000 ×g at 4 °C. Protein concentrations were determined by Bio-Rad assay. Glycerol velocity gradient centrifugation was performed as described by Hay et al.(31Hay J.C. Chao D. Kuo C.S. Scheller R.H. Cell. 1997; 89: 149-158Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Briefly, 1–2 mg of membrane proteins were loaded on a linear 5–25% (v/v) glycerol velocity gradient (31Hay J.C. Chao D. Kuo C.S. Scheller R.H. Cell. 1997; 89: 149-158Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) in gradient buffer (100 mm KCl, 20 mm HEPES, pH 7.2, 2 mmEGTA, 2 mm EDTA, 2 mm DTT, 0.2% CHAPS). Gradients were centrifuged at 40,000 rpm for 16 h at 4 °C in a SW40 rotor (Beckman L-70 ultracentrifuge). After centrifugation, 13 fractions of 1 ml were collected from bottom to top. Proteins were precipitated with an equal volume of 20% trichloroacetic acid, and the precipitated proteins were washed with 90% acetone. Protein pellets were solubilized in sample buffer containing 4 m urea and incubated for 10 min at 65 °C (32Walter J. Capell A. Grünberg J. Pesold B. Schindzielorz A. Prior R. Podlisny M.B. Fraser P. St. George-Hyslop P. Selkoe D.J. Haass C. Mol. Med. 1996; 2: 673-691Crossref PubMed Google Scholar). Proteins were separated on SDS-urea gels (32Walter J. Capell A. Grünberg J. Pesold B. Schindzielorz A. Prior R. Podlisny M.B. Fraser P. St. George-Hyslop P. Selkoe D.J. Haass C. Mol. Med. 1996; 2: 673-691Crossref PubMed Google Scholar) and transferred to polyvinylidene difluoride membranes. The polyclonal antibodies, 2953 and 3027, used in this study were described previously (29Walter J. Grünberg J. Capell A. Pesold B. Schindzielorz A. Citron M. Mendla K. St. George-Hyslop P. Multhaup G. Selkoe D.J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5349-5354Crossref PubMed Scopus (101) Google Scholar, 32Walter J. Capell A. Grünberg J. Pesold B. Schindzielorz A. Prior R. Podlisny M.B. Fraser P. St. George-Hyslop P. Selkoe D.J. Haass C. Mol. Med. 1996; 2: 673-691Crossref PubMed Google Scholar). The monoclonal antibody PS1N to the N terminus of PS-1 is described by Capell et al. (33Capell A. Saffrich R. Olivo J.-C. Meyn L. Walter J. Grünberg J. Dotti C. Haass C. J. Neurochem. 1997; 69: 2432-2440Crossref PubMed Scopus (75) Google Scholar). The monoclonal antibody APS 18 to the large loop of PS-1 was raised to a peptide corresponding to amino acids 314–334 of PS-1. Epitopes of all antibodies used are indicated in Fig. 1. Immunoblotting was carried out as described (32Walter J. Capell A. Grünberg J. Pesold B. Schindzielorz A. Prior R. Podlisny M.B. Fraser P. St. George-Hyslop P. Selkoe D.J. Haass C. Mol. Med. 1996; 2: 673-691Crossref PubMed Google Scholar). Bound antibodies were detected by enhanced chemiluminescence (Amersham Corp.) or the ECL-PLUS system (Amersham Corp.). For co-immunoprecipitation, membranes were prepared as described above and extracted either with 2% CHAPS, 1% Triton X-100, or 0.5% SDS. For SDS extraction, K+ was substituted by Na+ in the HEPES buffer. To remove undissolved membrane fragments, the extracts were pelleted by ultracentrifugation for 1 h at 100,000 × g at 4 °C. Incubation with PS-1 antibodies was performed as described (29Walter J. Grünberg J. Capell A. Pesold B. Schindzielorz A. Citron M. Mendla K. St. George-Hyslop P. Multhaup G. Selkoe D.J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5349-5354Crossref PubMed Scopus (101) Google Scholar, 32Walter J. Capell A. Grünberg J. Pesold B. Schindzielorz A. Prior R. Podlisny M.B. Fraser P. St. George-Hyslop P. Selkoe D.J. Haass C. Mol. Med. 1996; 2: 673-691Crossref PubMed Google Scholar). SDS extracts were diluted 10 × prior to antibody addition; CHAPS and Triton X-100 extracts were immunoprecipitated without further dilution. Immunoprecipitations of CHAPS-extracted proteins were washed 4 × for 20 min in CHAPS washing buffer (0.5% CHAPS, 200 mm NaCl, 50 mm HEPES, pH 7.6). Immunoprecipitations of SDS-extracted proteins were washed as described (30Haass C. Schlossmacher M. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B. Lieberburg I. Koo E. Schenk D. Teplow D. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1765) Google Scholar). Immunoprecipitations of Triton X-100-extracted proteins were washed 4 × for 20 min in STEN buffer only (30Haass C. Schlossmacher M. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B. Lieberburg I. Koo E. Schenk D. Teplow D. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1765) Google Scholar). To determine whether PS-1 fragments occur as a complex, we analyzed membrane protein fractions from human K293 cells. In most experiments, we specifically used untransfected K293 cells to allow the analysis of endogenous PS proteins under in vivo conditions. Moreover, this cell line is highly appropriate for the analysis of the biochemistry of the FAD-associated proteins (βAPP and PS-1/PS-2), since βAPP metabolism and the effects of βAPP and PS mutations originally sorted out in K293 cells (13Citron 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. Nat. Med. 1997; 3: 67-72Crossref PubMed Scopus