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Requirement of PEN-2 for Stabilization of the Presenilin N-/C-terminal Fragment Heterodimer within the γ-Secretase Complex

2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês

10.1074/jbc.m401789200

1083-351X

Stefan Prokop, Keiro Shirotani, Dieter Edbauer, Christian Haass, Harald Steiner,

Trace Elements in Health

γ-Secretase is a protease complex composed of presenilin (PS), nicastrin (NCT), APH-1, and PEN-2, which catalyzes intramembrane cleavage of several type I transmembrane proteins including the Alzheimer's disease-associated β-amyloid precursor protein. We generated stable RNA interference-mediated PEN-2 knockdown cells to probe mutant PEN-2 variants for functional activity. Knockdown of PEN-2 was associated with impaired NCT maturation and deficient PS1 endoproteolysis, which was efficiently rescued by wild type or N-terminally tagged PEN-2 but not by C-terminally tagged PEN-2 or by the C-terminally truncated PEN-2-ΔC mutant. Although the latter mutants rescued the PS1 holoprotein accumulation associated with the PEN-2 knockdown, they failed to restore normal levels of the PS1 N- and C-terminal fragments and to maturate NCT. PEN-2-ΔC was highly unstable and rapidly turned over by proteasomal degradation consistent with its failure to become stably incorporated into the γ-secretase complex. In addition, expression of PEN-2-ΔC caused a selective instability of the PS1 N-/C-terminal fragment heterodimer that underwent proteasomal degradation, whereas NCT and APH-1 were stable. Interestingly, when we knocked down PEN-2 in the background of the endoproteolysis-deficient PS1 Δexon9 mutant, immature NCT still accumulated, demonstrating that PEN-2 is also required for γ-secretase complex maturation when PS endoproteolysis cannot occur. Taken together, our data suggest that PEN-2 is required for the stabilization of the PS fragment heterodimer within the γ-secretase complex following PS endoproteolysis. This function critically depends on the PEN-2 C terminus. Moreover, our data show that PEN-2 is generally required for γ-secretase complex maturation independent of its activity in PS1 endoproteolysis. γ-Secretase is a protease complex composed of presenilin (PS), nicastrin (NCT), APH-1, and PEN-2, which catalyzes intramembrane cleavage of several type I transmembrane proteins including the Alzheimer's disease-associated β-amyloid precursor protein. We generated stable RNA interference-mediated PEN-2 knockdown cells to probe mutant PEN-2 variants for functional activity. Knockdown of PEN-2 was associated with impaired NCT maturation and deficient PS1 endoproteolysis, which was efficiently rescued by wild type or N-terminally tagged PEN-2 but not by C-terminally tagged PEN-2 or by the C-terminally truncated PEN-2-ΔC mutant. Although the latter mutants rescued the PS1 holoprotein accumulation associated with the PEN-2 knockdown, they failed to restore normal levels of the PS1 N- and C-terminal fragments and to maturate NCT. PEN-2-ΔC was highly unstable and rapidly turned over by proteasomal degradation consistent with its failure to become stably incorporated into the γ-secretase complex. In addition, expression of PEN-2-ΔC caused a selective instability of the PS1 N-/C-terminal fragment heterodimer that underwent proteasomal degradation, whereas NCT and APH-1 were stable. Interestingly, when we knocked down PEN-2 in the background of the endoproteolysis-deficient PS1 Δexon9 mutant, immature NCT still accumulated, demonstrating that PEN-2 is also required for γ-secretase complex maturation when PS endoproteolysis cannot occur. Taken together, our data suggest that PEN-2 is required for the stabilization of the PS fragment heterodimer within the γ-secretase complex following PS endoproteolysis. This function critically depends on the PEN-2 C terminus. Moreover, our data show that PEN-2 is generally required for γ-secretase complex maturation independent of its activity in PS1 endoproteolysis. The Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; APH, anterior pharynx defective; APP, β-amyloid precursor protein; NCT, nicastrin; PEN, presenilin enhancer; PS, presenilin; NTF, N-terminal fragment; CTF, C-terminal fragment; wt, wild type; swAPP, Swedish mutant APP; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; RNAi, RNA interference; HEK 293, human embryonic kidney 293.-associated γ-secretase is a high molecular weight complex with an aspartyl protease activity that catalyzes the second of two subsequent proteolytic cleavages of the β-amyloid precursor protein (APP) implicated in AD (1Haass C. Steiner H. Trends Cell Biol. 2002; 12: 556-562Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). This unusual, intramembranous cleavage liberates the neurotoxic amyloid-β peptide from the membrane. Amyloid-β peptide aggregates and is deposited in the brain of AD patients in amyloid plaques, an invariant pathological hallmark of AD (2Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5196) Google Scholar). The γ-secretase complex is composed of either of the two presenilins (PS), PS1 and PS2, polytopic membrane proteins that are endoproteolytically cleaved into stable heterodimers consisting of an N- and C-terminal fragment (NTF and CTF) (3Thinakaran 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), the type I transmembrane glycoprotein nicastrin (NCT) (4Goutte C. Hepler W. Mickey K.M. Priess J.R. Development. 2000; 127: 2481-2492Crossref PubMed Google Scholar, 5Yu G. Nishimura M. Arawaka S. Levitan D. Zhang L. Tandon A. Song Y.Q. Rogaeva E. Chen F. Kawarai T. Supala A. Levesque L. Yu H. Yang D.S. Holmes E. Milman P. Liang Y. Zhang D.M. Xu D.H. Sato C. Rogaev E. Smith M. Janus C. Zhang Y. Aebersold R. Farrer L.S. Sorbi S. Bruni A. Fraser P. St George-Hyslop P. Nature. 2000; 407: 48-54Crossref PubMed Scopus (827) Google Scholar), and the polytopic membrane proteins PEN-2 (6Francis R. McGrath G. Zhang J. Ruddy D.A. Sym M. Apfeld J. Nicoll M. Maxwell M. Hai B. Ellis M.C. Parks A.L. Xu W. Li J. Gurney M. Myers R.L. Himes C.S. Hiebsch R.D. Ruble C. Nye J.S. Curtis D. Dev. Cell. 2002; 3: 85-97Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar), APH-1a, and/or APH-1b (6Francis R. McGrath G. Zhang J. Ruddy D.A. Sym M. Apfeld J. Nicoll M. Maxwell M. Hai B. Ellis M.C. Parks A.L. Xu W. Li J. Gurney M. Myers R.L. Himes C.S. Hiebsch R.D. Ruble C. Nye J.S. Curtis D. Dev. Cell. 2002; 3: 85-97Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar, 7Goutte C. Tsunozaki M. Hale V.A. Priess J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 775-779Crossref PubMed Scopus (371) Google Scholar). Expression of the γ-secretase complex components is coordinately regulated. RNA interference (RNAi)-mediated knockdown studies in cultured Drosophila and mammalian cells as well as studies with cells derived from PS1, PS1/2, or NCT knockout mice have shown that γ-secretase complex formation is coordinately regulated. Down-regulation or genetic ablation of a given component typically causes a decrease in the level of the other components and/or in the maturation and trafficking of the γ-secretase complex to its functional sites (6Francis R. McGrath G. Zhang J. Ruddy D.A. Sym M. Apfeld J. Nicoll M. Maxwell M. Hai B. Ellis M.C. Parks A.L. Xu W. Li J. Gurney M. Myers R.L. Himes C.S. Hiebsch R.D. Ruble C. Nye J.S. Curtis D. Dev. Cell. 2002; 3: 85-97Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar, 8Hu Y. Ye Y. Fortini M.E. Dev. Cell. 2002; 2: 69-78Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 9Edbauer D. Winkler E. Haass C. Steiner H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8666-8671Crossref PubMed Scopus (218) Google Scholar, 10Leem J.Y. Vijayan S. Han P. Cai D. Machura M. Lopes K.O. Veselits M.L. Xu H. Thinakaran G. J. Biol. Chem. 2002; 277: 19236-19240Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 11Steiner H. Winkler E. Edbauer D. Prokop S. Basset G. Yamasaki A. Kostka M. Haass C. J. Biol. Chem. 2002; 277: 39062-39065Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 12Lee S.F. Shah S. Li H. Yu C. Han W. Yu G. J. Biol. Chem. 2002; 277: 45013-45019Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 13Gu Y. Chen F. Sanjo N. Kawarai T. Hasegawa H. Duthie M. Li W. Ruan X. Luthra A. Mount H.T. Tandon A. Fraser P.E. St. George-Hyslop P. J. Biol. Chem. 2003; 278: 7374-7380Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 14Luo W.J. Wang H. Li H. Kim B.S. Shah S. Lee H.J. Thinakaran G. Kim T.W. Yu G. Xu H. J. Biol. Chem. 2003; 278: 7850-7854Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 15Kimberly W.T. LaVoie M.J. Ostaszewski B.L. Ye W. Wolfe M.S. Selkoe D.J. J. Biol. Chem. 2002; 277: 35113-35117Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 16Herreman A. Van Gassen G. Bentahir M. Nyabi O. Craessaerts K. Mueller U. Annaert W. De Strooper B. J. Cell Sci. 2003; 116: 1127-1136Crossref PubMed Scopus (166) Google Scholar, 17Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar, 18Li T. Ma G. Cai H. Price D.L. Wong P.C. J. Neurosci. 2003; 23: 3272-3277Crossref PubMed Google Scholar). An exception is APH-1, which is stable in the absence of PS (13Gu Y. Chen F. Sanjo N. Kawarai T. Hasegawa H. Duthie M. Li W. Ruan X. Luthra A. Mount H.T. Tandon A. Fraser P.E. St. George-Hyslop P. J. Biol. Chem. 2003; 278: 7374-7380Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 19Nyabi O. Bentahir M. Horre K. Herreman A. Gottardi-Littell N. Van Broeckhoven C. Merchiers P. Spittaels K. Annaert W. De Strooper B. J. Biol. Chem. 2003; 278: 43430-43436Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 20Shirotani K. Edbauer D. Kostka M. Steiner H. Haass C. J. Neurochem. 2004; (in press)PubMed Google Scholar). Increased γ-secretase complex formation and activity have been observed when all four γ-secretase complex components are overexpressed in cultured Drosophila and mammalian cells (17Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar, 21Kimberly W.T. LaVoie M.J. Ostaszewski B.L. Ye W. Wolfe M.S. Selkoe D.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6382-6387Crossref PubMed Scopus (684) Google Scholar, 22Kim S.H. Ikeuchi T. Yu C. Sisodia S.S. J. Biol. Chem. 2003; 278: 33992-34002Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Moreover, reconstitution of γ-secretase complex formation and activity has recently been achieved in yeast, an organism that lacks any endogenous γ-secretase activity (23Edbauer D. Winkler E. Regula J.T. Pesold B. Steiner H. Haass C. Nat. Cell Biol. 2003; 5: 486-488Crossref PubMed Scopus (781) Google Scholar). This demonstrated that coexpression of all four components is necessary and sufficient for the reconstitution of γ-secretase activity (23Edbauer D. Winkler E. Regula J.T. Pesold B. Steiner H. Haass C. Nat. Cell Biol. 2003; 5: 486-488Crossref PubMed Scopus (781) Google Scholar). Overexpression and RNAi-mediated knockdown studies with cultured Drosophila cells have provided a first model of how the γ-secretase complex assembles (17Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar). These studies indicate that the PS holoprotein may assemble first with NCT and/or APH-1 to form a stable assembly intermediate (17Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar). Subsequent assembly of PEN-2 with this intermediate drives the conversion of the PS holoprotein into the active heterodimer (17Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar). Furthermore, a potential NCT/APH-1 assembly intermediate has been observed recently (24LaVoie M.J. Fraering P.C. Ostaszewski B.L. Ye W. Kimberly W.T. Wolfe M.S. Selkoe D.J. J. Biol. Chem. 2003; 278: 37213-37222Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), and NCT has been shown to stably interact with APH-1 in the absence of PS (20Shirotani K. Edbauer D. Kostka M. Steiner H. Haass C. J. Neurochem. 2004; (in press)PubMed Google Scholar, 25Morais V.A. Crystal A.S. Pijak D.S. Carlin D. Costa J. Lee V.M. Doms R.W. J. Biol. Chem. 2003; 278: 43284-43291Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Interestingly, γ-secretase complex assembly is associated with a conformational change of the NCT ectodomain, which adopts a protease-resistant conformation (26Shirotani K. Edbauer D. Capell A. Schmitz J. Steiner H. Haass C. J. Biol. Chem. 2003; 278: 16474-16477Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). At present, little is known about the subunit organization within the γ-secretase complex. However, recent studies (27Cervantes S. Gonzalez-Duarte R. Marfany G. FEBS Lett. 2001; 505: 81-86Crossref PubMed Scopus (39) Google Scholar, 28Schroeter E.H. Ilagan M.X. Brunkan A.L. Hecimovic S. Li Y.M. Xu M. Lewis H.D. Saxena M.T. De Strooper B. Coonrod A. Tomita T. Iwatsubo T. Moore C.L. Goate A. Wolfe M.S. Shearman M. Kopan R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13075-13080Crossref PubMed Scopus (181) Google Scholar) suggest that PS is a dimer within the γ-secretase complex. In addition, an interaction of the PS1 NTF with PEN-2 has been reported (29Fraering P.C. LaVoie M.J. Ye W. Ostaszewski B.L. Kimberly W.T. Selkoe D.J. Wolfe M.S. Biochemistry. 2004; 43: 323-333Crossref PubMed Scopus (119) Google Scholar). A large body of evidence suggests that PS carries the catalytic site of the γ-secretase complex. Mutagenesis of conserved aspartates in transmembrane domains 6 and 7 of PS (30Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar, 31Steiner 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 (273) Google Scholar) as well as aspartyl protease transition state analogues, which bind to the PS heterodimer (32Li Y.M. Xu M. Lai M.T. Huang Q. Castro J.L. DiMuzio-Mower J. Harrison T. Lellis C. Nadin A. Neduvelli J.G. Register R.B. Sardana M.K. Shearman M.S. Smith A.L. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (867) Google Scholar, 33Esler W.P. Kimberly W.T. Ostaszewski B.L. Diehl T.S. Moore C.L. Tsai J.-Y. Rahmati T. Xia W. Selkoe D.J. Wolfe M.S. Nat. Cell Biol. 2000; 2: 428-433Crossref PubMed Scopus (508) Google Scholar), inhibit γ-secretase activity. Moreover, PS belongs to a novel group of several polytopic aspartyl protease families (1Haass C. Steiner H. Trends Cell Biol. 2002; 12: 556-562Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 34Weihofen A. Martoglio B. Trends Cell Biol. 2003; 13: 71-78Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), which are defined by the presence of highly conserved, short GXGD active site motifs (35LaPointe C.F. Taylor R.K. J. Biol. Chem. 2000; 275: 1502-1510Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 36Steiner H. Kostka M. Romig H. Basset G. Pesold B. Hardy J. Capell A. Meyn L. Grim M.G. Baumeister R. Fechteler K. Haass C. Nat. Cell Biol. 2000; 2: 848-851Crossref PubMed Scopus (255) Google Scholar, 37Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar). Besides their requirement for γ-secretase complex assembly and maturation, the precise functional role of the other γ-secretase complex components is unclear. In particular, apart from its role in facilitation of PS endoproteolysis (14Luo W.J. Wang H. Li H. Kim B.S. Shah S. Lee H.J. Thinakaran G. Kim T.W. Yu G. Xu H. J. Biol. Chem. 2003; 278: 7850-7854Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 17Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar), functional information about the smallest subunit, the ∼10-kDa PEN-2 protein, is scarce. In order to gain further insight into its functional role, we thus initiated a structure-function analysis of this protein. Antibodies—The polyclonal antibody 1638 raised to the N terminus of human PEN-2 (11Steiner H. Winkler E. Edbauer D. Prokop S. Basset G. Yamasaki A. Kostka M. Haass C. J. Biol. Chem. 2002; 277: 39062-39065Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), the polyclonal and monoclonal antibodies against the PS1 C terminus (3027 and BI.3D7) (38Steiner H. Romig H. Grim M.G. Philipp U. Pesold B. Citron M. Baumeister R. Haass C. J. Biol. Chem. 1999; 274: 7615-7618Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), the N terminus (2953) (39Walter J. Grunberg J. Capell A. Pesold B. Schindzielorz A. Citron M. Mendla K. George-Hyslop P.S. Multhaup G. Selkoe D.J. Haass C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5349-5354Crossref PubMed Scopus (101) Google Scholar), and PS1N (40Capell A. Saffrich R. Olivo J.C. Meyn L. Walter J. Grunberg J. Mathews P. Nixon R. Dotti C. Haass C. J. Neurochem. 1997; 69: 2432-2440Crossref PubMed Scopus (75) Google Scholar), and the polyclonal antibody to the C terminus of APP (6687) (36Steiner H. Kostka M. Romig H. Basset G. Pesold B. Hardy J. Capell A. Meyn L. Grim M.G. Baumeister R. Fechteler K. Haass C. Nat. Cell Biol. 2000; 2: 848-851Crossref PubMed Scopus (255) Google Scholar) were described previously. The polyclonal antibodies 433 and 434 were generated against the APH-1aL C terminus (residues 245-265) and affinity-purified using a GST-APH-1aL (residues 207-265) fusion protein. The polyclonal antibody N1660 against the C terminus of NCT and the anti-β-actin antibody were obtained from Sigma, the anti-Xpress antibody was obtained from Invitrogen; the anti-β-catenin antibody was from Transduction Laboratories, and the anti-Myc antibody 9E10 was from Santa Cruz Biotechnology. cDNA Constructs—To stably knock down endogenous PEN-2 expression by RNAi, short hairpin RNA oligonucleotides (PEN-2-163) corresponding to the PEN-2-160 target region (11Steiner H. Winkler E. Edbauer D. Prokop S. Basset G. Yamasaki A. Kostka M. Haass C. J. Biol. Chem. 2002; 277: 39062-39065Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar) were cloned into the pSUPER vector (41Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3971) Google Scholar). Silencer mutations that do not alter the encoded amino acid sequence (5′-AAGGGATACGTGTGGCGATCTGC-3, the mutations are underlined) were introduced in all PEN-2 constructs to escape RNAi. PCR-mediated mutagenesis was used to generate mutant PEN-2 variants. Wild type and mutant PEN-2 variants were cloned into the pcDNA3.1/Zeo(+) vector (Invitrogen). N-terminal hexahistidine-Xpress (H6X) epitope-tagged and C-terminal myc-hexahistidine (mH6) epitope-tagged PEN-2 wild type or mutant PEN-2 variants were generated by cloning the respective cDNAs into the pcDNA4/HisC (Invitrogen) or pcDNA4/myc-HisA (Invitrogen) expression vectors. All constructs were verified by DNA sequencing. Cell Culture and Cell Lines—Stably transfected human embryonic kidney 293 (HEK 293) cells were cultured as described (9Edbauer D. Winkler E. Haass C. Steiner H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8666-8671Crossref PubMed Scopus (218) Google Scholar). A stable PEN-2 knockdown cell line was generated by cotransfection of HEK 293 cells stably expressing Swedish mutant APP (swAPP) (42Citron M. Oltersdorf T. Haass C. McConlogue L. Hung A.Y. Seubert P. Vigo-Pelfrey C. Lieberburg I. Selkoe D.J. Nature. 1992; 360: 672-674Crossref PubMed Scopus (1537) Google Scholar) with pSUPER/PEN-2-163 and pcDNA3.1/Hygro(-) (Invitrogen) and selection for hygromycin (150 μg/ml) resistance. The PEN-2 knockdown cell line was subsequently stably transfected with the indicated wt and mutant PEN-2 constructs. Likewise, PEN-2 was stably knocked down in HEK 293 cells stably coexpressing swAPP and PS1 Δexon9 (38Steiner H. Romig H. Grim M.G. Philipp U. Pesold B. Citron M. Baumeister R. Haass C. J. Biol. Chem. 1999; 274: 7615-7618Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Protein Analysis—Membrane fractions of HEK 293 cells were obtained by ultracentrifugation of postnuclear supernatant fractions from cell homogenates that were prepared as described (43Sastre M. Steiner H. Fuchs K. Capell A. Multhaup G. Condron M.M. Teplow D.B. Haass C. EMBO Rep. 2001; 2: 835-841Crossref PubMed Scopus (431) Google Scholar). For direct immunoblot analysis, membrane fractions were solubilized with STEN-lysis buffer (50 mm Tris, pH 7.6, 150 mm NaCl, 2 mm EDTA, 1% Nonidet P-40, protease inhibitors (Sigma)). After a clarifying spin, lysates were analyzed for PEN-2 by immunoblotting with antibody 1638, for PS1 by immunoblotting with antibodies PS1N or 3027, for NCT with antibody N1660, for APH-1aL by immunoblotting with antibody 433 or 434, and for APP and APP CTFs by immunoblotting with antibody 6687. For coimmunoprecipitation analysis, membrane fractions were solubilized in CHAPSO-lysis buffer (1% CHAPSO, 150 mm sodium citrate, pH 6.4, protease inhibitors (Sigma)), subjected to a clarifying spin by ultracentrifugation, and incubated with preimmune serum, N1660 or 2953 antibody, and protein G-Sepharose for 2 h at 4 °C. Following two washes with CHAPSO-wash buffer (0.5% CHAPSO, 150 mm sodium citrate, pH 6.4), the immunoprecipitates were subjected to immunoblot analysis as above. In order to identify functional domains of PEN-2 required for γ-secretase complex formation, maturation, and activity, we generated a HEK 293 cell line stably overexpressing Swedish mutant APP (HEK 293/sw) in which PEN-2 expression is stably knocked down by RNAi (Fig. 1A). This PEN-2 knockdown cell line was stably transfected with cDNA constructs encoding RNAi-resistant wt and mutant PEN-2 variants to assess their capability to rescue PEN-2 deficiency. We first transfected the PEN-2 knockdown cells with cDNA constructs encoding untagged wt PEN-2 and N-terminal hexahistidine-Xpress (H6X) epitope-tagged and C-terminal myc-hexahistidine (mH6) epitope-tagged wt PEN-2. All three proteins were stably expressed at robust levels, and as expected, the tagged PEN-2 variants migrated at higher molecular weight than untagged PEN-2 (Fig. 1A). Consistent with previous results (6Francis R. McGrath G. Zhang J. Ruddy D.A. Sym M. Apfeld J. Nicoll M. Maxwell M. Hai B. Ellis M.C. Parks A.L. Xu W. Li J. Gurney M. Myers R.L. Himes C.S. Hiebsch R.D. Ruble C. Nye J.S. Curtis D. Dev. Cell. 2002; 3: 85-97Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar, 17Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar, 24LaVoie M.J. Fraering P.C. Ostaszewski B.L. Ye W. Kimberly W.T. Wolfe M.S. Selkoe D.J. J. Biol. Chem. 2003; 278: 37213-37222Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), the PEN-2 knockdown was associated with reduced γ-secretase activity as manifested by the accumulation of APP CTFs, which was rescued by the expression of wt PEN-2 and H6X-PEN-2 (Fig. 1B). APP CTFs also accumulated when PEN-2-mH6 was expressed indicating that the authentic C terminus of PEN-2 is critical for γ-secretase activity. We next sought to investigate why expression of PEN-2-mH6 caused a defect in γ-secretase activity, and we asked whether a defect in γ-secretase complex maturation was responsible for the observed loss of γ-secretase function. To confirm further the functional importance of the PEN-2 C terminus, we also constructed a PEN-2 variant with a C-terminal truncation of 17 amino acids (PEN-2-ΔC), and we expressed it stably in the PEN-2 knockdown cells (Fig. 1C). We first investigated the maturation of PS. Consistent with previous results (11Steiner H. Winkler E. Edbauer D. Prokop S. Basset G. Yamasaki A. Kostka M. Haass C. J. Biol. Chem. 2002; 277: 39062-39065Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 14Luo W.J. Wang H. Li H. Kim B.S. Shah S. Lee H.J. Thinakaran G. Kim T.W. Yu G. Xu H. J. Biol. Chem. 2003; 278: 7850-7854Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 24LaVoie M.J. Fraering P.C. Ostaszewski B.L. Ye W. Kimberly W.T. Wolfe M.S. Selkoe D.J. J. Biol. Chem. 2003; 278: 37213-37222Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), down-regulation of PEN-2 was associated with reduced levels of PS fragments and an accumulation of the PS1 holoprotein (Fig. 1D). Expression of untagged PEN-2 allowed efficient recovery of PS1 holoprotein endoproteolysis (Fig. 1D). In contrast, expression of PEN-2-mH6 and of PEN-2-ΔC caused an intriguing, unexpected biochemical phenotype. Although the PS1 holoprotein did not accumulate anymore, the levels of the PS1 NTF and CTF were strongly reduced similar to those observed in the PEN-2 knockdown cells (Fig. 1D). Thus, although the PEN-2 C terminus appears to be dispensable for PS1 endoproteolysis, it is required for the maintenance of normal levels of the PS fragments. As reported previously (11Steiner H. Winkler E. Edbauer D. Prokop S. Basset G. Yamasaki A. Kostka M. Haass C. J. Biol. Chem. 2002; 277: 39062-39065Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 24LaVoie M.J. Fraering P.C. Ostaszewski B.L. Ye W. Kimberly W.T. Wolfe M.S. Selkoe D.J. J. Biol. Chem. 2003; 278: 37213-37222Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), the PEN-2 knockdown caused impaired NCT maturation, which was efficiently restored by the expression of untagged PEN-2 (Fig. 1E). Interestingly, the levels of APH-1aL were largely unaffected by the knockdown of PEN-2 (Fig. 1E). Neither PEN-2-mH6 nor PEN-2-ΔC rescued the maturation defect of NCT (Fig. 1D). Consequently, as observed for PEN-2-mH6 (Fig. 1B), expression of PEN-2-ΔC was associated with impaired γ-secretase activity as judged from the accumulation of APP CTFs (Fig. 1F). Thus, either addition of an epitope tag to the C terminus or a large deletion of it abrogated PEN-2 function in γ-secretase complex formation, stability, maturation, and activity. Taken together, these data suggest that the C terminus is a functionally important domain of PEN-2 required for the stabilization of PS fragments. We next asked why the C-terminal PEN-2 variants failed to stabilize PS1 fragments. To address this question we sought to investigate the stability of PEN-2-ΔC and treated cells with cycloheximide to block de novo protein synthesis. We first investigated the stability of the γ-secretase complex components in control and PEN-2 knockdown cells. As expected, endogenous PEN-2, PS1 fragments, NCT, and APH-1aL were stable in control cells (Fig. 2A). Similarly, residual endogenous PEN-2 in the knockdown cells was stable as were the PS1 holoprotein, NCT, and APH-1aL in these cells (Fig. 2A), consistent with previous results (17Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar). We then analyzed the stability of PEN-2 or PEN-2-ΔC expressed in the background of PEN-2 knockdown cells. wt PEN-2 occurred as a stable protein (Fig. 2B). In contrast, cycloheximide treatment revealed that PEN-2-ΔC was highly unstable and almost completely turned over within 3 h (Fig. 2B). We next investigated the stability of PS1, NCT, and APH-1aL in the background of wt PEN-2 and PEN-2-ΔC expression. The PS1 fragments, NCT and APH-1aL, were stable in the PEN-2 knockdown cells stably expressing wt PEN-2. In contrast, in the PEN-2-ΔC-expressing knockdown cells, the already low levels of PS1 fragments dropped further during the time course of the cycloheximide treatment. This indicates that the PS1 fragments were not stabilized and thus turned over (Fig. 2B). Interestingly, immature NCT and APH-1aL as well as the low levels of residual mature NCT (because of the incomplete PEN-2 knockdown) remained stable (Fig. 2B), consistent with the observation that these two proteins can independently stabilize each other (13Gu Y. Chen F. Sanjo N. Kawarai T. Hasegawa H. Duthie M. Li W. Ruan X. Luthra A. Mount H.T. Tandon A. Fraser P.E. St. George-Hyslop P. J. Biol. Chem. 2003; 278: 7374-7380Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 20Shirotani K. Edbauer D. Kostka M. Steiner H. Haass C. J. Neurochem. 2004; (in press)PubMed Google Scholar, 24LaVoie M.J. Fraering P.C. Ostaszewski B.L. Ye W. Kimberly W.T. Wolfe M.S. Selkoe D.J. J. Biol. Chem. 2003; 278: 37213-37222Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 25Morais V.A. Crystal A.S. Pijak D.S. Carlin D. Costa J. Lee V.M. Doms R.W. J. Biol. Chem. 2003; 278: 43284-43291Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Taken together, these data demonstrate that PEN-2-ΔC is highly unstable. Furthermore, these data indicate that expression of PEN-2-ΔC might cause a selective destabilization of the PS1 NTF/CTF heterodimer within the γ-secretase complex. We next asked which proteolytic pathway might be involved in the degradation of PEN-2-ΔC and the destabilized PS1 fragments. Because we had shown previous