A Conserved GXXXG Motif in APH-1 Is Critical for Assembly and Activity of the γ-Secretase Complex
2004; Elsevier BV; Volume: 279; Issue: 6 Linguagem: Inglês
10.1074/jbc.m309745200
ISSN1083-351X
AutoresSheu-Fen Lee, Sanjiv Shah, Cong Yu, W. Christian Wigley, Harry Li, Myungsil Lim, Kia Pedersen, Weiping Han, Philip Thomas, Johan Lundkvist, Yi-Heng Hao, Gang Yu,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoThe multipass membrane protein APH-1, found in the γ-secretase complex together with presenilin, nicastrin, and PEN-2, is essential for Notch signaling in Caenorhabditis elegans embryos and is required for intramembrane proteolysis of Notch and β-amyloid precursor protein in mammalian and Drosophila cells. In C. elegans, a mutation of the conserved transmembrane Gly123 in APH-1 (mutant or28) leads to a notch/glp-1 loss-of-function phenotype. In this study, we show that the corresponding mutation in mammalian APH-1aL (G122D) disrupts the physical interaction of APH-1aL with hypoglycosylated immature nicastrin and the presenilin holoprotein as well as with mature nicastrin, presenilin, and PEN-2. The G122D mutation also reduced γ-secretase activity in intramembrane proteolysis of membrane-tethered Notch. Moreover, we found that the conserved transmembrane Gly122, Gly126, and Gly130 in the fourth transmembrane region of mammalian APH-1aL are part of the membrane helix-helix interaction GXXXG motif and are essential for the stable association of APH-1aL with presenilin, nicastrin, and PEN-2. These findings suggest that APH-1 plays a GXXXG-dependent scaffolding role in both the initial assembly and subsequent maturation and maintenance of the active γ-secretase complex. The multipass membrane protein APH-1, found in the γ-secretase complex together with presenilin, nicastrin, and PEN-2, is essential for Notch signaling in Caenorhabditis elegans embryos and is required for intramembrane proteolysis of Notch and β-amyloid precursor protein in mammalian and Drosophila cells. In C. elegans, a mutation of the conserved transmembrane Gly123 in APH-1 (mutant or28) leads to a notch/glp-1 loss-of-function phenotype. In this study, we show that the corresponding mutation in mammalian APH-1aL (G122D) disrupts the physical interaction of APH-1aL with hypoglycosylated immature nicastrin and the presenilin holoprotein as well as with mature nicastrin, presenilin, and PEN-2. The G122D mutation also reduced γ-secretase activity in intramembrane proteolysis of membrane-tethered Notch. Moreover, we found that the conserved transmembrane Gly122, Gly126, and Gly130 in the fourth transmembrane region of mammalian APH-1aL are part of the membrane helix-helix interaction GXXXG motif and are essential for the stable association of APH-1aL with presenilin, nicastrin, and PEN-2. These findings suggest that APH-1 plays a GXXXG-dependent scaffolding role in both the initial assembly and subsequent maturation and maintenance of the active γ-secretase complex. Regulated intramembrane proteolysis of the Notch receptor, the β-amyloid precursor protein, and select type I membrane polypeptides represents a novel mechanism of signal transduction (1Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1151) Google Scholar). It is now generally believed that the enzyme responsible for the intramembrane proteolysis of these substrates is the high molecular mass, multiprotein γ-secretase complex, which consists of a heterodimer of the presenilin amino- and carboxyl-terminal endoproteolytic fragments (NTF 1The abbreviations used are: NTFamino-terminal fragmentCTFcarboxyl-terminal fragmentTMRtransmembrane regionHAhemagglutininCHAPSO3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acidNi-NTAnickel-nitrilotriacetic acidPIPES1,4-piperazinediethanesulfonic acidWTwild-type. and CTF, respectively) as the putative catalytic subunit (2De Strooper B. Neuron. 2003; 38: 9-12Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar, 3Selkoe D. Kopan R. Annu. Rev. Neurosci. 2003; 26: 565-597Crossref PubMed Scopus (564) Google Scholar). A single transmembrane glycoprotein (nicastrin) has also been identified as a critical component of the γ-secretase complex (4Yu 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). Recent genetic studies in Caenorhabditis elegans have uncovered two additional genes, aph-1 and pen-2, both of which encode multipass membrane proteins that are required for γ-secretase activity and notch/glp-1 signal transduction (5Goutte 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, 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. Ruble C. Nye J.S. Curtis D. Dev. Cell. 2002; 3: 85-97Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar). Subsequent experiments in mammalian cells have indicated that the functionally conserved APH-1 and PEN-2 proteins physically associate with nicastrin and the presenilin NTF/CTF heterodimer and are essential for Notch and β-amyloid precursor protein intramembrane proteolysis (7Lee 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, 8Steiner 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). Overexpression of presenilin, nicastrin, APH-1, and PEN-2 together produces or enhances γ-secretase activity in yeast, insect cells, and mammalian cells, further supporting the hypothesis that these four proteins are essential for γ-secretase activity (9Edbauer D. Winkler E. Regula J.T. Pesold B. Steiner H. Haass C. Nat. Cell Biol. 2003; 5: 486-488Crossref PubMed Scopus (781) Google Scholar, 10Hu Y. Fortini M.E. J. Cell Biol. 2003; 161: 685-690Crossref PubMed Scopus (127) Google Scholar, 11Kimberly 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, 12Takasugi 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, 13Kim S.H. Ikeuchi T. Yu C.J. Sisodia S.S. J. Biol. Chem. 2003; 278: 33992-34002Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Aside from the putative role of presenilin as the catalytic subunit of the γ-secretase complex, the specific functions of nicastrin, APH-1, and PEN-2 remain unclear. Recent studies indicate that PEN-2 may be important in initiating presenilin endoproteolysis, whereas APH-1 may play a role in stabilizing the γ-secretase complex (12Takasugi 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, 13Kim S.H. Ikeuchi T. Yu C.J. Sisodia S.S. J. Biol. Chem. 2003; 278: 33992-34002Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 14Gu 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, 15Luo 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). Recent studies also suggest that APH-1 and nicastrin may interact to form a subcomplex prior to the assembly of the γ-secretase complex (10Hu Y. Fortini M.E. J. Cell Biol. 2003; 161: 685-690Crossref PubMed Scopus (127) Google Scholar, 16LaVoie M.J. Fraering P.C. Ostaszewski B.L. Ye W.J. 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). Despite these advances in research, the mechanism of γ-secretase assembly in the lipid bilayers to form and maintain an active membrane protein complex capable of performing intramembrane proteolysis within a hydrophobic environment remains an enigma. amino-terminal fragment carboxyl-terminal fragment transmembrane region hemagglutinin 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid nickel-nitrilotriacetic acid 1,4-piperazinediethanesulfonic acid wild-type. It has been reported that mutation of Gly123 to aspartic acid within the fourth putative transmembrane region (TMR) of C. elegans APH-1 (mutant or28) inhibits notch/glp-1 signal transduction and leads to the "anterior-pharynx-defective" phenotype associated with the loss of aph-1, aph-2 (nicastrin), or sel-12 plus hop-1(presenilins) (5Goutte 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). This glycine residue in C. elegans APH-1 has been conserved during evolution and corresponds to Gly122 in mammalian APH-1aL and APH-1aS and Gly121 in mammalian APH-1b. APH-1aL and APH-1aS are alternative spliced forms of APH-1a that differ at the carboxyl termini (7Lee 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). Because it is known that the role of the APH-1 proteins in Notch signaling is conserved during evolution and is associated with γ-secretase function, we set out to investigate the biochemical function of the conserved glycine residue in the assembly and activity of the γ-secretase complex. In this study, we used human APH-1aL as an example to demonstrate that the equivalent mutation in human APH-1aL (G122D) affects the ability of APH-1 to associate with the immature as well as the mature γ-secretase complex and inhibits the intramembrane proteolysis of Notch. Moreover, we found that Gly122, Gly126, and Gly130 in APH-1aL belong to a conserved GXXXGXXXG motif (where X represents any amino acid) generally accepted as a major determinant in transmembrane helix-helix protein interactions (17Lemmon M.A. Treutlein H.R. Adams P.D. Brunger A.T. Engelman D.M. Nat. Struct. Biol. 1994; 1: 157-163Crossref PubMed Scopus (298) Google Scholar, 18MacKenzie K.R. Prestegard J.H. Engelman D.M. Science. 1997; 276: 131-133Crossref PubMed Scopus (877) Google Scholar, 19Popot J.L. Engelman D.M. Annu. Rev. Biochem. 2000; 69: 881-922Crossref PubMed Scopus (541) Google Scholar, 20Russ W.P. Engelman D.M. J. Mol. Biol. 2000; 296: 911-919Crossref PubMed Scopus (790) Google Scholar, 21Senes A. Gerstein M. Engelman D.M. J. Mol. Biol. 2000; 296: 921-936Crossref PubMed Scopus (515) Google Scholar, 22Fleming K.G. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14340-14344Crossref PubMed Scopus (159) Google Scholar) and show that these glycine residues are critical for γ-secretase assembly and activity. cDNA Constructs—Human full-length APH-1aL and PEN-2 cDNAs were obtained as described (7Lee 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, 15Luo 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). Subsequent site-directed mutagenesis studies were performed using the QuikChange kit (Stratagene), and the identities of all clones were confirmed by DNA sequence analyses. Cell Lines—Cells were maintained in Dulbecco's modified Eagle's medium and 10% fetal bovine serum (Invitrogen) with 5% CO2 at 37 °C. For transient expression, plasmids were transfected into the appropriate cell lines in 10-cm dishes using LipofectAMINE 2000 (Invitrogen), and samples were collected 48 h after transfection. HEK293 cells stably or transiently transfected with Myc/His-tagged APH-1aL and its glycine mutants were used to evaluate the interaction of APH-1 with presenilin, nicastrin, and PEN-2. Antibodies and Chemicals—The antibodies used in this study include anti-Myc monoclonal antibody 9E10 (American Type Culture Collection); anti-Myc polyclonal antibody A14 (Santa Cruz Biotechnology); anti-FLAG antibody M2 (Sigma); antibodies against presenilin-1-(21–80) (Chemicon International, Inc.), presenilin-1-(263–378) (Chemicon International, Inc.), and presenilin-2-(7–24) (Oncogene); antibody against nicastrin-(693–709) (Sigma); anti-PEN-2 antibody PNT2 (15Luo 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); and antibody against the hemagglutinin (HA) epitope (Santa Cruz Biotechnology). Inhibitors of γ-secretase activity (N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (23Dovey H.F. John V. Anderson J.P. Chen L.Z. de Saint A.P. Fang L.Y. Freedman S.B. Folmer B. Goldbach E. Holsztynska E.J. Hu K.L. Johnson-Wood K.L. Kennedy S.L. Kholodenko D. Knops J.E. Latimer L.H. Lee M. Liao Z. Lieberburg I.M. Motter R.N. Mutter L.C. Nietz J. Quinn K.P. Sacchi K.L. Seubert P.A. Shopp G.M. Thorsett E.D. Tung J.S. Wu J. Yang S. Yin C.T. Schenk D.B. May P.C. Altstiel L.D. Bender M.H. Boggs L.N. Britton T.C. Clemens J.C. Czilli D.L. Dieckman-McGinty D.K. Droste J.J. Fuson K.S. Gitter B.D. Hyslop P.A. Johnstone E.M. Li W.Y. Little S.P. Mabry T.E. Miller F.D. Audia J.E. J. Neurochem. 2001; 76: 173-181Crossref PubMed Scopus (803) Google Scholar) and N-(2-naphthoyl)-Val-phenylalaninal (inhibitor IV) (24Sinha S. Lieberburg I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11049-11053Crossref PubMed Scopus (426) Google Scholar)) were from Calbiochem. Coprecipitation Studies—Co-immunoprecipitations were performed as described previously (4Yu 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, 25Yu G. Chen F. Levesque G. Nishimura M. Zhang D.M. Levesque L. Rogaeva E. Xu D. Liang Y. Duthie M. St George-Hyslop P.H. Fraser P.E. J. Biol. Chem. 1998; 273: 16470-16475Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 26Yu G. Chen F. Nishimura M. Steiner H. Tandon A. Kawarai T. Arawaka S. Supala A. Song Y.Q. Rogaeva E. Holmes E. Zhang D.M. Milman P. Fraser P.E. Haass C. St George-Hyslop P. J. Biol. Chem. 2000; 275: 27348-27353Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, proteins extracted using lysis buffer (1% digitonin or 1% CHAPSO in 50 mm sodium phosphate (pH 8.0), protease inhibitors (Roche Applied Sciences) and 300 mm NaCl) were pre-absorbed with preimmune serum and combined with anti-Myc antibody A14 overnight and then with protein A-agarose beads for 2 h at 4 °C. The beads were washed four times for 15 min each with lysis buffer. Immunoprecipitated proteins were eluted with 0.1 m glycine HCl (pH 2.5) and 0.25% detergent, neutralized with 1.0 m Tris, and subjected to Western blot analysis. Ni-NTA-agarose affinity pulldown experiments were performed as described previously (7Lee 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) in 1% digitonin or 1% CHAPSO lysis buffer. Each binding experiment contained equal amounts of proteins with similar expression of Myc/His-tagged proteins. γ-Secretase Activity Assay—The appropriate cell lines were assayed for γ-cleavage of Notch using the NΔE-GV luciferase reporter as described (7Lee 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, 27Karlstrom H. Bergman A. Lendahl U. Naslund J. Lundkvist J. J. Biol. Chem. 2002; 277: 6763-6766Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) in an adapted in vitro γ-secretase activity assay (28Esler W.P. Kimberly W.T. Ostaszewski B.L. Ye W. Diehl T.S. Selkoe D.J. Wolfe M.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2720-2725Crossref PubMed Scopus (345) Google Scholar). The in vitro activity assay involved lysis of membrane proteins with 1% CHAPSO, 50 mm PIPES (pH 7), 5 mm MgCl2, 5 mm CaCl2, and protease inhibitors (Roche Applied Science) and immunoprecipitation of Myc-tagged proteins with immobilized anti-Myc antibody 9E10. The beads were then washed with CHAPSO lysis buffer and subjected to incubation at 37 °C in the presence of 0.25% CHAPSO, 50 mm PIPES (pH 7), 5 mm MgCl2, 5 mm CaCl2, 0.0125% phosphatidylethanolamine, 0.1% phosphatidylcholine, and N100 substrate. This N100 substrate, which was overexpressed and purified from Sf9 cells, harbors Val1711–Glu1809 of the mouse Notch-1 receptor and a carboxyl-terminal FLAG/His tag. The γ-secretase-cleaved N100-FLAG/His fragment was detected with anti-FLAG antibody. Miscellaneous—All experiments are repeated at least four times, and representative data are presented. Proteins were separated by SDS-PAGE, immunoblotted with the appropriate antibodies, and processed using ECL reagents (Amersham Biosciences). Digitonin-solubilized membranes were fractionated using a 10–30% linear glycerol velocity gradient as described (25Yu G. Chen F. Levesque G. Nishimura M. Zhang D.M. Levesque L. Rogaeva E. Xu D. Liang Y. Duthie M. St George-Hyslop P.H. Fraser P.E. J. Biol. Chem. 1998; 273: 16470-16475Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar), and 1-ml fractions collected from the top of the gradient were subjected to Western blot analysis. Immunofluorescence was analyzed using anti-Myc antibody A14 and Alexa Fluor® 568-conjugated secondary antibody (Molecular Probes, Inc.) on a Leica TCS SP2 laser scanning spectral confocal microscope. Gly122 in APH-1aL Is Critical for γ-Secretase Assembly—To understand the effect of the G122D mutation on the γ-secretase complex, we analyzed the ability of human APH-1aL (either wild-type (WT) or G122D mutant) to interact with the predominant in vivo active presenilin species, the endogenous presenilin endoproteolytic fragments. To assist in this objective, we generated HEK293 cells stably expressing APH-1aLWT-Myc/His or APH-1aLG122D-Myc/His and examined cell lines that expressed a comparable level of either wild-type or mutant Myc/His-tagged proteins. No obvious difference was observed in the level of endogenous presenilin-1 NTF between the APH-1aLWT-Myc/His and APH-1aLG122D-Myc/His cells (Fig. 1A, lanes 1–4). In Ni-NTA-agarose pull-down experiments, we observed that endogenous presenilin-1 NTF was able to coprecipitate with APH-1aLWT-Myc/His, an observation consistent with a previous report (7Lee 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). However, the amount of presenilin-1 NTF that coprecipitated with APH-1aLWT-Myc/His was much higher than the amount that co-isolated with APH-1aLG122D-Myc/His (Fig. 1A, lanes 5–8). Using a co-immunoprecipitation approach, we similarly observed that a minute amount of presenilin-1 NTF or presenilin-2 NTF coprecipitated with APH-1aLG122D-Myc/His compared with the ample amount of presenilin-1 and -2 fragments that coprecipitated with APH-1aLWT-Myc/His (Fig. 1B). Based on these observations, we concluded that the G122D mutation abrogates the association of APH-1aL with the mature presenilin species. Next, we tested whether Gly122 in human APH-1aL is required for interaction with the immature presenilin holoprotein. Given that the endogenous presenilin holoprotein is usually maintained at a low or undetectable steady-state level, we transiently overexpressed full-length presenilin-1 in cell lines stably expressing APH-1aLWT-Myc/His and APH-1aLG122D-Myc/His and performed Ni-NTA-agarose pull-down experiments. We found that significantly less full-length presenilin-1, as well as presenilin-1 endoproteolytic fragments, coprecipitated with APH-1aLG122D-Myc/His compared with APH-1aLWT-Myc/His (Fig. 2, lanes 1–8). These findings suggest that the G122D mutation disrupts the ability of APH-1aL to interact with both the mature presenilin endoproteolytic fragments and the immature presenilin holoprotein.Fig. 2Mutation G122D disrupts the interaction of APH-1aL with the presenilin holoprotein and nicastrin. Full-length presenilin-1 was transiently transfected into HEK293 cells stably overexpressing LacZ-Myc/His (lanes 1 and 5), APH-1aLWT-Myc/His (lanes 2 and 6), or APH-1aLG122D-Myc/His clone 1 (lanes 3 and 7) or clone 2 (lanes 4 and 8), and the cells were solubilized in 1% digitonin. After having determined that the presenilin-1 holoprotein and Myc/His-tagged proteins were expressed at similar levels in each set of cells, we subjected equal amounts of proteins (lanes 1–4) to Ni-NTA pull-down experiments. The resultant Ni-NTA pull-down products (lanes 5–8) were resolved by SDS-PAGE and probed with antibody against amino-terminal presenilin-1 to identify the presenilin-1 holoprotein (PS1-holo) and presenilin-1 NTF (PS1-NTF) and with antibodies against nicastrin (NCT) and the Myc epitope as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our next investigation focused on the ability of immature and mature nicastrin to coprecipitate with APH-1aL. According to published reports, hypoglycosylated immature nicastrin is mainly associated with immature full-length presenilin, whereas fully glycosylated mature nicastrin is associated with presenilin NTF/CTF fragments (29Tomita T. Katayama R. Takikawa R. Iwatsubo T. FEBS Lett. 2002; 520: 117-121Crossref PubMed Scopus (63) Google Scholar, 30Yang D.S. Tandon A. Chen F. Yu G. Yu H. Arawaka S. Hasegawa H. Duthie M. Schmidt S.D. Ramabhadran T.V. Nixon R.A. Mathews P.M. Gandy S.E. Mount H.T. St George-Hyslop P. Fraser P.E. J. Biol. Chem. 2002; 277: 28135-28142Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). In our study, we observed that APH-1aLWT-Myc/His coprecipitated with more of the immature than the mature species of nicastrin in cells overexpressing APH-1aL and the presenilin-1 holoprotein (Fig. 2, lane 6), an observation consistent with earlier studies (10Hu Y. Fortini M.E. J. Cell Biol. 2003; 161: 685-690Crossref PubMed Scopus (127) Google Scholar, 14Gu 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). Moreover, we found that much less immature and mature nicastrin co-isolated with APH-1aLG122D-Myc/His compared with APH-1aLWT-Myc/His (Fig. 2, lanes 5–8). We also observed that cells stably expressing APH-1aLWT-Myc/His contained slightly more immature nicastrin than cells stably expressing APH-1aLG122D-Myc/His (Fig. 2, lanes 1–4), suggesting that the G122D mutation may also affect the reported ability of wild-type APH-1 to stabilize immature nicastrin (10Hu Y. Fortini M.E. J. Cell Biol. 2003; 161: 685-690Crossref PubMed Scopus (127) Google Scholar, 16LaVoie M.J. Fraering P.C. Ostaszewski B.L. Ye W.J. 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). Based on these findings, we conclude that Gly122 mediates the association of APH-1aL with both immature and mature nicastrin either directly or indirectly during the assembly process of the γ-secretase complex. To test the effect of the G122D mutation on the interaction between APH-1aL and PEN-2, we transiently expressed HA-tagged PEN-2 in either the APH-1aLWT-Myc/His or APH-1aLG122D-Myc/His cells. Detergent extracts of these cells were then subjected to Ni-NTA pull-down experiments. We observed that the association between APH-1aL and overexpressed HAPEN-2 was not significantly affected by the G122D mutation, even though the interactions between APH-1aL and both presenilin-1 and nicastrin were disrupted (Fig. 3A). To examine whether the G122D mutation affects the association of endogenous PEN-2 with APH-1aL, we next performed the same Ni-NTA pull-down experiments with detergent-solubilized HEK293 cells stably expressing APH-1aLWT-Myc/His or APH-1aLG122D-Myc/His. In contrast to overexpressed HA-PEN-2, endogenous PEN-2 did not strongly coprecipitate with mutant APH-1aL compared with wild-type APH-1aL (Fig. 3B). The reason that the G122D mutation affected binding of APH-1aL to endogenous (but not overexpressed) PEN-2 is unclear. One possibility is that overexpressed PEN-2 exists in a state parallel to immature nicastrin and the presenilin holoprotein, whereas endogenous PEN-2 exists mostly in a "mature" state similar to the presenilin endoproteolytic fragments and mature nicastrin. In this context, "immature" overexpressed PEN-2 and APH-1aL can directly interact with each other in the absence of both presenilin-1 and nicastrin, perhaps via a site or sites other than Gly122 in APH-1aL. Because the active γ-secretase complex is not stable when any one of the four components is missing (2De Strooper B. Neuron. 2003; 38: 9-12Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar), the failure of mature PEN-2 to interact with G122D mutant APH-1aL might be an indirect consequence of the disruption of the association of APH-1 with presenilin and nicastrin (see "Discussion"). Mutation G122D in APH-1aL Inhibits γ-Secretase Activity— Having established that the G122D mutation in APH-1aL disrupted the structural assembly of the γ-secretase complex, we proceeded to investigate whether this structural defect could lead to deleterious effects on γ-secretase activity. We tested the effect of the G122D mutation in APH-1aL on the production of the Notch intracellular domain (a product of γ-secretase activity) in a cell-based Gal4/VP16-dependent luciferase transactivation assay. This assay, which indirectly measures γ-secretase activity, utilizes a γ-secretase substrate consisting of a chimeric membrane-tethered Notch receptor trimmed in the extracellular domain (NΔE) and Gal4 and VP16 domains (GV) inserted at the junction between the transmembrane and intracellular domains of NΔE (7Lee 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, 27Karlstrom H. Bergman A. Lendahl U. Naslund J. Lundkvist J. J. Biol. Chem. 2002; 277: 6763-6766Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). In HEK293 cells stably expressing robust amounts of APH-1aLG122D-Myc/His, we observed a 50–60% reduction in luciferase reporter activity compared with control cells expressing comparable amounts of APH-1aLWT-Myc/His (Fig. 4A). No analysis could be performed on cells expressing higher levels of APH-1aLG122D-Myc/His because these cells did not grow well (possibly because of toxic effects of the G122D mutation). However, the modest reduction in γ-secretase activity in cells stably expressing APH-1aLG122D-Myc/His obtained in the transactivation assay contrasts with the dramatic effect of the G122D mutation imposed on the structural assembly of the γ-secretase complex and the loss-of-function notch/glp-1 phenotype observed in the C. elegans or28 mutant. A likely explanation for this discrepancy could be that the effect of the G122D mutation on γ-secretase activity is masked by the presence of endogenous APH-1 proteins in the cells. To examine the effect of the G122D mutation on γ-secretase activity in the absence of endogenous APH-1, we immunoprecipitated the γ-secretase complex containing either APH-1aLWT-Myc/His or APH-1aLG122D-Myc/His from the respective CHAPSO-solubilized membranes and measured the ability of the immunoprecipitate to cleave a purified Notch substrate containing the transmembrane S3/γ-secretase-like cleavage site (N100) in an in vitro γ-secretase activity assay. We observed a much more robust N100 cleavage in the immunoprecipitate containing wild-type APH-1aL compared with mutant APH-1aL. The presence of the N100 proteolytic intracellular fragment was also sensitive to two different γ-secretase inhibitors (Fig. 4B). Taken together, these findings show that the G122D mutation in APH-1aL has an inhibitory effect on γ-secretase activity. Our studies suggest that the G122D mutation perturbs the ability of APH-1aL to participate in the initial assembly process of the γ-secretase complex and to associate with and stabilize the active γ-secretase complex to directly modulate the intramembrane proteolysis of Notch. However, we currently cannot rule out the possibility that the G122D mutation affects proper trafficking of APH-1aL within cells and thus the formation of the γ-secretase complex. We attempted to address this issue by assessing the cellular distribution of wild-type and G122D mutant APH-1aL. Immunofluorescence data revealed similar intracellular punctate/vesicular staining patterns for both wild-type and G122D mutant APH-1aL (Fig. 5A), suggesting that G122D mutant APH-1aL does not have a gross trafficking defect that could account for its effect on γ-secretase assembly and activity. We also used linear glycerol density centrifugation to examine the native state of wild-type and G122D mutant APH-1aL and found that the G122D mutation did not significantly alter the native state of APH-1aL (Fig. 5B). Interestingly, G122D mutant APH-1aL, like wild-type APH-1aL, existed in similar high molecular mass glycerol density fractions (Fig. 5B
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