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• W2079184536 abstract "Understanding the intracellular transport of the β-amyloid precursor protein (APP) is a major key to elucidate the regulation of APP processing and thus β-amyloid peptide generation in Alzheimer disease pathogenesis. APP and its two paralogues, APLP1 and APLP2 (APLPs), are processed in a very similar manner by the same protease activities. A putative candidate involved in APP transport is protein interacting with APP tail 1 (PAT1), which was reported to interact with the APP intracellular domain. We show that PAT1a, which is 99.0% identical to PAT1, binds to APP, APLP1, and APLP2 in vivo and describe their co-localization in trans-Golgi network vesicles or endosomes in primary neurons. We further demonstrate a direct interaction of PAT1a with the basolateral sorting signal of APP/APLPs. Moreover, we provide evidence for a direct role of PAT1a in APP/APLP transport as overexpression or RNA interference-mediated knockdown of PAT1a modulates APP/APLPs levels at the cell surface. Finally, we show that PAT1a promotes APP/APLPs processing, resulting in increased secretion ofβ-amyloid peptide. Taken together, our data establish PAT1a as a functional link between APP/APLPs transport and their processing. Understanding the intracellular transport of the β-amyloid precursor protein (APP) is a major key to elucidate the regulation of APP processing and thus β-amyloid peptide generation in Alzheimer disease pathogenesis. APP and its two paralogues, APLP1 and APLP2 (APLPs), are processed in a very similar manner by the same protease activities. A putative candidate involved in APP transport is protein interacting with APP tail 1 (PAT1), which was reported to interact with the APP intracellular domain. We show that PAT1a, which is 99.0% identical to PAT1, binds to APP, APLP1, and APLP2 in vivo and describe their co-localization in trans-Golgi network vesicles or endosomes in primary neurons. We further demonstrate a direct interaction of PAT1a with the basolateral sorting signal of APP/APLPs. Moreover, we provide evidence for a direct role of PAT1a in APP/APLP transport as overexpression or RNA interference-mediated knockdown of PAT1a modulates APP/APLPs levels at the cell surface. Finally, we show that PAT1a promotes APP/APLPs processing, resulting in increased secretion ofβ-amyloid peptide. Taken together, our data establish PAT1a as a functional link between APP/APLPs transport and their processing. Amyloid plaques, the major hallmark of Alzheimer disease, are mainly composed of the β-amyloid peptide (Aβ), 6The abbreviations used are: Aβ, β-amyloid peptide; APP, β-amyloid precursor protein; sAPP, secreted APP; APLP, β-amyloid precursor-like protein; BaSS, basolateral sorting sequence; RNAi, RNA interference; BACE, β-site APP cleaving enzyme; CTF, C-terminal fragment; ICD, intracellular domain; AICD, APP intracellular domain; KLC, kinesin light chain; IB, immunoblotting; IC, immunocapture; HA, hemagglutinin; GST, glutathione S-transferase; ELISA, enzyme-linked immunosorbent assay; SNP, single nucleotide polymorphism; aa, amino acids; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; sh, short hairpin; scr, scrambled. which is proteolytically derived from the β-amyloid precursor protein (APP) (1Kang J. Lemaire H.G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3957) Google Scholar). APP belongs to a protein family with two mammalian paralogues, the β-amyloid precursor-like proteins (APLP) 1 and 2 (2Wasco W. Bupp K. Magendantz M. Gusella J.F. Tanzi R.E. Solomon F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10758-10762Crossref PubMed Scopus (322) Google Scholar, 3Paliga K. Peraus G. Kreger S. Durrwang U. Hesse L. Multhaup G. Masters C.L. Beyreuther K. Weidemann A. Eur. J. Biochem. 1997; 250: 354-363Crossref PubMed Scopus (49) Google Scholar, 4Wasco W. Gurubhagavatula S. Paradis M.D. Romano D.M. Sisodia S.S. Hyman B.T. Neve R.L. Tanzi R.E. Nat. Genet. 1993; 5: 95-100Crossref PubMed Scopus (322) Google Scholar, 5Sprecher C.A. Grant F.J. Grimm G. O'Hara P.J. Norris F. Norris K. Foster D.C. Biochemistry. 1993; 32: 4481-4486Crossref PubMed Scopus (162) Google Scholar). APP/APLPs share highly conserved protein domain organization (6Coulson E.J. Paliga K. Beyreuther K. Masters C.L. Neurochem. Int. 2000; 36: 175-184Crossref PubMed Scopus (161) Google Scholar), form homo- and heterotypic interactions (7Soba P. Eggert S. Wagner K. Zentgraf H. Siehl K. Kreger S. Lower A. Langer A. Merdes G. Paro R. Masters C.L. Muller U. Kins S. Beyreuther K. EMBO J. 2005; 24: 3624-3634Crossref PubMed Scopus (235) Google Scholar), and are proteolytically processed in a similar manner (8Eggert S. Paliga K. Soba P. Evin G. Masters C.L. Weidemann A. Beyreuther K. J. Biol. Chem. 2004; 279: 18146-18156Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The extracellular domain of APP/APLPs can be cleaved by α-secretases or, alternatively, by the β-secretase β-site APP cleaving enzyme 1 (BACE 1) (8Eggert S. Paliga K. Soba P. Evin G. Masters C.L. Weidemann A. Beyreuther K. J. Biol. Chem. 2004; 279: 18146-18156Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 9Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3922-3927Crossref PubMed Scopus (986) Google Scholar, 10Buxbaum J.D. Liu K.N. Luo Y. Slack J.L. Stocking K.L. Peschon J.J. Johnson R.S. Castner B.J. Cerretti D.P. Black R.A. J. Biol. Chem. 1998; 273: 27765-27767Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar, 11Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Crossref PubMed Scopus (3327) Google Scholar, 12Li Q. Sudhof T.C. J. Biol. Chem. 2004; 279: 10542-10550Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). The resulting membrane-retained C-terminal fragments (CTFs) are subsequently processed by cleavage within the transmembrane domain by the γ-secretase complex (13Takasugi 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, 14Edbauer D. Winkler E. Regula J.T. Pesold B. Steiner H. Haass C. Nat. Cell Biol. 2003; 5: 486-488Crossref PubMed Scopus (781) Google Scholar). Consecutive β- and γ-cleavage of APP/APLPs results in the release of Aβ/Aβ-like peptides, whereas α- and γ-cleavage generate p3/p3-like fragments, respectively. Concomitantly, both processing pathways liberate the corresponding intracellular domains (ICDs) (8Eggert S. Paliga K. Soba P. Evin G. Masters C.L. Weidemann A. Beyreuther K. J. Biol. Chem. 2004; 279: 18146-18156Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 15Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. Biochemistry. 2002; 41: 2825-2835Crossref PubMed Scopus (322) Google Scholar, 16Walsh D.M. Fadeeva J.V. LaVoie M.J. Paliga K. Eggert S. Kimberly W.T. Wasco W. Selkoe D.J. Biochemistry. 2003; 42: 6664-6673Crossref PubMed Scopus (91) Google Scholar). A function in nuclear signaling was proposed for the APP/APLP ICDs (16Walsh D.M. Fadeeva J.V. LaVoie M.J. Paliga K. Eggert S. Kimberly W.T. Wasco W. Selkoe D.J. Biochemistry. 2003; 42: 6664-6673Crossref PubMed Scopus (91) Google Scholar, 17Scheinfeld M.H. Matsuda S. D'Adamio L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1729-1734Crossref PubMed Scopus (63) Google Scholar, 18Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1058) Google Scholar), suggesting that processing of APP/APLPs is a crucial step in the pathology of Alzheimer disease and central to the physiological function of APP/APLPs. For APP, a number of intracellular interaction partners, such as Fe65 (19Sabo S.L. Lanier L.M. Ikin A.F. Khorkova O. Sahasrabudhe S. Greengard P. Buxbaum J.D. J. Biol. Chem. 1999; 274: 7952-7957Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), Fe65L1 (20Chang Y. Tesco G. Jeong W.J. Lindsley L. Eckman E.A. Eckman C.B. Tanzi R.E. Guenette S.Y. J. Biol. Chem. 2003; 278: 51100-51107Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), or X11α and X11β (21Sastre M. Turner R.S. Levy E. J. Biol. Chem. 1998; 273: 22351-22357Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), are known to affect its processing. All of these proteins interact with the NPTY motif in the intracellular domain of APP, APLP1, and APLP2 via their phosphotyrosine binding (PTB) domain (22King G.D. Scott Turner R. Exp. Neurol. 2004; 185: 208-219Crossref PubMed Scopus (139) Google Scholar). Protein interacting with APP tail 1 (PAT1) binds to the basolateral sorting sequence (BaSS) of APP and is associated with microtubules. Further, an influence on APP cleavage at the cell surface has been proposed (23Zheng P. Eastman J. Vande Pol S. Pimplikar S.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14745-14750Crossref PubMed Scopus (108) Google Scholar). Therefore, a kinesin light chain (KLC)-like function has been proposed (23Zheng P. Eastman J. Vande Pol S. Pimplikar S.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14745-14750Crossref PubMed Scopus (108) Google Scholar). However, the structural and primary sequence homology between PAT1 and KLC is low. Additionally, PAT1 was shown not to interact with kinesin heavy chain (23Zheng P. Eastman J. Vande Pol S. Pimplikar S.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14745-14750Crossref PubMed Scopus (108) Google Scholar), and its association to microtubules is elevated in the presence of ATP (23Zheng P. Eastman J. Vande Pol S. Pimplikar S.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14745-14750Crossref PubMed Scopus (108) Google Scholar), indicating that PAT1 has no KLC-like function. It has also been reported that PAT1 affects the half-life time of the APP intracellular domain (AICD), suggesting a regulatory function of PAT1 in the putative nuclear signaling of the AICD (24Gao Y. Pimplikar S.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14979-14984Crossref PubMed Scopus (212) Google Scholar). In this study, AICD fragments with a length of 57 (C57) and 59 (C59) amino acids were shown to be affected by PAT1. However, the majority of AICD released to the cytoplasm results from ϵ-cleavage, and it is not clear whether C57 and C59 are released to the cytosol in vivo (15Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. Biochemistry. 2002; 41: 2825-2835Crossref PubMed Scopus (322) Google Scholar, 25Gu Y. Misonou H. Sato T. Dohmae N. Takio K. Ihara Y. J. Biol. Chem. 2001; 276: 35235-35238Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 26Sastre 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, 27Yu C. Kim S.H. Ikeuchi T. Xu H. Gasparini L. Wang R. Sisodia S.S. J. Biol. Chem. 2001; 276: 43756-43760Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 28Zhao G. Cui M.Z. Mao G. Dong Y. Tan J. Sun L. Xu X. J. Biol. Chem. 2005; 280: 37689-37697Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 29Zhao G. Mao G. Tan J. Dong Y. Cui M.Z. Kim S.H. Xu X. J. Biol. Chem. 2004; 279: 50647-50650Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Additionally, Ara67, which is 99.6% identical to PAT1, was reported as an androgen receptor-interacting protein, interrupting androgen receptor cytoplasmic-nuclear import and modulating androgen receptor signaling. These studies suggest that PAT1 might be a regulatory factor that controls the subcellular localization of multiple proteins with divergent functions. Here we show that PAT1a, which is 99.0% identical to PAT1, exists in a common complex with APP, APLP1, and APLP2 in vivo and co-localizes with APP/APLPs bearing intracellular vesicles. Moreover, we demonstrate that PAT1a regulates the levels of APP family members at the cell surface. Finally, we provide evidence that PAT1a increases the proteolytic conversion of APP/APLPs and promotes the generation of Aβ, suggesting an important regulatory role of PAT1a in the transport of APP/APLPs with possible implications for Alzheimer disease pathogenesis. Cloning of Human PAT1a—The PAT1a cDNA was amplified from a human brain cDNA library (Clontech) using open reading frame-flanking primers (sense, 5′-gaa gga aga tgg cgg ccg tgg-3′; antisense, 5′-cct cag cag ctc ggt ccc tcg aca ttc tg-3′) and cloned into the pCRII-TOPO vector (Invitrogen). For recombinant expression of PAT1a, the encoding cDNA was subcloned into pcDNA3.1 (Invitrogen). Alternatively, a hemagglutinin (HA) tag was fused to the 3′-end of the PAT1a cDNA by PCR and cloned into pcDNA3.1. For this purpose, the following oligonucleotides were used: sense, 5′-aat tgg tac cgc cgc cac cat ggc ggc cgt gga act aga gtg-3′, and antisense, 5′-gta att gcg gcc gct caa gcg tct tag ggg acg tcg tat ggg tag cag cac ggt ccc tcg aca ttc tg-3′. Cell Fractionation and Carbonate Extraction—COS-7 or SH-SY5Y cells were co-transfected with PAT1a-HA and APP, APLP1, or APLP2 cDNAs. 28 h after transfection, cells were lysed in homogenization buffer (HOM) (250 mm sucrose, 5 mm HEPES, pH 7.4, 1 mm MgCl2, 10 mm KCl, 1 mm phenylmethylsulfonyl fluoride) by passing 20 times through a 27-gauge needle and centrifuged at 100 × g for 5 min at 4 °C. The supernatant was added to a 9-fold higher volume of carbonate buffer (0.1 m Na2CO3, pH 11) and incubated for 15 min on ice. The postnuclear homogenate was then layered on a 1/3 volume of sucrose/carbonate buffer (carbonate buffer containing 0.25 m sucrose) and centrifuged at 100,000 × g for 45 min. Soluble proteins in the supernatant and membrane fractions in the pellet were analyzed by SDS-PAGE followed by Western blotting. The amounts of PAT1a associated with APP/APLPs in the membrane fraction or present in the fraction containing soluble proteins were quantified using Advanced Image Data Analyzer (AIDA) software, version 3.20.116 (Raytest Isotopen-Messgeräte GmbH). GST Pulldown Analysis—The generation of GST fusion proteins containing the intracellular domain of APP (AICD, aa 649-695), APLP1 (ALICD1, aa 604-650), and APLP2 (ALICD2 aa 717-763) have been described previously (30Lazarov O. Morfini G.A. Lee E.B. Farah M.H. Szodorai A. DeBoer S.R. Koliatsos V.E. Kins S. Lee V.M. Wong P.C. Price D.L. Brady S.T. Sisodia S.S. J. Neurosci. 2005; 25: 2386-2395Crossref PubMed Scopus (196) Google Scholar). The cDNAs encoding ALICD1 and ALICD2 lacking the BaSS (aa 604-620 and aa 733-763) (ALICD1ΔBaSS and ALICD2ΔBaSS) were amplified by PCR and cloned by BamHI/NotI in-frame into pGEX4T-1 (Amersham Biosciences). The sequence was verified by sequencing of both strands (Agowa). Expression of GST and GST fusion proteins and loading of glutathione-Sepharose beads (Amersham Biosciences) was performed as described previously (31Fuhrmann J.C. Kins S. Rostaing P. El Far O. Kirsch A. Sheng M. Triller A. Betz H. Kneussel M. J. Neurosci. 2002; 22: 5393-5402Crossref PubMed Google Scholar). PAT1a was in vitro-translated in the presence of [35S]methionine (TnT7 quick-coupled transcription/translation system (Promega)) and incubated with the loaded beads in buffer H (50 mm Tris, pH 6.8, 50 mm KCl, 100 mm NaCl, 2 mm CaCl2,2mm MgCl2, 0,1% (w/v) Triton X-100, 5 mm dithiothreitol) for 90 min. After three times of washing with buffer H, the proteins were eluted and analyzed by 12% SDS-PAGE. Subsequently, gels were dried and subjected to autoradiography (MR films, Amersham Biosciences) overnight. Quantification is based on densitometric measurements using Image Gauge (Fuji Systems). Antibodies—The monoclonal anti-HA and rat anti-c-Myc antibody were purchased from Roche Applied Science and the mouse anti-c-Myc (9E10) antibody was purchased from Santa Cruz Biotechnology. The β-tubulin antibody (mouse) was from Sigma. Monoclonal antibody against APP (22C11) has been described (32Hilbich C. Monning U. Grund C. Masters C.L. Beyreuther K. J. Biol. Chem. 1993; 268: 26571-26577Abstract Full Text PDF PubMed Google Scholar). The rabbit polyclonal antibodies against APLP1 (CT-11) and APLP2 (DII-2) were obtained from Calbiochem. For generation of an anti-PAT1a antibody, rabbits were immunized with synthesized peptide corresponding to the C-terminal residues 542-572 of human PAT1a. The resulting antiserum was affinity-purified using the same peptide (SulfoLink kit, Pierce). Primary antibodies were used at the following dilutions: anti-APP (22C11) 1:10,000 (IB); anti-PAT1a 1:500 (IB), 1:50 (IC); anti-APLP1 (CT11) 1:10,000 (IB); anti-APLP2 (D2-II) 1:10,000 (IB); anti-HA 1:2000 (IB), 1:200 (IC); anti c-Myc 1:200 (IC). For immunoblotting, secondary anti-rabbit and anti-mouse IgG antibodies (1:10,000) (Promega) and anti-rat IgG antibodies (1:10,000) (DAKO Diagnostic) conjugated to horseradish peroxidase were used. For immunofluorescence analysis, secondary anti-mouse, anti-rat, and anti-rabbit antibodies (1:500) were purchased from Molecular Probes (Alexa Fluor series). Immunocytochemical Assays—For immunocytochemical analysis, primary neurons were fixed with 4% paraformaldehyde/phosphate-buffered saline for 10 min and permeabilized with 0.1% Nonidet P-40 in phosphate-buffered saline for 10 min. Staining of cells was performed using standard procedures (33Kins S. Kurosinski P. Nitsch R.M. Gotz J. Am. J. Pathol. 2003; 163: 833-843Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 34Kins S. Betz H. Kirsch J. Nat. Neurosci. 2000; 3: 22-29Crossref PubMed Scopus (231) Google Scholar). The fluorescent signals were visualized with an Axiovert 200 m Inverted Microscope (Zeiss) supplied with a CCD Camera (Hamamatsu), and images were acquired and enhanced using the MetaMorph Imaging System (Universal Imaging Co.). Cell Culture and Transfection—COS-7, HEK293, and SH-SY5Y cells were cultured under standard conditions. Primary neurons were prepared as described (35Tienari P.J. Ida N. Ikonen E. Simons M. Weidemann A. Multhaup G. Masters C.L. Dotti C.G. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4125-4130Crossref PubMed Scopus (145) Google Scholar) with the exception that mouse cortical neurons of embryonic day 14 were used. Neurons were grown on poly-l-lysine-coated 15-mm coverslips (Marienfeld) in serum-free neurobasal medium (Invitrogen) with B-27 supplement (Invitrogen), 25 μm glutamate, and 0.5 mm glutamin. Cells were transfected with plasmid DNA using Lipofectamine Plus (Invitrogen) or Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions and collected for analysis 20-72 h after transfection. Silencing of PAT1a Expression—For the down-regulation of PAT1a, a short hairpin (sh) construct (shPAT1a) was cloned into pSilencer1.0-U6 (Ambion) using the sequence previously described by Zhang et al. (36Zhang Y. Yang Y. Yeh S. Chang C. Mol. Cell. Biol. 2004; 24: 1044-1057Crossref PubMed Scopus (32) Google Scholar). A scrambled RNAi sequence directed against PAT1a (scrPAT1a) was cloned accordingly and served as a control. For cassette cloning of the shPAT1a construct, the following two oligonucleotides were used: forward, 5′-cga agg cag aac agt taa ttt tca aga gaa att aac tgt tct gcc ttc ttt ttt g-3′, and reverse, 5′-aat tca aaa aag aag gca gaa cag tta att tct ctt gaa aat taa ctg ttc tgc ctt cgg gcc-3′. The non-functional scrPAT1a construct was generated by the same strategy, using the following oligonucleotides: forward, 5′-cga agg tag atc agc taa ttt tca aga gaa att aac tgt tct gcc ttc ttt ttt g-3′, and reverse, 5′-aat tca aaa aag aag gca gaa cag tta att tct ctt gaa aat tag ctg atc tac ctt cgg gcc-3′. The identity of the construct was confirmed by sequencing. SH-SY5Y cells were transfected with pSilencer sh-PAT1a and src-PAT1a using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions and collected for analysis 48-72 h after transfection. Co-Immunoprecipitation Assay—Adult mouse brains were homogenized in ice-cold co-immunoprecipitation buffer (10 mm HEPES, pH 7.2, 143 mm KCl, 5 mm MgCl2,1mm EGTA, 1% Triton X-100, protease inhibitor mix (Roche Applied Science). Before immunoprecipitation, either anti-PAT1a antibody or preimmune serum as a control was incubated with protein A-Sepharose beads. Alternatively, antibodies were covalently coupled to an agarose gel (ProFundTM co-immunoprecipitation kit (Pierce)). For IPs, brain lysates of young adult mice (C57Bl/6) containing 400 μg of protein were incubated with beads loaded with anti-PAT1/PAT1a antibody or preimmune serum for 4 h at 4°C. After extensive washing with co-immunoprecipitation buffer, immunocaptured protein samples were collected in sample buffer containing β-mercaptoethanol and analyzed by SDS-PAGE and Western blotting. The antigen/antibody-complexes were visualized using ECL detection (Amersham Biosciences). Analyses of APP/APLPs Processing—SH-SY5Y cells were transfected as described previously. For the analysis of secreted sAPP/APLPs and Aβ, fresh growth medium was added to the cells after transfection (2.5 ml for each 6-cm dish and 1 ml for each well of a 6-well plate), conditioned for 30 h, and subsequently assayed for Aβ (ELISA) or analyzed by Western blotting. For the analysis of the full-length APP/APLPs and CTFs, cells were lysed 30 h after transfection for 30 min in ice-cold lysis buffer (50 mm Tris/HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, 5 mm EDTA, and Complete™ protease inhibitor mixture (Roche Applied Science)) and centrifuged at 10,000 × g. The supernatants were denatured in sample buffer and analyzed by SDS-PAGE on 8% Tris-glycine or 15% Tris-Tricine gels and Western blotting. Quantification of Aβ Using Sandwich ELISA Assay—Secreted Aβ levels were measured by a Sandwich ELISA assay (hAmyloid ELISA High Sensitive, The Genetics Company, Zürich, Switzerland) as described (37Duering M. Grimm M.O. Grimm H.S. Schroder J. Hartmann T. Neurobiol. Aging. 2005; 26: 785-788Crossref PubMed Scopus (88) Google Scholar). Mouse monoclonal antibodies specific to Aβ40 (G2-10) or Aβ42 (G2-13) were used as capture antibodies, and an N-terminal Aβ antibody (W0-2) was used for detection. Aβ levels in the samples were determined by comparison with the signal from media spiked with known quantities of Aβ40 and Aβ42. Surface Biotinylation— 48 h after transfection with control plasmid, PAT1a cDNA, or shPAT1a, SH-SY5Y cells were washed with cold phosphate-buffered saline containing 0.2 mm CaCl2 and 2 mm MgCl2 followed by incubation with sulfo-NHS-LC-biotin (0.5 mg/ml, Pierce) for1hat4 °C. The reaction was stopped by extensive washing with phosphate-buffered saline and quenched with 50 mm NH4Cl, 1% bovine serum albumin in 50 mm Tris, pH 8.0. Cell lysates were prepared and precipitated with streptavidin-Sepharose (Amersham Biosciences). The precipitated biotinylated proteins were subjected to Western blot analysis. For detection of PAT1a or APP/APLPs, the indicated antibodies were used and visualized using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence technique (Amersham Biosciences). Polymorphism of the PAT1/APPBP2 Gene—As a prerequisite for our studies of the PAT1 protein, we amplified the coding region of PAT1 cDNA from a human brain cDNA library. Surprisingly, we consistently found seven nucleotide exchanges in eight independent clones, resulting in six amino acid substitutions (F296L, K325R, A337A, S340C, L354V, P369R, N544K) (Fig. 1). Data base analyses of the PAT1 sequence (cDNA, AF017782; aa, AAC83973.1) revealed that besides PAT1, a sequence (APPBP2 cDNA, NM_006380; aa, NP_006371.2) completely identical with the sequence we amplified from the brain cDNA is annotated in the data base. Furthermore, we found that the genomic sequence, which is located on chromosome 17, is also identical to our brain cDNA PCR product and APPBP2 (LocusID: 10513). The Ensembl website gives no indication for a duplication of this gene in the human genome. We analyzed 53 expressed sequence tags covering the region of PAT1 cDNA containing the nucleotide exchanges and found that all tested expressed sequence tag sequences were identical with our brain cDNA PCR products and APPBP2, but none was equal to PAT1. These data indicate that the PAT1 sequence used in previous analyses (23Zheng P. Eastman J. Vande Pol S. Pimplikar S.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14745-14750Crossref PubMed Scopus (108) Google Scholar, 24Gao Y. Pimplikar S.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14979-14984Crossref PubMed Scopus (212) Google Scholar, 36Zhang Y. Yang Y. Yeh S. Chang C. Mol. Cell. Biol. 2004; 24: 1044-1057Crossref PubMed Scopus (32) Google Scholar) carries six amino acid exchanges. The PAT1 amino acid sequence is thus 99% identical to APPBP2. We conclude that the PAT1 sequence represents a rare variant of APPBP2 rather than a sequencing error as independently of the PAT1 sequence, six of seven nucleotide exchanges in the PAT1 sequence are annotated as single nucleotide polymorphisms (SNPs) in the genomic sequence. In addition, Ara67 has been described to be 99.6% identical with PAT1 (36Zhang Y. Yang Y. Yeh S. Chang C. Mol. Cell. Biol. 2004; 24: 1044-1057Crossref PubMed Scopus (32) Google Scholar), suggesting that Ara67 is a further variant of PAT1/APPBP2. Thus, PAT1, Ara67, and APPBP2 likely represent different alleles of one single gene, of which APPBP2 is the most common form. For simplification, we propose to rename APPBP2 to PAT1a. Co-localization and Interaction of PAT1a with APP, APLP1, and APLP2 in Neurons—To test whether PAT1a interacts with APP and possibly also with APLP1 and APLP2 in vivo, we performed co-fractionation, co-immunoprecipitation, and co-localization experiments. In a cell fractionation assay, we found that about 50% of PAT1a co-fractionated with membranes containing APP/APLPs. Moreover, PAT1a can be released into the cytosolic fraction by carbonate extraction, showing that it is not a transmembrane protein nor coupled covalently to the membrane (data not shown). Together these data suggest that PAT1a is associated with membrane compartments containing APP, APLP1, and APLP2. To perform co-immunoprecipitation analysis, we generated a polyclonal anti-PAT1a antibody directed against an epitope in the C terminus of human PAT1a (amino acids 542-572). This region contains only one of the amino acid substitutions (N544K) when compared with PAT1/Ara67. Since we have not tested whether the antibody recognizes PAT1 and Ara67, we cannot exclude the possibility of cross-reactivity. However, we show that this anti-PAT1a antibody can be used for Western analyses and immunoprecipitation studies of recombinant HA-tagged PAT1a and endogenous PAT1a (possibly also PAT1 and Ara67) (Fig. 2 and see also Fig. 5). Co-immunoprecipitation experiments with mouse brain extracts using the anti-PAT1a antibody revealed that APP, APLP1, and APLP2 can be co-immunoprecipitated with PAT1a, suggesting that all three APP family members are interacting with PAT1a in vivo (Fig. 2). Co-immunoprecipitations with PAT1a preimmune serum served as control (Fig. 2). Noteworthily, the mature forms of APP, APLP1, and APLP2 were selectively co-immunoprecipitated with PAT1a, indicating specific interaction in post-Golgi compartments.FIGURE 5Cell surface localization of APP depends on PAT1a expression. A, to determine the endogenous PAT1a protein levels, an anti-PAT1a antibody was generated. Depending on the gel system and degree of separation, it recognizes a very close double band (B)at ∼67 kDa in protein extracts (20 μg) of SH-SY5Y cells expressing recombinant PAT1a tagged with a HA tag (PAT1a-HA). The difference in the apparent molecular weight may be due to different post-translational modifications of PAT1a, such as phosphorylation. On the blot in panel A, the proteins were less well separated when compared with the blot in panel B. Thus, PAT1a appears as a single band or as a double band, respectively. Probing of the same extract with a HA antibody revealed an identical signal. After loading of higher amounts of protein extracts (60 μg), the anti-PAT1a antibody also detected endogenous PAT1a in non-transfected cells with the same apparent molecular weight. The overexpression rate of PAT1a was about 3-5-fold higher than endogenous PAT1a protein levels. B, SH-SY5Y cells were treated with transfection reagent only, with shPAT1a, or with a non-functional RNAi construct (scrPAT1a). 48 h after transfection, cell lysates were analyzed on a 8% Tris-glycine gel and subjected to Western analysis using anti-PAT1a and anti-β-tubulin antibodies. C, biotinylation assay 48 h after transfection with PAT1a cDNA, for control pCDNA3.1 or shPAT1a, and for control scrPAT1a. Cells were incubated at 4 °C for 1 h with a biotinylation reagent. Cell extracts were prepared, and biotinylated surface proteins were immunoprecipitated. Western analysis was performed with anti-APP, anti-APLP1, or anti-APLP2 antibodies. D, for quantification, densitometric measurements of three independent experiments were performed. The data were normalized to the amount of total APP, APLP1, and APLP2. The mean ± S.E. is indicated. Statistically significant differences in comparison with control cells transfected with pCDNA3.1 (served as control for PAT1a overexpression) or the scrPAT1a construct (served as control for PAT1a knockdown) (Mann-Whitney U-test) with a p value <0.05 are marked by an asterisk.View Large Image Figure ViewerDownload Hi-res" @default.
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• W2079184536 date "2006-12-01" @default.
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• W2079184536 title "PAT1a Modulates Intracellular Transport and Processing of Amyloid Precursor Protein (APP), APLP1, and APLP2" @default.
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