Two Endoplasmic Reticulum (ER)/ER Golgi Intermediate Compartment-based Lysine Acetyltransferases Post-translationally Regulate BACE1 Levels
2008; Elsevier BV; Volume: 284; Issue: 4 Linguagem: Inglês
10.1074/jbc.m804901200
ISSN1083-351X
Autores Tópico(s)Amino Acid Enzymes and Metabolism
ResumoWe have recently identified a novel form of post-translational regulation of BACE1 (β-site amyloid precursor protein-cleaving enzyme 1), a membrane protein that acts as the rate-limiting enzyme in the generation of the Alzheimer disease amyloid β-peptide. Specifically, nascent BACE1 is transiently acetylated in seven lysine residues clustered in a highly disordered region of the protein that faces the lumen of the endoplasmic reticulum (ER)/ER Golgi intermediate compartment (ER/ERGIC). The acetylation protects the nascent protein from degradation by PCSK9/NARC-1 in the ERGIC and allows it to reach the Golgi apparatus. Here we report the identification of two ER/ERGIC-based acetyltransferases, ATase1 and ATase2. Both proteins display acetyl-CoA:lysine acetyltransferase activity, can interact with and acetylate BACE1, and display an ER/ERGIC localization with the catalytic site facing the lumen of the organelle. Both ATase1 and ATase2 regulate the steady-state levels of BACE1 and the rate of amyloid β-peptide generation. Finally, their transcripts are up-regulated by ceramide treatment. In conclusion, our studies have identified two new enzymes that may be involved in the pathogenesis of late-onset Alzheimer disease. The biochemical characterization of the above events could lead to the identification of novel pharmacological strategies for the prevention of this form of dementia. We have recently identified a novel form of post-translational regulation of BACE1 (β-site amyloid precursor protein-cleaving enzyme 1), a membrane protein that acts as the rate-limiting enzyme in the generation of the Alzheimer disease amyloid β-peptide. Specifically, nascent BACE1 is transiently acetylated in seven lysine residues clustered in a highly disordered region of the protein that faces the lumen of the endoplasmic reticulum (ER)/ER Golgi intermediate compartment (ER/ERGIC). The acetylation protects the nascent protein from degradation by PCSK9/NARC-1 in the ERGIC and allows it to reach the Golgi apparatus. Here we report the identification of two ER/ERGIC-based acetyltransferases, ATase1 and ATase2. Both proteins display acetyl-CoA:lysine acetyltransferase activity, can interact with and acetylate BACE1, and display an ER/ERGIC localization with the catalytic site facing the lumen of the organelle. Both ATase1 and ATase2 regulate the steady-state levels of BACE1 and the rate of amyloid β-peptide generation. Finally, their transcripts are up-regulated by ceramide treatment. In conclusion, our studies have identified two new enzymes that may be involved in the pathogenesis of late-onset Alzheimer disease. The biochemical characterization of the above events could lead to the identification of novel pharmacological strategies for the prevention of this form of dementia. The efficiency of folding, conformational maturation, and molecular stability of nascent membrane and secretory proteins is greatly affected in the endoplasmic reticulum (ER) 2The abbreviations used are: ER, endoplasmic reticulum; ERGIC, ER Golgi intermediate compartment; APP, amyloid precursor protein; BACE1, β-site APP-cleaving enzyme 1; AD, Alzheimer disease; Aβ, amyloid β-peptide; siRNA, short interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; CHO, Chinese hamster ovary; HAT, histone acetyltransferase. by post-translational events that modify, either temporally or definitively, the protein (1Trombetta E.S. Parodi A.J. Annu. Rev. Cell Dev. Biol.. 2003; 19: 649-676Google Scholar). One of the best characterized forms of transient modification is the attachment of a glucose residue to improperly folded nascent glycoproteins by the UDP-glucose:glycoprotein glucosyltransferase (2Dempski Jr., R.E. Imperiali B. Curr. Opin. Chem. Biol.. 2002; 6: 844-850Google Scholar, 3Kleizen B. Braakman I. Curr. Opin. Cell Biol.. 2004; 16: 343-349Google Scholar). This transient event regulates the interaction of the nascent protein with the chaperone calnexin and its ability to leave the early secretory pathway. The mechanism works in such a way that successfully folded proteins dissociate from the calnexin/calreticulin cycle and advance toward the Golgi apparatus, whereas misfolded intermediates are directed toward the ER-associated degradation system (1Trombetta E.S. Parodi A.J. Annu. Rev. Cell Dev. Biol.. 2003; 19: 649-676Google Scholar, 4Meusser B. Hirsch C. Jarosch E. Sommer T. Nat. Cell Biol.. 2005; 7: 766-772Google Scholar). We have recently reported the identification of a novel form of post-translational regulation of BACE1 (β-site APP-cleaving enzyme 1) (5Costantini C. Ko M.H. Jonas M.C. Puglielli L. Biochem. J.. 2007; 407: 383-395Google Scholar), a membrane protein that acts as the rate-limiting enzyme in the generation of the Alzheimer disease amyloid β-peptide (Aβ) from the amyloid precursor protein (APP). Specifically, nascent BACE1 is transiently acetylated in seven lysine residues clustered in a highly disordered region of the protein that faces the lumen of the ER and ER Golgi intermediate compartment (ERGIC). The lysine acetylation of nascent BACE1 regulates its ability to advance toward the Golgi apparatus, where a Golgi-based deacetylase removes the acetyl groups. Nonacetylated intermediates of the nascent protein are retained in the early secretory pathway and degraded by a mechanism that involves the serine protease PCSK9/NARC-1 (proprotein convertase subtilisin kexin-type 9/neural apoptosis-regulated convertase-1) (6Jonas M.C. Costantini C. Puglielli L. EMBO Rep.. 2008; 9: 916-922Google Scholar). The acetylation of nascent BACE1 in the lumen of the ER and/or ERGIC requires a membrane transporter that translocates acetyl-CoA, the donor of the acetyl group, from the cytoplasm to the lumen of the ER, and one or more ER/ERGIC-based acetyl-CoA:lysine acetyltransferases (5Costantini C. Ko M.H. Jonas M.C. Puglielli L. Biochem. J.. 2007; 407: 383-395Google Scholar). The acetylation/deacetylation process is tightly regulated by the lipid second messenger ceramide (5Costantini C. Ko M.H. Jonas M.C. Puglielli L. Biochem. J.. 2007; 407: 383-395Google Scholar, 7Puglielli L. Ellis B.C. Saunders A.J. Kovacs D.M. J. Biol. Chem.. 2003; 278: 19777-19783Google Scholar, 8Costantini C. Weindruch R. Della Valle G. Puglielli L. Biochem. J.. 2005; 391: 59-67Google Scholar) and is under the control of the general aging program mediated by the insulin-like growth factor 1 receptor (IGF-1R) (9Costantini C. Scrable H. Puglielli L. EMBO J.. 2006; 25: 1997-2006Google Scholar, 10Puglielli L. Neurobiol. Aging.. 2008; 29: 795-811Google Scholar). Ceramide, the last output of the above pathway, regulates both efficiency of acetylation in the lumen of the ER/ERGIC and rate of deacetylation in the Golgi apparatus (5Costantini C. Ko M.H. Jonas M.C. Puglielli L. Biochem. J.. 2007; 407: 383-395Google Scholar). As a result, the hyperactivation of ceramide signaling that occurs during normal aging (8Costantini C. Weindruch R. Della Valle G. Puglielli L. Biochem. J.. 2005; 391: 59-67Google Scholar) or in progeroid-like animal models (9Costantini C. Scrable H. Puglielli L. EMBO J.. 2006; 25: 1997-2006Google Scholar) leads to increased acetylation and steady-state levels of BACE1 and to increased production of Aβ (8Costantini C. Weindruch R. Della Valle G. Puglielli L. Biochem. J.. 2005; 391: 59-67Google Scholar, 9Costantini C. Scrable H. Puglielli L. EMBO J.. 2006; 25: 1997-2006Google Scholar). The relevance of the above events for AD neuropathology is stressed by the fact that aging is the single most important risk factor for late-onset AD and that AD patients have very high levels of ceramide in the brain, when compared with age-matched controls (reviewed in Ref. 10Puglielli L. Neurobiol. Aging.. 2008; 29: 795-811Google Scholar). Here we report the identification of two ER/ERGIC-based acetyltransferases, which we named ATase1 and ATase2. Both proteins display acetyl-CoA:lysine acetyltransferase activity in vitro and under native conditions can interact with and acetylate BACE1 in vitro and in vivo, and show an ER/ERGIC localization. They both regulate the steady-state levels of BACE1 and the generation of Aβ, and are up-regulated by ceramide treatment of cultured cells. The following experimental procedures, which have been extensively described in our previous work, are in the supplemental Methods: cell cultures and cell treatment; cell extraction, immunoprecipitation, and affinity purification; subcellular fractionation; Aβ determination; and siRNA treatment. cDNA, Antibodies, and Western Blot Analysis—The cDNA for ATase1 and ATase2 was obtained from Origene (catalog number SC311351 and catalog number SC108791, respectively). Western blotting was performed on a 4-12% BisTris SDS-PAGE system (NuPAGE; Invitrogen) as described (5Costantini C. Ko M.H. Jonas M.C. Puglielli L. Biochem. J.. 2007; 407: 383-395Google Scholar, 7Puglielli L. Ellis B.C. Saunders A.J. Kovacs D.M. J. Biol. Chem.. 2003; 278: 19777-19783Google Scholar, 8Costantini C. Weindruch R. Della Valle G. Puglielli L. Biochem. J.. 2005; 391: 59-67Google Scholar, 9Costantini C. Scrable H. Puglielli L. EMBO J.. 2006; 25: 1997-2006Google Scholar, 11Puglielli L. Konopka G. Pack-Chung E. Ingano L.A. Berezovska O. Hyman B.T. Chang T.Y. Tanzi R.E. Kovacs D.M. Nat. Cell Biol.. 2001; 3: 905-912Google Scholar, 12Ko M.H. Puglielli L. J. Biol. Chem.. 2007; 282: 19742-19752Google Scholar). The following antibodies were used in this study: anti-BACE1 N-terminal (monoclonal, R&D Systems); anti-BACE1 C-terminal (polyclonal, Abcam); anti-acetylated lysine (monoclonal, Abcam; polyclonal, Cell Signaling); anti-calreticulin (ER marker; polyclonal, Abcam); anti-ERGIC-53 (ERGIC marker; polyclonal, Sigma); anti-syntaxin (Golgi marker; monoclonal, Abcam); anti-EEA1 (endosomal marker; monoclonal, BD Transduction Laboratories); anti-Myc (polyclonal, Sigma); anti-C99 (monoclonal, MBL); flotillin 2 (polyclonal; Cell Signaling); and anti-actin (polyclonal, Cell Signaling). Secondary antibodies (Amersham Biosciences) were used at a 1:6000 dilution. Binding was detected by chemiluminescence (LumiGLO kit; Kirkegaard & Perry Laboratories, Gaithersburg, MD). In some cases, the bands corresponding to BACE1 were also validated with the BACE1-Cat1 antibody (generous gift from Dr. Robert Vassar (13Zhao J. Fu Y. Yasvoina M. Shao P. Hitt B. O'Connor T. Logan S. Maus E. Citron M. Berry R. Binder L. Vassar R. J. Neurosci.. 2007; 27: 3639-3649Google Scholar)). Pixel densities (for signal area) of scanned images were calculated with Adobe Photoshop; densitometry (for signal-density) was analyzed with the EpiChemi3 Darkroom™ (UVP Bioimaging Systems) using Labworks Image Acquisition and Analysis Software 4.5. Additionally, samples were also imaged and quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). For infrared imaging, IRDye 680 conjugated goat anti-rabbit IgG (catalog number 926-32221, LI-COR Biosciences) and IRDye 800CW conjugated goat anti-mouse IgG (catalog number 926-32210, LI-COR Biosciences) were used instead of horseradish peroxidase-conjugated secondary antibodies. For quantification, values were normalized to appropriate loading controls. Acetyl-CoA:Lysine Acetyltransferase Activity—For the analysis of the acetyltransferase activity, we employed affinity-purified BACE1 as acceptor of the acetyl group and [3H]acetyl-CoA as donor of the acetyl group. The acetyl-CoA:lysine acetyltransferases (ATase1 and/or ATase2) were provided either as pure enzymes following affinity purification with the ProFound kit (see supplemental Methods) or as membrane extracts of stably transfected CHO cells. The reaction was performed in 150-200 μl of acetylation buffer (50 mm Tris-HCl (pH 8.0), 0.1 mm EDTA, 1 mm dithiothreitol, 10% glycerol, 20 μm acetyl-CoA) and let run for 1 h at 30 °C. The reaction was stopped by adding an equal volume of ice-cold buffer and immediate immersion in ice; BACE1 was immunoprecipitated with an anti BACE1 N-terminal monoclonal antibody and then counted on a liquid scintillation counter. The experimental controls are described in the appropriate figure legends. Additionally, the acetyltransferase activity of affinity-purified ATase1 and ATase2 was assayed with commercially available colorimetric (catalog number K332-100, BioVision Inc.) and fluorescent kits (catalog number 10006515, Cayman Chemicals). The assays were performed as recommended by the manufacturer. Real Time PCR—Total RNA from H4 (human neuroglioma) and SH-SY5Y (human neuroblastoma) cells was extracted and purified using the RNeasy plus mini kit (Qiagen). cDNA was synthesized using the SuperScript double-stranded cDNA synthesis system (Invitrogen) and then PCR-amplified. Primers used for RT-PCR were as follows: GAPDH, forward 5′-GAAGGTGAAGGTCGGAGTC-3′, reverse 5′-GAAGATGGTGATGGGATTTC-3′; ATase1, forward 5′-CGATTACTGAAGCTGCCTCGA-3′, reverse 5′-GGTTTTTTGGCAAGGAACCA-C-3′; and ATase2, forward 5′-TCCTTGCCAAAAAACCCTGG-3′, reverse 5′-ATGCCCACCACCTTCTCTTCA-3′. RNA quantification was performed by real time PCR in a ABI PRISM 7000 sequence detection system using SYBR Green PCR master mix (Applied Biosystems). Amplifications were generated at 2 min at 52 °C and 3 min at 95 °C, followed by 40 cycles of denaturations at 95 °C for 15 s, annealing, and synthesis (30 s at 61 °C and 30 s at 72 °C). ΔΔCt values (14Livak K.J. Schmittgen T.D. Methods (San Diego).. 2001; 25: 402-408Google Scholar) were normalized with those obtained from the amplification of GAPDH and were expressed as fold-change over control. The assay was repeated three times, with each assay containing triplicate reactions and with each assay including an independent amplification of the probes. Statistical Analysis—Results are always expressed as mean ± S.D. of the indicated number of determinations. The data were analyzed by analysis of variance and Student's t test comparison, using GraphPad InStat3 software. Statistical significance was reached at p < 0.05. Identification of ATase1 and ATase2 as Two Putative ER/ER-GIC Acetyl-CoA:Lysine Acetyltransferases—Our studies previously demonstrated the presence of acetyl-CoA:lysine acetyltransferase activity in the ER/ERGIC. They also demonstrated that the catalytic site of the putative ER/ERGIC-based acetyltransferase(s) faced the lumen of the organelle (5Costantini C. Ko M.H. Jonas M.C. Puglielli L. Biochem. J.. 2007; 407: 383-395Google Scholar). To identify the corresponding gene(s), we searched in the human genome for proteins displaying structural similarities to the histone acetyltransferase catalytic domain. The search yielded 46 possible candidates, 2 of which were predicted to function in the ER/ERGIC. The candidates had unknown biochemical function and were initially named Camello-like 2 (accession number NM_016347; also called putative N-acetyltransferase 8B) and Camello-like 1 (accession number NM_003960; also called putative N-acetyltransferase 8). They appear to have orthologs in Xenopus laevis (15Popsueva A.E. Luchinskaya N.N. Ludwig A.V. Zinovjeva O.Y. Poteryaev D.A. Feigelman M.M. Ponomarev M.B. Berekelya L. Belyavsky A.V. Dev. Biol.. 2001; 234: 483-496Google Scholar) but not in Caenorhabditis elegans or Drosophila melanogaster. The biochemical activity of both proteins was explored, and based on the results reported here they were re-named acetyltransferase 1 (ATase1) and 2 (ATase2) to indicate their biochemical function rather than the domain organization. Both ATase1 and ATase2 have a high probability of being ER/ERGIC-resident proteins (k-NN prediction using the PSORT II software of the University of Tokyo, Japan and available on line) (16Horton P. Park K.J. Obayashi T. Fujita N. Harada H. Adams-Collier C.J. Nakai K. Nucleic Acids Res.. 2007; 35: W585-W587Google Scholar, 17Nakai K. Horton P. Trends Biochem. Sci.. 1999; 24: 34-36Google Scholar). Alignment of the amino acid sequences of ATase1 and ATase2 reveals an 88% sequence identity between the two proteins (Fig. 1A); the Kyte-Doolittle hydrophilicity/hydrophobicity plot (Fig. 1B) indicates the presence of one single hydrophobic segment, corresponding to the membrane-spanning domain. In both cases the putative catalytic site is situated in the C-terminal domain and is predicted to face the lumen of the ER/ERGIC. ATase1 Is an ER/ERGIC Resident Acetyl-CoA:Lysine Acetyltransferase—We initially targeted ATase1, which was cloned and inserted into a mammalian expression vector containing a Myc tag at the C terminus, to allow subcellular localization and affinity purification for the in vitro studies. Stable transfection in CHO cells yielded several colonies overexpressing ATase1 (Fig. 2A). Membrane extracts from nontransfected control and ATase1-expressing cells were analyzed in vitro for acetyl-CoA:lysine acetyltransferase activity. The assay was performed in the presence of affinity-purified BACE1, which served as acceptor of the acetyl group, and radiolabeled acetyl-CoA, which served as donor of the acetyl group. Fig. 2B shows that the expression of ATase1 increased the acetyltransferase activity recovered from membrane extracts by ∼2-fold. No activity was observed when the assay was performed in the absence of extracts (source of the enzymatic activity) or in the presence of extracts that had been boiled prior to the incubation (Fig. 2B). Subcellular fractionation studies indicated a predominant ERGIC localization that extended to ER, but not to Golgi, fractions (Fig. 2C), which is consistent with our previous localization of the ER/ERGIC-based acetyl-CoA:lysine acetyltransferase activity (5Costantini C. Ko M.H. Jonas M.C. Puglielli L. Biochem. J.. 2007; 407: 383-395Google Scholar). The subcellular localization of ATase1 overlaps with the compartments where the acetylation of nascent BACE1 occurs and the nonacetylated mutants of BACE1 are retained (5Costantini C. Ko M.H. Jonas M.C. Puglielli L. Biochem. J.. 2007; 407: 383-395Google Scholar). In fact, we have previously demonstrated that the Lys-to-Ala (BACE1Ala) and the Lys-to-Arg (BACE1Arg) mutant forms of BACE1 cannot be acetylated in vivo and in vitro, are retained in the ER/ERGIC system, and rapidly degraded by a proteasome-independent system (5Costantini C. Ko M.H. Jonas M.C. Puglielli L. Biochem. J.. 2007; 407: 383-395Google Scholar, 6Jonas M.C. Costantini C. Puglielli L. EMBO Rep.. 2008; 9: 916-922Google Scholar). Next, we analyzed the acetyl-CoA:lysine acetyltransferase activity of the individual fractions from the gradient shown in Fig. 2C and found that the distribution pattern of ATase1 completely overlapped with the acetyl-CoA:lysine acetyltransferase activity (Fig. 2D). Finally, when compared with control (nontransfected) cells, ERGIC fractions from ATase1-expressing cells displayed increased (∼2-fold) acetyltransferase activity (Fig. 2E). The above results indicate that ATase1 is localized in the compartments where the acetylation of nascent BACE1 normally occurs. They also indicate that the overexpression of ATase1 results in increased acetyl-CoA:lysine acetyltransferase activity. However, they do not rule out the possibility that ATase1 is only a co-factor in the reaction of lysine acetylation. To address this issue, we used affinity-purified ATase1 (instead of membrane extracts) for the in vitro acetylation of BACE1. As control, we used BACE1Arg, which does not act as acceptor of the acetyl groups in vitro or in vivo (5Costantini C. Ko M.H. Jonas M.C. Puglielli L. Biochem. J.. 2007; 407: 383-395Google Scholar). Fig. 3A shows that purified ATase1 was able to acetylate BACE1WT but not BACE1Arg proving that it is an acetyl-CoA:lysine acetyltransferase. The results obtained with BACE1Arg are particularly important because they indicate that ATase1 can only acetylate the lysine residues that are found acetylated under physiological conditions (5Costantini C. Ko M.H. Jonas M.C. Puglielli L. Biochem. J.. 2007; 407: 383-395Google Scholar). Next, we incubated ATase1 with BACE1 in the presence of nonradiolabeled acetyl-CoA. Both BACE1 and ATase1 were purified by affinity chromatography prior to the incubation. BACE1 was then immunoprecipitated at the end of the reaction and analyzed by classical immunoblotting. In the absence of ATase1 only the immature form of BACE1 could be detected with an anti-acetylated lysine antibody (Fig. 3B, left panel,-ATase1, indicated by 1). However, incubation with ATase1 allowed detection of two additional bands corresponding to the fully mature and a biosynthetic intermediate form of BACE1 (Fig. 3B, left panel, +ATase1, indicated by 2 and 3). These results confirm those performed with radiolabeled acetyl-CoA (Fig. 3A) and clearly indicate that ATase1 can acetylate BACE1. The normal migration of the mature and biosynthetic intermediate forms of BACE1 is shown in the middle panel of Fig. 3B and in supplemental Fig. 1. The anti-acetylated lysine antibody also detected a lower band migrating with the predicted molecular mass of the cleaved ectodomain of BACE1 (Fig. 3B, left panel, indicated by *). The same band could be detected with an antibody against the N-terminal domain of BACE1 (data not shown) but not with antibodies raised against the C-terminal domain of BACE1 or the Myc tag (Fig. 3B, middle and right panels, indicated by *). The band was never observed immediately after purification of BACE1 and appeared only after the 6-h incubation required for the in vitro acetylation assay, suggesting that it corresponds to the cleaved N-terminal ectodomain of BACE1 and most likely results from in vitro autocatalytic cleavage, as described previously (18Benjannet S. Elagoz A. Wickham L. Mamarbachi M. Munzer J.S. Basak A. Lazure C. Cromlish J.A. Sisodia S. Checler F. Chretien M. Seidah N.G. J. Biol. Chem.. 2001; 276: 10879-10887Google Scholar). Surprisingly, the anti-acetylated lysine antibody recognized a band of ∼28 kDa, which corresponds to ATase1 (Fig. 3B, left panel, +ATase1). In fact, the same band could be observed with anti-Myc antibodies (Fig. 3B, right panel, +ATase1) but not with antibodies against the N-terminal (data not shown) or C-terminal domain (Fig. 3B, middle panel) of BACE1. Therefore, these results indicate that ATase1 can interact with BACE1 in vitro and can undergo acetylation in one or more lysine residues, most likely through an autocatalytic mechanism (discussed below). ATase2 Is an ER/ERGIC Resident Acetyl-CoA:Lysine Acetyltransferase—We next cloned ATase2 into a mammalian expression vector containing a Myc tag at the C terminus. This construct was used to generate cells stably expressing ATase2 and ATase1 + ATase2 (Fig. 4A). Similarly to ATase1, the expression of ATase2 alone increased (by ∼3-fold) the acetyl-CoA:lysine acetyltransferase activity recovered from total membrane extracts (Fig. 4B). In addition, the subcellular distribution of ATase2 (Fig. 4C), even though slightly different, appeared to overlap with ATase1 (Fig. 2C) and with the normal distribution of the acetyl-CoA:lysine acetyltransferase activity (Fig. 2D). Finally, ER/ERGIC fractions generated from ATase2-expressing cells displayed increased acetyltransferase activity, when compared with corresponding fractions from control nontransfected cells (Fig. 4D). As with ATase1-expressing cells (Fig. 2D), no activity was found in fractions corresponding to the Golgi apparatus (data not shown, see also Ref. 5Costantini C. Ko M.H. Jonas M.C. Puglielli L. Biochem. J.. 2007; 407: 383-395Google Scholar). When affinity-purified ATase2 was incubated with BACE1 and nonradiolabeled acetyl-CoA, we found a marked increase in the acetylation of both biosynthetic intermediates of BACE1 (Fig. 5A, left panel). Interestingly, the acetylation pattern produced by ATase2 seems somehow different from the one observed with ATase1 (compare Fig. 5A to Fig. 3B) suggesting potential differences in substrate recognition activities of the two enzymes. Similarly to ATase1, the immunoprecipitation of BACE1 resulted in the pulldown of ATase2, suggesting physical interaction, at least in vitro (Fig. 5A, right panel). However, in contrast to ATase1, we could not clearly resolve a band corresponding to ATase2 with an anti-acetylated lysine antibody (Fig. 5A, left panel). This occurred even though ATase2 could clearly be identified with an anti-Myc antibody (Fig. 5A, right panel). These results might indicate potential differences in the biochemical properties and functional regulation of ATase1 and ATase2 (discussed below). Finally, incubation of ATase2 with BACE1 and radiolabeled acetyl-CoA resulted in acetylation of BACE1WT but not BACE1Arg (Fig. 5B) proving that ATase2 can only acetylate the lysine residues that are found acetylated under physiological conditions (5Costantini C. Ko M.H. Jonas M.C. Puglielli L. Biochem. J.. 2007; 407: 383-395Google Scholar). We next used a commercially available colorimetric assay that employs a recombinant peptide corresponding to the N-terminal tail of the histone protein, as acceptor of the acetyl group, and a highly concentrated nuclear histone acetyltransferase (HAT) preparation, as enzyme. The assay was partially modified to employ our affinity-purified ATases, instead of HAT, as source of the enzymatic activity. Fig. 5C shows that both ATase1 and ATase2 displayed robust acetyl-CoA:lysine acetyltransferase activity, which was in the same range of that observed with HAT. Finally, when ATase1 and ATase2 were included together in the assay, we detected a clear additive effect indicating that the two enzymes can act independently of each other, at least in vitro. It remains to be determined whether the additive effect is caused by their ability to target different lysine residues of the substrate-peptide or by the presence of excess substrate in the assay. The ability of ATase1 and ATase2 to acetylate an appropriate substrate was also confirmed by using a fluorescent assay that employs the histone H3-(5-23) peptide as acceptor and acetyl-CoA as donor (supplemental Fig. 2). Catalytic C-terminal Domain of ATase1 and ATase2 Faces the Lumen of the ER/ERGIC System—We have previously shown that the transient acetylation of the N-terminal and globular domain of nascent BACE1 occurs in the lumen of the ER/ERGIC system (5Costantini C. Ko M.H. Jonas M.C. Puglielli L. Biochem. J.. 2007; 407: 383-395Google Scholar). We also demonstrated that the acetyltransferase activity responsible for the lysine acetylation of the nascent protein was positioned in the luminal face of the secretory compartment (5Costantini C. Ko M.H. Jonas M.C. Puglielli L. Biochem. J.. 2007; 407: 383-395Google Scholar). The hydrophilic/hydrophobic plot (Fig. 1B) together with the apparent topology (using the MTOP prediction model (19Hartmann E. Rapoport T.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A.. 1989; 86: 5786-5790Google Scholar)) of ATase1 and ATase2 predicts the C-terminal domain, which contains the acetyl-CoA:lysine acetyltransferase homology domain, to face the lumen of the ER/ERGIC system. We decided to assess whether this was indeed the case by assaying in vitro the acetyl-CoA:lysine acetyltransferase activity recovered from ERGIC vesicles of ATase1- or ATase2-expressing cells under two different experimental conditions as follows: in the presence or absence of mild concentrations (0.2-0.5%) of Triton X-100 (Fig. 6A). When prepared, vesicles are sealed and of the same membrane topographical orientation as in vivo (5Costantini C. Ko M.H. Jonas M.C. Puglielli L. Biochem. J.. 2007; 407: 383-395Google Scholar, 20Carey D.J. Hirschberg C.B. J. Biol. Chem.. 1981; 256: 989-993Google Scholar, 21Puglielli L. Mandon E.C. Hirschberg C.B. J. Biol. Chem.. 1999; 274: 12665-12669Google Scholar, 22Puglielli L. Mandon E.C. Rancour D.M. Menon A.K. Hirschberg C.B. J. Biol. Chem.. 1999; 274: 4474-4479Google Scholar). Therefore, under normal conditions (Fig. 6A, Sealed) the acetylation of affinity-purified BACE1 can occur only if the catalytic site of the enzyme (ATase1 or ATase2) is facing the outside/cytosolic face of the vesicles. Conversely, if the catalytic site of the enzyme resides in the luminal face of the vesicles, the reaction will only occur in the presence of mild concentrations of detergent, which will allow access of both the acceptor (purified BACE1) and donor (radiolabeled acetyl-CoA) to ATase1 or ATase2 (Fig. 6A, Opened). Fig. 6B clearly shows that the acetyltransferase activity of native ATase1 and ATase2 could only be observed in the presence of Triton X-100, indicating that the catalytic site is facing the lumen of the organelle in its native conditions. The activity recovered in the absence of Triton X-100 was in the normal range of latency (3-5%) observed with the glucose-6-phosphatase and sialyltransferase methods (see supplemental Methods). Next, we took advantage of the fact that both ATase1 and ATase2 have a Myc tag at the C terminus, which is predicted to face the lumen of the ER/ERGIC system. Therefore, the above vesicles were incubated with an anti-Myc antibody covalently attached to aldehyde-activated agarose beads (ProFound system) for immunoprecipitation. The experiment was performed under both the “opened” and “sealed” conditions (Fig. 6A) to determine the topology of the enzymes. Fig. 6C shows that ATase1 and ATase2 could be immunoprecipitated only when the vesicles were used under the opened condition, which allowed the anti-Myc antibody to interact with the C-terminal Myc tag. Therefore, when taken together, the above results indicate that both ATase1 and ATase2 have the predicted topology, with the catalytic C-terminal domain facing the lumen of the ER/ERGIC system, where the lysine acetylation of nascent BACE1 normally occurs. ATase1 and ATase2 Influence the Steady-state Levels of BACE1 and the Generation of Aβ—The results shown in Figs. 3B and 5A indicate th
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