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A Transcriptively Active Complex of APP with Fe65 and Histone Acetyltransferase Tip60
Xinwei Cao and Thomas C. Südhof
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Supplementary Material
1. Transactivation Assays
1.1 General design
PC12, COS, HeLa, and HEK293 cells were cotransfected at 50 to 80% confluency in 6-well plates using Fugene6 (Roche), and 4 to 5 plasmids (1.0 to 3.0
g DNA/well depending on cell types; see plasmid list below for description of all constructs). All transfections included one of the following: (i) Gal4 (pG5E1B-luc) or LexA (pL8G5-luc) reporter plasmids, (ii) constitutively expressed
-galactosidase expression plasmid (pCMV-LacZ) to control for transfection efficiency, (iii) the Gal4- or LexA-fusion protein vectors, and (iv) plasmids encoding various cofactors of pCDNA4.1 to make up equal amounts of total DNA transfected. Cells were harvested 48 hours posttransfection in 0.2 ml/well reporter lysis buffer (Promega), and their luciferase and
-galactosidase activities were determined with the Promega luciferase assay kit and the standard
O-nitrophenyl-
D-galacto-pyranoside method, respectively. The luciferase activity was standardized by the
-galactosidase activity to control for transfection efficiency and general effects on transcription, and in most experiments further normalized for the transactivation observed in cells expressing Gal4 or LexA alone. Values shown are averages of transactivation assays carried out in duplicate, and repeated at least three times for each cell type and constructs. Most constructs were assayed in three or four cell lines, but usually only representative results for one cell line are shown. To confirm expression of transfected proteins and secretase cleavage of the various APP constructs, transfected cells were also analyzed by immunoblotting using antibodies to the respective proteins and/or antibodies to the epitope tags attached to the proteins.
1.2 Amounts of DNA used
1.21 APP-Gal4 and APP-LexA assays with and without various Fe65 and Mint1 constructs. For transactivation by APP-Gal4 and APP-LexA constructs, cells were cotransfected with the following: (i) pG5E1B-luc (Gal4 reporter plasmid) or pL8G5-luc (LexA-reporter plasmid) (HEK293 cells, HeLa cells, and COS cells = 0.3 g DNA; PC12 cells = 1.0 g); (ii) pCMV-LacZ (-galactosidase control plasmid. HEK293 cells, HeLa cells, and COS cells = 0.05 g DNA; PC12 cells = 0.2 to 0.5 g DNA); (iii) pMst (Gal4), pMst-APP (APP-Gal4), pMst-APP* (APP*-Gal4), pMst-APP
(APP-Gal4), pMst-APP* (APP*-Gal4), pMst-AN-APPc32 (APP-G-NRX-APPc32), pMst-AN (APPe-G-NRXc), pML (LexA), pML-APP (APP-LexA), pML-APP* (APP*-LexA), pML-APPct (APPct-LexA), pML-APPct* (APPct*-LexA) (HEK293 cells, HeLa cells, and COS cells = 0.3-0.5 g DNA; PC12 cells = 1.0-1.5 g); and (iv) pCMV-Mint1 (mint1) or pCMV5-Fe65 (Fe65) (HEK293 cells, HeLa cells, and COS cells = 0.3-0.5 g DNA; PC12 cells = 1.0-1.5 g) where indicated.
For transactivation assays of Fe65 mutants, cells were cotransfected with the following: (i) pG5E1B-luc (Gal4 reporter plasmid) or pL8G5-luc (LexA-reporter plasmid); (ii) pCMV-LacZ (-galactosidase control plasmid); (iii) pMst (Gal4), pMst-APP (APP-Gal4), pML (LexA), or pML-APP (APP-LexA); and (iv) pCMV5-Fe65 (Fe65), pCMVMyc-Fe65(128-711) [Fe65(128-711)], pCMVMyc-Fe65(242-711) [Fe65(242-711)], pCMVMyc-Fe6(287-711) [Fe65(287-711)], pCMV5-Fe65(1-553) (Fe65
PTB2), pCMVMyc-Fe65PTB1 (Fe65PTB1), pCMV5-Fe65mW1(Fe65mW1), pCMV5-Fe65mW2 (Fe65mW2), pCMV5-Fe65mW3 (Fe65mW3), pCMV5-Fe65mW4 (Fe65mW4), or pCMV5-Fe65mW5 (Fe65mW5) where indicated. The amounts of DNA in (i) and (ii) were the same as under 1.21. (iii) and (iv):0.3-0.5 g DNA for COS, HeLa, and HEK293 cells; 1.0-1.5 g DNA for PC12 cells.
1.22 Gal4-Tip60 assays. For transactivation assays of Gal4-Tip60, COS, HeLa, and HEK293 cells were cotransfected with the following: (i) pG5E1B-luc (Gal4 reporter plasmid, 0.3 g DNA); (ii) pCMV-LacZ (-galactosidase control plasmid, 0.05 g DNA); (iii) pMst (Gal4); pM-Tip60 (rat Gal4-Tip60ß residues 63 to 454); pM-Tip60* (mutant rat Gal4-Tip60ß residues 63 to 454); pM-hTip60 (full-length wild-type human Gal4-Tip60ß); or pM-hTip60* (full-length mutant human Gal4-Tip60ß) (0.4 g DNA); (iv) pCMV5-Fe65 (Fe65), pCMVMyc-Fe65(242-711) [Fe65(242-711)], pCMV5-Fe65(1-553) (Fe65PTB2), or pCMV5-Fe65mW4 (Fe65mW4) (0.3 g DNA) where indicated; and (v) pCMV5-APP (human APP695) or pCMV5-APP* (mutant human APP695) (0.3 g DNA). All transfections contained one of the plasmids listed in (i to iii), whereas (iv) and (v) were variable.
2. Yeast Two-Hybrid Screens for APP- and Fe65-Binding Proteins
2.1 APP screens
Bait: pBTM116-APP
Yeast strain: L40
Library: P8 rat brain library constructed in prey vector pVP16-3.
Screening condition: 250 ml mid-log phase yeasts harboring the bait vector pBTM116-APP were transformed with 125 g of P8 rat brain library plasmids, and plated on CSM-Trp-Leu-His plates supplemented with 5 mM 3-amino-1,2,4-triazole. Total transformants (2 × 107) were obtained. Around 250 positives appeared after 3 days incubation at 30°C. 80 positives were recovered and identified by sequencing or Southern blotting. Among them, 72 are Fe65 and one is Fe65-like protein 2 (Fe65LP2).
Summary of Fe65 prey clones that were sequenced
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| Prey clones | Insert size (kb) | Residues | Extra residues |
| P29a | 3.1 | 1-711 | 43 |
| P50, P64, P65, P69 | 2.9 | 1-711 | 43 |
| P42 | 3.0 | 48-711 | 0 |
| P60a | 2.6 | 128-711 | 0 |
| P6a, P7, P18 | 1.9 | 242-711 | 8 |
| P57 | 2.3 | 242-711 | 50 |
| P19, P25b | 1.0 | 242-? | 21 |
| P15, P27a | 1.6 | 287-711 | 0 |
| P21 | 1.5 | 301-711 | 0 |
| P4, P22 | 1.4 | 313-711 | 0 |
| P9, P16, P17 | 1.9 | 321-711 | 0 |
| P39 | 1.4 | 339-711 | 0 |
aP29, P60, P6, P27 were used to generate pCMV5-Fe65, pCMVMyc-Fe65(128-711), pCMVMycFe65(242-711), and pCMVMyc-Fe65(287-711), respectively.
bP25 insert (1.0 kb) was used as Southern blotting probe to identify the rest of the recovered prey clones.
2.2 Fe65 screens
Bait: pLexN-Fe65(287-711)
Yeast strain: L40
Library: P8 rat brain library constructed in prey vector pVP16-3.
Screening condition: 250 ml mid-log phase yeasts harboring the bait vector pLexN-Fe65(287-711) were transformed with 125 g of P8 rat brain library plasmids, and plated on CSM-Trp-Leu-His plates supplemented with 25 mM 3-amino-1,2,4-triazole. 7 × 107 total transformants were obtained, and ~300 positives selected after 3 days incubation at 30°C.
Summary of prey clones that were sequenced: Tip60 (residues 63 to 454) = 8 clones; APLP1 = 9 clones.
3. Quantitative Yeast Two-Hybrid Assays
For quantitative measurements of yeast two-hybrid interactions, various bait and prey plasmids were cotransfected into yeast strain L40 and plated on CSM-Trp-Leu (Bio101) plates. Single colonies from the CSM-Trp-Leu plates were cultured in CSM-Trp-Leu liquid medium after 3 days, and re-inoculated into YPAD medium at OD600 = 0.3 on the following day. When the yeast cultures reached mid-log phase (OD600 = 0.6-1.0), cells were collected by centrifugation, washed in Z buffer (16.1 g/l Na2HPO4·7H2O, 5.5 g/l NaH2PO4·H2O, 0.75 g/l KCl, 0.246 g/l MgSO4·7H2O pH 7.0), and resuspended in 100 l Z buffer. Cells were lysed by three freeze-thawing cycles, 0.8 ml reaction solution (0.5 g/l ONPG and 0.024% -mercaptoethanol in Z buffer) was added, and reactions were stopped with 0.4 ml 1M Na2CO3at the appropriate time points depending on color development. After centrifugation (14,000g for 10 min), OD420 was measured, and the relative -galactosidase activity was calculated as 1000 × OD420/(min × vol. yeast in ml × OD600).
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| 3.1 | APP & Fe65 | 02/22/2001 |
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| # | Bait | Prey | OD600 | Time(min) | OD420 | ß -gal unit | Mean | SD |
| 1 | APPcta | P29 [Fe65(1-711)]a | 0.899 | 15 | 5.025 | 372.6363 | 423.8138 | 48.30529 |
| | | | 0.764 | 15 | 4.93 | 430.192 | | |
| | | | 0.685 | 15 | 4.815 | 468.6131 | | |
| 2 | APPcta | Fe65mW5c | 0.657 | 15 | 4.895 | 496.7022 | 449.4991 | 71.701238 |
| | | | 0.68 | 15 | 4.945 | 484.8039 | | |
| | | | 0.822 | 15 | 4.525 | 366.9911 | | |
| 3 | APPcta | Vector | 0.813 | 15 | 0.004 | 0.328003 | 0.370065 | 0.0425321 |
| | | | 0.903 | 15 | 0.005 | 0.36914 | | |
| | | | 0.807 | 15 | 0.005 | 0.413052 | | |
| 4 | APPct*b | P29 [Fe65(1-711)]a | 0.776 | 15 | 0.005 | 0.429553 | 0.506697 | 0.0674611 |
| | | 0.601 | 15 | 0.005 | 0.554631 | | |
| | | 0.622 | 15 | 0.005 | 0.535906 | | |
| 5 | APPct*b | Fe65mW5c | 0.631 | 15 | 0.004 | 0.42261 | 0.44523 | 0.0467628 |
| | | 0.644 | 15 | 0.004 | 0.414079 | | |
| | | 0.668 | 15 | 0.005 | 0.499002 | | |
aAPPct and P29(Fe65 1-711) are bait and prey clones described in section 2.1.
bAPPct* was expressed from pBTM116-APP*, which was generated by QuickChange site-directed mutagenesis kit (Stratagene) to mutate
684NPTY to
684NATA with pBTM116-APP as the template.
cFe65mW5 was expressed from pVP16-3-Fe65mW5, which encodes full-length rat Fe65 (1 to 711 residues) with
Y270A,
Y271A,
W272A,
W281F, and
P284A mutations.
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| 3.2 | Fe65 & LBP-1c | 02/22/2001 |
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| # | Bait | Prey | OD600 | Time(min) | OD420 | ß-gal unit | Mean | SD |
| 1 | Fe65(287-711)a | LBP-1cb | 0.747 | 60 | 0.037 | 0.825524 | 0.772376 | 0.1010779 |
| | | 0.737 | 60 | 0.029 | 0.655812 | | |
| | | 0.678 | 60 | 0.034 | 0.835792 | | |
| 2 | Fe65(287-531)a | LBP-1cb | 0.686 | 60 | 0.167 | 4.057337 | 4.438847 | 0.3744772 |
| | | 0.704 | 60 | 0.203 | 4.805871 | | |
| | | 0.625 | 60 | 0.167 | 4.453333 | | |
| 3 | Fe65(287-711)a | vector | 0.709 | 60 | 0.006 | 0.141044 | 0.15965 | 0.0262405 |
| | | 0.703 | 60 | 0.008 | 0.189663 | | |
| | | 0.787 | 60 | 0.007 | 0.148242 | | |
| 4 | Fe65(287-531)a | vector | 0.725 | 60 | 0.022 | 0.505747 | 0.5543 | 0.0714302 |
| | | 0.681 | 60 | 0.026 | 0.636319 | | |
| | | 0.672 | 60 | 0.021 | 0.520833 | | |
| 5 | Lamina | LBP-1cb | 0.626 | 60 | 0.017 | 0.452609 | 0.721477 | 0.3382954 |
| | | 0.819 | 60 | 0.03 | 0.610501 | | |
| | | 0.454 | 60 | 0.03 | 1.101322 | | |
aFe65(287-711) and Fe65(287-531) were expressed from pLexN-Fe65(287-711) and pLexN-Fe65(287-531), respectively. Lamin was expressed from pBTM116-Lamin.
bLBP-1c was expressed from pVP16-3-LBP-1c, which encodes full-length human LBP-1c (1 to 450 residues). References: Z. Bing, S. A. G. Reddy, Y. Ren, J. Qin, W. S. L. Liao,
J. Biol. Chem.
274, 24649 (1999). N. Zambrano, G. Minopoli, P. de Candia, T. Russo,
J. Biol. Chem.
273, 20128 (1998).
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| 3.3 | Fe65 & hTip60 | 02/22/2001 |
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| # | Bait | Prey | OD600 | Time(min) | OD420 | ß-gal unit | Mean | SD |
| 1 | Fe65(287-711)a | hTip60b | 0.806 | 15 | 4.905 | 405.7072 | 431.7927 | 45.395309 |
| | | | 0.818 | 15 | 4.975 | 405.4605 | | |
| | | | 0.665 | 15 | 4.83 | 484.2105 | | |
| 2 | Fe65(287-531)a | hTip60b | 0.695 | 15 | 3.895 | 373.6211 | 391.1748 | 52.914479 |
| | | | 0.774 | 15 | 4.055 | 349.2679 | | |
| | | | 0.682 | 15 | 4.61 | 450.6354 | | |
| 3 | Fe65(287-711)a | vector | 0.709 | 60 | 0.006 | 0.141044 | 0.15965 | 0.0262405 |
| | | | 0.703 | 60 | 0.008 | 0.189663 | | |
| | | | 0.787 | 60 | 0.007 | 0.148242 | | |
| 4 | Fe65(287-531)a | vector | 0.725 | 60 | 0.022 | 0.505747 | 0.5543 | 0.0714302 |
| | | | 0.681 | 60 | 0.026 | 0.636319 | | |
| | | | 0.672 | 60 | 0.021 | 0.520833 | | |
| 5 | Lamina | hTip60b | 0.594 | 15 | 0.011 | 1.234568 | 1.751572 | 0.5012274 |
| | | | 0.635 | 15 | 0.017 | 1.784777 | | |
| | | | 0.507 | 15 | 0.017 | 2.235371 | | |
aFe65(287-711) and Fe65(287-531) were expressed from pLexN-Fe65(287-711) and pLexN-Fe65(287-531), respectively. Lamin was expressed from pBTM116-Lamin.
bhTip60 was expressed from pVP16-3-hTip60, which encodes full-length human Tip60ß (residues 1 to 461). Reference: Q. Ran and O. M. Pereira-Smith,
Gene 258, 141 (2000).
4. Plasmid List
4.1. Standard plasmids
pCMV-LacZ: Transfection control plasmid encoding bacterial -galactosidase under control of the CMV promoter.
pG5E1B-luc: Gal4 reporter reporter plasmid [J. W. Lillie, M. R. Green, Transcription activation by the adenovirus E1a protein. Nature 338, 39 (1989)] in which luciferase mRNA is driven by five copies of GAL4 UAS.
pL8G5-luc: LexA reporter plasmid in which luciferase mRNA is driven by eight copies of the LexA binding site and five copies of GAL4 UAS. [S. M. Hollenberg, R. Sternglanz, P. F. Cheng, H. Weintraub, Identification of a new family of tissue-specific basic Helix-Loop-Helix proteins with a two-hybrid system. Mol. Cell. Biol. 15, 3813 (1995)].
pMst: Gal4 expression vector driven by the SV40 promoter derived from pM (Clontech) by mutating the stop codon before the Gal4 DNA binding domain.
pML: LexA expression vector generated by replacing the Nhe I/Eco RI fragment of pM (Clontech) with the PCR amplified LexA-coding sequence.
4.2. APP plasmids
4.2.1 Mammalian expression plasmids
pMst-APPct, encodes APPct-Gal4. Generated by cloning the cytoplasmic tail of human APP695 (APPct, residues 652 to 695) into the Bam HI/Sal I sites of pMst (linker sequence between Gal4 and APPct = QLTVSPEFPGIPPGQYTSI).
pMst-APPct*, encodes APPct*-Gal4 corresponding to APPct-Gal4 with the NPTY mutation. Generated by cloning the mutant cytoplasmic tail from pMst-GV-APPct* into the Bam HI/Sal I sites of pMst.
pMst-APP, encodes APP-Gal4. Generated by cloning a PCR fragment containing the extracellular and transmembrane region of human APP695 (APPe, residues 1 to 651) into the Nhe I site of pMst-APPct (linker sequence between APPe and Gal4 = MLKKKPLASSRMKLLS).
pMst-APP*, encodes APP*-Gal4. Obtained as pMst-APP, but cloned into the Nhe I site of pMst-APPct*.
pMst-APP, encodes APP-Gal4. Generated by cloning a PCR fragment coding for a methionine and residues 639 to 651 of human APP695 into the Bgl II/Nhe I sites of pMst-APPct (linker sequence between TMR and Gal4 = MLKKKPLASSRMKLLS).
pMst-APP*, encodes APP*-Gal4. Obtained as pMst-APP, but cloned into pMst-APPct*.
pMst-AN encodes APPe-G-NRXc. Generated as pMst-GV-AN, but into pMst (linker sequences between APPe and Gal4 = MLKKKPLASSRMKLLS, and between Gal4 and NRXct = QLTVSPEFPGIPPGYKYRN.
pMst-AN-APPc32, encodes APP-G-NRX-APPc32. Generated from pMst-AN by inserting a PCR fragment encoding the COOH-terminal 32 residues of human APP695 (APPc32) into the rat neurexin Iß cytoplasmic tail (NRXct) between residue 425 and 427, with V426 deleted during the cloning (linker sequences between APPc32 and NRXct = EGSYHIDDAAVT, and between APPc32 and NRXct = EQMQNIDESRN.
pML-APPct, encodes APPct-LexA. Generated by replacing the Gal4 sequence (the Nhe I/Eco RI fragment) in pMst-APPct with the LexA-sequence (linker sequence between LexA and APPct = NGDWLEFPGIPPGQYTSI).
pML-APPct*, encodes APPct*-LexA. Generated as pML-APPct in pMst-APPct*.
pML-APP, encodes APP-LexA. Generated by cloning the extracellular and transmembrane region of human APP695 (APPe, residues 1 to 651) into the Nhe I site of pML-APPct (linker sequence between APPe and LexA = MLKKKPLAKMKALT).
pML-APP*, encodes APP*-LexA. Generated as pML-APP with pML-APPct*.
pCMV5-APP, encodes full-length human APP695 inserted into the blunted-Eco RI/Xba I sites of pCMV5.
pCMV5-APP*, encodes full length human APP695 containing point mutations in the cytoplasmic NPTY sequence. Generated by QuickChange site-directed mutagenesis (Stratagene) with pCMV5-APP.
4.2.2 Yeast two-hybrid plasmids
pBTM116-APP, encodes residues 648 to 695 of human APP695 cloned into the Bam HI/Sal I sites of the yeast two-hybrid bait vector pBTM116 using a PCR fragment.
pBTM116-APP* = pBTM116-APP in which the codons encoding the NPTY sequence in the APP cytoplasmic tail were mutated to NATA using QuickChange site-directed mutagenesis (Stratagene).
4.3. Fe65 plasmids
4.31 Mammalian expression plasmids.
pCMV5-Fe65: encodes full-length rat Fe65 (711 residues). Constructed by sub-cloning the 3 kb Sal I fragment from the yeast two-hybrid prey clone #P29 into the Sal I site of pCMV5.
pCMVMyc-Fe65(128-711): encodes residues 128 to 711 of Fe65. Generated by cloning the blunt-ended fragment from the rat Fe65 cDNA into the blunted Eco RI site in pCMVMyc.
pCMVMyc-Fe65(242-711): encodes residues 242 to 711 of Fe65. Generated by cloning the blunt-ended fragment from the rat Fe65 cDNA into the blunted Eco RI site in pCMVMyc.
pCMVMyc-Fe65(287-711): encodes residues 287 to 711 of Fe65. Generated by cloning the blunt-ended fragment from the rat Fe65 cDNA into the blunted Eco RI site in pCMVMyc.
pCMV5-Fe65(1-553): encodes Fe65PTB2, which lacks residues 554 to 711 of Fe65. Generated by introducing a stop condon into pCMV5-Fe65 after residue 553 with the QuickChange site directed mutagenesis kit (Stratagene).
pCMVMyc-Fe65PTB1: encodes Fe65PTB1 which lacks residues 314 to 440. Generated by sequentially cloning the PCR fragments encoding residues 441 to 711 and residues 1 to 313 of rat Fe65 into the Cla I and Mlu I sites, respectively, of pCMVMyc.
pCMV5-Fe65mW1: encodes Fe65mW1 point mutant in carrying substitutions W281F and P284A. Generated by QuickChange site directed mutagenesis (Stratagene) with pCMV5-Fe65 as template.
pCMV5-Fe65mW2: encodes Fe65mW2 point mutant in carrying substitution W260F. Generated by QuickChange site directed mutagenesis (Stratagene) with pCMV5-Fe65 as template.
pCMV5-Fe65mW3: encodes Fe65mW3 point mutant in carrying substitutions W260F, W281F and P284A. Generated by QuickChange site directed mutagenesis (Stratagene) with pCMV5-Fe65mW1 as template.
pCMV5-Fe65mW4: encodes Fe65mW4 point mutant in carrying substitutions Y270A, Y271A, and W272A. The insert (2.1kb) can be cut out by Hind III + Sal I double digestion.
pCMV5-Fe65mW5: encodes Fe65mW4 point mutant in carrying substitutions Y270A, Y271A, W272A, W281F, and P284A. The insert (2.1kb) can be cut out by Hind III + Sal I double digestion.
pcDNA3.1-N-HA-Fe65: encodes full-length rat Fe65 preceded by a hemagglutinin (HA) epitope. Obtained by subcloning the rat Fe65 cDNA into the blunted-Eco RI/Xba I sites of pcDNA3.1-N-HA.
4.3.2 Yeast two-hybrid plasmids
pLexN-Fe65(287-711), encodes residues 287 to 711 of Fe65 in the Sal I/blunted-Pst I sites in pLexN.
pLexN-Fe65(287-531), encodes residues 287 to 531 of Fe65 in the Bam HI/blunted-Sal I sites in pLexN. Insert (740 bp) can be cut out by Bam HI + Pst I double digestion.
pVP16-3-Fe65mW5, encodes the mW5 mutant of Fe65 (see pCMV vectors above). Was generated by cloning the blunted 2.1 kb Hind III/Xba I fragment from pCMV5-Fe65mW5 into the blunted-Not I/Xba I sites of the yeast prey vector pVP16-3. The insert can be cut out by Sal I.
4.4. Tip60 plasmids
4.4.1 pCMV expression plasmids
pCMVMyc-Tip60(63-454), encodes residues 63 to 454 of rat Tip60ß. Generated by cloning the 1.3 kb Eco RI fragment from yeast two-hybrid prey clone #B36 into the Eco RI site of pCMVMyc.
pCMVMyc-Tip60(63-454)*, encodes residues 63 to 454 of rat Tip60ß with a mutation in residues 257 to 260 (sequences: wild-type = NKSY; mutant = NASA). Generated by QuickChange site directed mutagenesis kit (Stratagene) with pCMVMyc-Tip60(63-454) as template.
pM-Tip60 encodes rat Tip60ß residues 63 to 454 preceded by the Gal4 DNA binding domain. Generated by subcloning the 1.3 kb Bam HI/Xba I fragment from prey clone #36 into the Bam HI/Xba I sites of pM.
pM-Tip60* encodes the same protein as pM-Tip60 with the inactivating mutation in residues 257 to 260. Generated by cloning the 1.3 kb Bam HI/Xba I fragment from pVP16-3-Tip60* into the Bam HI/Xba I sites of pM.
pCMVMyc-hTip60 encodes Myc-tagged full-length human Tip60. Obtained by subcloning the insert of EST IMAGE clone 2901054 into the Mlu I/Xba I sites of pCMVMyc.
pM-hTip60 encodes full-length human Tip60ß preceded by the Gal4-DNA binding domain. Obtained by cloning the blunted 1.6 kb Eco RI/Not I fragment from EST clone 2901054 into the blunted Eco RI site of pM. Insert can be cut out by Sal I.
pM-hTip60*, same as pM-hTip60 but with the inactivating mutation in residues 257 to 260. Generated by QuickChange site directed mutagenesis (Stratagene) with pM-hTip60 as template.
4.4.2 Yeast two-hybrid plasmids
Prey clone #B36 in pVP16-3; identified in yeast two-hybrid screens with pLexN-Fe65(287-711) as the bait in a P8 rat brain library. B36 encodes rat Tip60 corresponding to residues 63 to 454 of human Tip60ß, with a single amino acid change between human and rat sequences.
pVP16-3-Tip60* encoding mutant rat Tip60 with the inactivating mutation in residues 257 to 260 (sequences: wild-type = NKSY; mutant = NASA).Generated by cloning the Eco RI fragment from pCMVMyc-Tip60(63-454)* into the Eco RI site of yeast prey vector pVP16-3.
pVP16-3-hTip60, full-length human Tip60ß cloned into the Eco RI/Not I sites of pVP16-3.
4.4.3 GST-fusion protein plasmids
pGEX-KG-Tip60(63-454), residues 63 to 454 Tip60 fused to GST. Generated by cloning the 1.3 kb Eco RI fragment (1.3kb) of the yeast two-hybrid prey clone B36 into the Eco RI site of pGEX-KG.
pGEX-KG-Tip60(63-454)*, encodes residues 63 to 454 Tip60 fused to GST. Generated as pGEX-KG-Tip60(63-454), but from pVP16-3-Tip60(63-454)*.
4.5 Miscellaneous plasmids
4.5.1 pCMV expression plasmids
pCMV5-Mint-1: rat Mint1 cloned into the Eco RI site of pCMV5 [M. Okamoto, T. C. Südhof, J. Biol. Chem. 272, 31459-31464 (1997)].
pCS2 + MT-SEF: Myc-tagged full-length human LBP-1c (1 to 450 residues) was expressed from control of the CMV promoter [gift from W. S. L. Liao, University of Texas MD Anderson Cancer Center, Houston TX; reference: Z. Bing, S. A. G. Reddy, Y. Ren, J. Qin, W. S. L. Liao, J. Biol. Chem. 274, 24649 (1999)].
pcDNA3.1-PS2D366A (kind gift of C. Haass, Munich): encodes a dominant negative mutant of human presenilin 2 in pcDNA3.1.
4.5.2 Yeast two-hybrid plasmids
pVP16-3-LBP-1c, encodes full-length human LBP-1c. Generated by cloning the blunted 1.4 kb Xho I fragment from pCS2 + MT-SEF into the blunted Not I site of the yeast prey vector pVP16-3. Insert can by cut out by Xho I.
5. Immunoblotting and Immunoprecipitation Experiments
(see Web figs. 1 and 2)
COS7 cells were transfected in 60- or 100-mm dishes using DEAE-dextran or Fugene6 (Roche) with single or combinations of expression vectors encoding wild-type and mutant APPct-Gal4, APP-Gal4, and APP-Gal4, Myc-tagged or HA-tagged wild-type or mutant Fe65, and wild-type and mutant Tip60, and harvested 72 hours after transfection. Cell extracts were immunoblotted with polyclonal antibodies to the COOH-terminus of APP (U955) or to Fe65, and with monoclonal antibodies to Gal4 (Clontech) or to the Myc- (Santa Cruz Biotechnology Co.) or HA-epitope (BAbCo) to examine the size of APP cleavage products. For the immunoprecipitation experiments, cells were washed twice with cold PBS, harvested in 1 ml lysis buffer (50 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 10% glycerol, 1% IGEPAL CA-630, 1.5 mM MgCl2, 1 mM EGTA, 1 mM DTT, 0.1 g/L PMSF, 10 mg/liter Leupeptin, 10 mg/liter aprotinin, 1 mg/liter pepstatin A), and passed through a 28 guage needle 5×. Cell extracts were clarified by centrifugation at 20,800g for 10 min. The supernatants (~1 ml) were incubated with 10 l of a polyclonal antibody raised against the COOH-terminus of APP (U955) or monoclonal antibodies to Myc-tag (Santa Cruz) for 2 hours at 4°C, 60 l of a 50% slurry of protein A- or protein G-Sepharose (Pharmacia) were added, and the beads were incubated with the reactions for 1 hour at 4°C on a rotator and then collected by centrifugation. Beads were washed 3× with lysis buffer, resuspended in 0.1 ml SDS-PAGE sample buffer, and 20 l of the protein solutions were resolved on 10% SDS-PAGE, and detected by immunoblotting with antibodies to APP, Gal4, or the Myc-epitope.
Supplemental Figure 1. Immunoblotting analysis of COS cells transfected with APP-Gal4 fusion proteins. Gal4-fusion proteins with the cytoplasmic tail of APP starting either at the -cleavage site in the transmembrane region (APP-Gal4) or immediately after the transmembrane region (APPct-Gal4) and the full-length APP-Gal4 fusion protein (APP-Gal4) were expressed by transfection in COS cells. Proteins were then analyzed by immunoblotting using antibodies to the C-terminus of APP (anti-APPc) or to Gal4 (anti-Gal4). The positions of full-length APP-Gal4, of the 
/- and -cleavage products of APP-Gal4 (APP/-Gal4 and APP-Gal4, respectively) and of the cytoplasmic tail of APP-Gal4 (APPct-Gal4) are indicated on the right; numbers on the left show positions of molecular weight markers.
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Supplemental Figure 2. Fe65 lacking the first PTB domain that is inactive in transactivation assays (see Fig. 2) still binds to APP. Immunoprecipitations from COS cells cotransfected with Myc-tagged Fe65 lacking the first PTB domain (Myc-Fe65PTB1) and full-length wild-type APP-Gal4 or APP-Gal4 carrying a NPTY to NATA mutation in the cytoplasmic tail (APP*-Gal4) were performed with antibodies to the Myc-epitope (A) or to APP (B PIS, preimmune serum). Immunoprecipitates were analyzed by immunoblotting with antibodies to APP (A) and lower part of (B) or to Myc (upper part of B). The positions of the various proteins are indicated on the right of the immunoblots, and locations of molecular weight standards are shown on the left.
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Supplemental Figure 3. Fe65 stimulates transactivation by the -cleavage product of APP-Gal4 and by full-length APP-Gal4 as assayed in two different cell types. A comparison of the same experiment performed in two different cell lines is shown to illustrate the reproducibility of results between different types of cells. Note inhibition of stimulation by mutation of the Fe65-binding site in all instances.
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