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Supplemental Fig. S1 a) b) PsAPY1 (AB071369) MtAPY1;1 (AY180377) 1:MEFLIKLIT-FLLFSMPAITSSQYLGNNLLTSRKIFLKQEEISSYAVVFDAGSTGSRIHV 59 1:MEFLITLITTVLLLLMPAITSSQYLGNNLLTNRKIFQKQETISSYAVVFDAGSTGSRIHV 60 ***** *** ** **************** **** *** ******************* PsAPY1 (AB071369) MtAPY1;1 (AY180377) 60:YHFNQNLDLLHIGKGVEYYNKITPGLSSYANNPEQAAKSLIPLLEQAEDVVPDDLQPKTP 119 61:YHFDQNLDLLHIGKDVEFFNKITPGLSSYANDPEQAAKSLIPLLQQAENVVPIDLHHKTP 120 *** ********** ** ************ ************ *** *** ** *** PsAPY1 (AB071369) 120:VRLGATAGLRLLNGDASEKILQSVRDMLSNRSTFNVQPDAVSIIDGTQEGSYLWVTVNYA 179 MtAPY1;1 (AY180377) 121:IRLGATAGLRLLNGDASEKILQAVRDMFSNRSTFNVQPDAVSIIDGTQEGSYLWVTVNYA 180 ********************* **** ******************************** 50 kDa - * PsAPY1 (AB071369) 180:LGNLGKKYTKTVGVIDLGGGSVQMAYAVSKKTAKNAPKVADGDDPYIKKVVLKGIPYDLY 239 MtAPY1;1 (AY180377) 181:LGNLGKKYTKTVGVMDLGGGSVQMAYAVSKKTAKNAPKVADGVDPYIKKLVLKGKPYDLY 240 ************** *************************** ****** **** ***** PsAPY1 (AB071369) 240:VHSYLHFGREASRAEILKLTPRSPNPCLLAGFNGIYTYSGEEFKATAYTSGANFNKCKNT 299 MtAPY1;1 (AY180377) 241:VHSYLHFGREASRAEIMKVTRSSPNPCLLAGFDGTYTYAGEEFKAKAPASGANFNGCKKI 300 **************** * * ********** * *** ****** * ****** ** PsAPY1 (AB071369) 300:IRKALKLNYPCPYQNCTFGGIWNGGGGNGQKNLFASSSFFYLPEDTGMVDASTPNFILRP 359 MtAPY1;1 (AY180377) 301:IRKALKLNYPCPYQNCTFGGIWNGGGGNGQKHLFASSSFFYLPEDVGMVDPKTPNFKIRP 360 ******************************* ************* **** **** ** PsAPY1 (AB071369) 360:VDIETKAKEACALNFEDAKSTYPFLDKKNVASYVCMDLIYQYVLLVDGFGLDPLQKITSG 419 MtAPY1;1 (AY180377) 361:VDLVSEAKKACALNFEDAKSTYPFLAKKNIASYVCMDLIYQYVLLVDGF--DPLQEITSG 418 ** ** **************** *** ******************* **** **** PsAPY1 (AB071369) 420:KEIEYQDAIVEAAWPLGNAVEAISALPKFERLMYFV MtAPY1;1 (AY180377) 419:KEIEYQDAVLEAAWPLGNAVEAISSLPKFERMMYFV ******** ************** ****** **** 455 454 Fig. 1 Apyrase protein can be detected with the antibody generated against a mixture of synthetic peptides conserved in PsAPY1 and MtAPY1;1. a) Alignment of PsAPY1 (AB071369) and MtAPY1;1 (AY180377) proteins. Synthesized peptides (underlined) were injected into a rabbit to produce an antigen-specific antibody. b) Cell wall proteins from pea epicotyl tissues were separated on 12% SDS-PAGE gel and subjected to a western blot with the antibody. The blot was visualized with a ChemiDoc XRS (Bio-Rad), using ECL Plus Western Blotting Detection Reagents (GE Healthcare Life Science). Supplemental Fig. S2 a) 2138.1500 1412.7600 990.4900 1591.9000 2472.2500 1861.1000 2272.3100 1260.6200 b) 1 51 101 151 201 251 301 351 401 451 501 551 601 651 MASTTTMRLA QNKYPISNNR QTHEILINLR KKRGLNLSEI TIVADLDLMK PQGPGFQING KGYISELFVP IDTYVHSANG LIVRTIVTVG EDLHGKLVSA SKRKSYWTTE IPAIPAHPLL DTLAVWTKQN NFFERNPVLK LFSVLTLLSF LAFHYIGLDD IRSIVSDNIH VCSSFTMGWF IVEYHDRDIE HSVSWANWKF YQDPTEEFYF TPILLKNAIC NYDNVIDWEF NSIGIYHDHF TQTAKTESDA TEDDYPQIRG REIVNKDIVM TLSPRDVAWP HAVVSVTPLH PEKDHVLRYE NGYGFPILSV GEEKNVRTVR AVPTAENTEY HIGFDVRAGI KTFFDSGEFG VFEQYGNIMW KASGSIKPSI YIYYLDFDID KITIGLAPAE AFTNYNVWVT WHVVGIHHVP GCSN VQHPLDPLTK THPTLVSIPR DEQSLAIKLP LDCFMKESTV QVSKQSPPFG VISLASIYDL FGLSTVSLIP RHTENGIPNE ALSGILEIKG GTHNSFEKTS LVVVNPNIKT AYNRTEKWAG AQEDFPIMPL R.LAFHYIGLDDPEKDHVLR.Y R.YETHPTLVSIPR.K K.FHIGFDVR.A R.AGIVISLASIYDLEK.H K.GYISELFVPYQDPTEEFYFK.T K.ITIGLAPAELVVVNPNIK.T R.LIPAIPAHPLLTEDDYPQIR.G K.WAGGLYVDHSR.G EEFLAVQTIV KIFVVAIINS LKYPPFIDSV NIYVRPITGI PKQHSLTSHQ EKHKSRRVLY NRDCPPHAQF SIEESRTEVN TNIKHKDEIK LKTVRIKDGS AVGNEVGYRL GLYVDHSRGD LSTSFELRPT Fig. 2 Identification of p74 protein as a copper amine oxidase. a) MALDI-TOF-MS spectrum data for the tryptic peptides. b) Amino acid sequence of copper amine oxidase (CuAO) of pea. Matched peptides are underlined. Supplemental Fig. S3 Bait Prey PsAPY1 PsCuAO PsCuAO PsAPY1 PsAPY1 - PsAPY1 PsCuAO - PsCuAO SD (-His/-Lue/-Trp) YPDA Fig. 3 Ecto-apyrase PsAPY1 from pea interacts with a copper amine oxidase PsCuAO in yeast. The coding regions for PsAPY1 (GenBank accession AB071369; Kawahara et al. 2003) and PsCuAO (GenBank/ENBL data bank accession L39931; Tipping and McPherson 1996) were amplified by PCR and ligated into pGBKT7 as a bait and pGADT7 as a prey, respectively, and tested for the interaction using Saccharomyces cerevisiae strain AH109, according to the manufacturer’s protocol. The transformed S. cerevisiae expressing both PsAPY1 and PsCuAO successfully grows on selective media (SD/-His/-Lue/-Trp). Note that the interaction was detected by reciprocal bait–prey pairing.