Download a) b) Fig. 1 - Springer Static Content Server

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.