Supplemental Table 1. List of PCR primers

Transcription

1 Supplemental Table 1. List of PCR primers Primer Sequences (5 to 3 ) 1 TATCCATGGCGCCGGCGGCGAGGGCGGAG 2 TATAAGCTTCTTGGCGTGTCCAGCCCACGGGGCGTAGAACTCGAC 3 ATAAAGCTTGCTCCAGAGTATGAGAAAGCT 4 ATACTCGAGGAGCTCATCCTTGAGAGGCTC 5 GAATTCTATGCACCTTGGGCCGGACACGCCAAGAAGCTGGCTCC 6 GGAGCCAGCTTCTTGGCGTGTCCGGCCCAAGGTGCATAGAATTC 7 TAATACGACTCACTATAGG 8 CCATGGGCTGGGCACGCACAGCAGCTGACGCCGATT 9 TGCGTGCCCAGCCCATGGCGCGAAGAACTCCACCAG 10 CCGCTCGAGCGGTCACAACTCGTCATTTACTGGA 11 TGCGTGCCCAGCCCACGGTGCAAAGAACTCAACAAT 12 CCGTGGGCTGGGCACGCAAAGAAATTGGCACCTGAA 13 CTGCTGGAGTTCGTGACC 14 GCAATGGCAGCAGAAGCAGCTTTCTCTTCCATGGCGT 15 GCTGCTTCTGCTGCCATTGCCTTTGTATCTTTCCTTC 16 GCAACGCAATTGCTTGCAAT PDIL1;1 a domain mutagenesis Two PCR reactions were conducted with the primers 1 and 2, and primers 3 and 4 using an EST clone (accession No. C62870) encoding PDIL1;1 as template. The two PCR products were purified and inserted into an appropriate vector by a three-fragment-ligation reaction. PDIL1;1 aʹ domain mutagenesis Two mutagenic primers 5 and 6 were used to introduce mutations at the CGHC site in the aʹ domain by QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer s instructions. PDIL2;3 a domain mutagenesis Two PCR reactions were conducted with the primers 7 and 8, and primers 9 and 10 using an expression vector (pet23d) that contains wild-type PDIL2;3 as template. The two PCR products were purified, mixed, and combined by another PCR reaction with the primers 7 and 10. PDIL2;3 aʹ domain mutagenesis Two PCR reactions were conducted with the primers 7 and 11 and with primers 12 and 10 using an expression vector (pet23d) that contains wild-type PDIL2;3 as template. The two PCR products were purified, mixed, and combined by another PCR reaction with the primers 7 and 10. PDIL2;3 b domain mutagenesis Two PCR reactions were conducted with the primers 13 and 14 and with primers 15 and 16 using a vector that contains spgfp-pdil2;3 and the Gt1 terminator as template. The two PCR products were purified, mixed, and combined by another PCR reaction with the primers 13 and 16. 1

2 Hs PDI At PDIL1;2 Os PDIL1;2 Os PDIL1;3 Hs ERp57 At PDIL1;1 At PDIL1;3 At PDIL1;4 Os PDIL1;1 Zm PDIL1;2 Zm PDIL1;1 Os PDIL1;4 Zm PDIL1;3 Zm PDIL1;4 At PDIL2;3 At PDIL2;2 Zm PDIL2;3 Os PDIL2;3 At PDIL2;1 Os PDIL2;1 Zm PDIL2;2 Os PDIL2;2 Zm PDIL2;1 Hs P5 0.2 Supplemental Figure 1. Phylogenetic tree of PDI proteins. The amino acid sequences of representative PDI family oxidoreductases in Homo sapience (Hs), Arabidopsis thaliana (At), Zea mays (Zm), and Oryza sativa (Os) were aligned with the CLUSTALW program using the BLOSUM matrix method. The phylogenetic tree was constructed by the Neighbor-Joining method using MEGA version 4 (Tamura et al., 2007; The reliability of different phylogenetic groupings was evaluated by the bootstrap test (1000 replicates). The corresponding alignment is provided in Supplemental Dataset 1. Hs PDI, NM_000918; Hs ERp57, NM_005315; Hs P5, NM_005742; At PDIL1;1, NM_102024; At PDIL1;2, NM_106400; At PDIL1;3, NM_115353; At PDIL1;4, NM_180903; At PDIL2;1, NM_130315; At PDIL2;2, NM_100376; At PDIL2;3, NM_128852; Zm PDIL1;1, AY739284; Zm PDIL1;2, AY739285; Zm PDIL1;3, AY739286; Zm PDIL1;4, AY739287; Zm PDIL2;1, AY739288; Zm PDIL2;2, AY739289; Zm PDIL2;3, AY739290; Os PDIL1;1, AK068268; Os PDIL1;2, AY739308; Os PDIL1;3, AK073161; Os PDIL1;4, AK071514; Os PDIL2;1, AK103944; Os PDIL2;2, NM_183927; Os PDIL2;3, AK

3 Supplemental Data. Onda et al. (2011). Plant Cell /tpc B WT PDIL2;3-KD W T PD IL 2; 3KD A PDIL2;3 PDIL1;1 BiP Supplemental Figure 2. Immunolabeling of crp10 and cpp13 in PB-I of the wild-type and PDIL2;3-KD endosperm. (A) Immunoblot analyses of proteins extracted from mature seeds of wild-type (WT) or PDIL2;3 knockdown (PDIL2;3-KD). Proteins were separated by SDSPAGE and subjected to immunoblotting with the indicated antibodies. (B) The left and right panels show PB-I in the wild-type and PDIL2;3-KD endosperm, respectively. The indicated parts in the upper panels are enlarged (lower panels). The black and white arrowheads indicate crp10 (5-nm gold particles) and cpp13 (15-nm gold particles), respectively. Bars = 200 nm.

4 Hs P5 MALLVLGLVSCTFFLAVNGLYSSSDDVIELTPSNFNREVIQSDSLWLVEFY 51 PDIL2;3 MRPAVAAALLLVAAAVAASPVSALYSAGSPVLQFNPNNFKSKVLNSNGVVLVEFF 55 Hs P5 APWCGHCQRLTPEWKKAATALKDVVKVGAVDADKHHSLGGQYGVQGFPTIKIFGS 106 PDIL2;3 APWCGHCQQLTPIWEKAAGVLKGVATVAALDADAHKELAQEYGIRGFPTIKVFVP 109 **** Hs P5 NKNRPEDYQGGRTGEAIVDAALSALRQLVKDRLGGRSGGYSSGKQ--GRSDSSSK 159 PDIL2;3 GK-PPVDYQGARDVKPIVEFALSQVKALLRDRLNGKTSAGSGGKKSGGSSEKTEP 164 Hs P5 KDVIELTDDSFDKNVLDSEDVWMVEFYAPWCGHCKNLEPEWAAAASEVKEQTKGK 214 PDIL2;3 SASIELNSQNFDKLVTKSKDLWIVEFFAPWCGHCKKLAPEWKKAA----KNLKGQ 215 **** Hs P5 VKLAAVDATVNQVLASRYGIRGFPTIKIFQKG-ESPVDYDGGRTRSDIVSRALDL 268 PDIL2;3 VKLGHVDCDAEKSLMSKYKVEGFPTILVFGADKESPFPYQGARVASAIESFALEQ 270 Hs P5 FSDNAPPPELLEIINEDIAKRTCEEHQLCVVAVLPHILDTGAAGRNSYLEVLLKL 323 PDIL2;3 LEANAAPPEVSELTGPDAMEEKCASAAICFVSFLPDILDSKAEGRNKYLELLLSV 325 Hs P5 ADKYKKKMWGWLWTEAGAQSELETALGIGGFGYPAMAAINARKMKFALLKGSFSE 378 PDIL2;3 AEKFKKSPYSFVWTAAGKQADLEKQVGVGGYGYPAMVALNVKKGAYAPLRSAFQL 380 Hs P5 QGINEFLRELSFGRGSTAPVGGGAFPTIVEREPWDGRDGELPVEDDIDLSDVELD 433 PDIL2;3 DEITEFVREAGRGGKGNLPLDG--TPTIVQSEPWDGKDGEVIEEDEFSLEELMAD 433 Hs P5 DLG-KDEL 440 PDIL2;3 NSPVNDEL 441 Supplemental Figure 3. Sequence alignment of human P5 and rice PDIL2;3. The sequences of rice PDIL2;3 (AK062254) and human P5 (NM_005742) were aligned using the CLUSTALW program. The sequences of TRX-like domains, a (24-128, pink), a' ( , purple), and b ( , light blue) of human P5 are indicated (Ferrari and Soling, 1999). The redox-active sites (CGHC) are indicated by asterisks. The conserved Arg residues that may modulate the pk a values of the active Cys residues of P5 are indicated with arrows (Lappi et al., 2004; Karala et al., 2010). Acidic amino acid residues in the C-terminal tail are highlighted in red. Arrowheads show the predicted cleavage sites of the signal peptides.

5 Os PPEVSELTGPDAMEEKCASAAICFVSFLPDILDSKAEGRNKYLELLLSVAEKFKKSPYSF 336 Zm PAEVSELTGPDVMEEKCASAAICFVSFLPDILDSKAEGRNKYLELLLSVAEKFKKSPYSF 334 Ta PPEVSELTSADVMEEKCASAAICFVSFLPDILDSKAEGRNKYLELLLSVAEKFKKSPYSF 335 Gm PPEVTELHSPDVLEEKCGSAAICFVAFLPDILDSKAEGRNIYLQQLLSVAEKFKRSPYSY 333 At PVEVTELTGPDVMEKKCGSAAICFISFLPDILDSKAEGRNKYLEMLLSVAEKFKKQPYSF 333 Pp APEVVELVGQDVLDKECGSAAICFVSFLPDILDSKAEGRNKYLATLRNVAEKYKRNAY Rn PPELLEIINEDIAKKTCEEHQLCVVAVLPHILDTGATGRNSYLEVLLKLADKYKKKMWGW 339 Mm PPELLEIINEDIAKKTCEEHQLCVVAVLPHILDTGAAGRNSYLEVLLKLADKYKKKMWGW 339 Hs PPELLEIINEDIAKRTCEEHQLCVVAVLPHILDTGAAGRNSYLEVLLKLADKYKKKMWGW 334 Xl PPEINEILNGDIVKKTCDEHQLCIVAVLPHILDTGAAGRNSYLEVMLKMAEKYKKKMWGW 336 Dm APELIEIINESTFETACEGKPLCVVSVLPHILDCDAKCRNKFLDTLRTLGEKFKQKQWGW 330 Ce APEVFEGINQQVVEDACKEKQLCIFAFLPHILDCQSECRNNYLAMLKEQSEKFKKNLWGW 337 Os VWTAAGKQADLEKQVGVGGYGYPAMVALNVKKGAYAPLRSAFQLDEITEFVRE 389 Zm VWTAAGKQANLENQVGVGGYGYPAMVALNVKKGAYAPLRSAFQRDEIIEFVKE 387 Ta VWAGAGKQADLEKQVGVGGYGYPAMVALNVKKGAYAPLRSAFELAEITEFVKE 388 Gm VWVAAGNQPDLEKNVGVGGYGYPALVALNLKKAVYAPLKSAFELDQIIEFVKE 386 At MWVAAVTQMDLEKRVNVGGYGYPAMVAMNVKKGVYAPLKSAFELQHLLEFVKD 386 Pp RQPDLEKAVGVGGFGYPAMVALNVKKAVYAPLRGAFEQEPVMKFVAE 375 Rn LWTEAGAQYELENALGIGGFGYPAMAAINARKMKFALLKGSFSEQGINEFLRE 392 Mm LWTEAGAQYELENALGIGGFGYPAMAAINARKMKFALLKGSFSEQGINEFLRE 392 Hs LWTEAGAQSELETALGIGGFGYPAMAAINARKMKFALLKGSFSEQGINEFLRE 387 Xl LWTEAGAQMDLETSLGIGGFGYPAMAAINARKIKFALLKGSFSEQGINEFLRE 389 Dm AWAEGGQQLALEESLEVGGFGYPAMAVVNFKKMKFSVLKGSFSKDGINEFLRD 383 Ce IWVEGAAQPALEESFEVGGFGYPAMTALNFRKNKYAVLKGSFGKDGIHEFLRD 390 Supplemental Figure 4. Sequence alignment of the b domain of the P5 subfamily oxidoreductases. The absolutely conserved Cys residues of CX 5 C motif are indicated with a red line above the sequence. The listed 12 sequences are from Oryza sativa (Os, AK062254), Zea mays (Zm, AY739290), Triticum aestivum (Ta, AK334521), Glycine max (Gm, AK244958), Arabidopsis thaliana (At, NM_128852), Physcomitrella patens (Pp, XM_ ), Rattus norvegicus (Rn, NM_ ), Mus musculus (Mm, AK168279), Homo sapiens (Hs, NM_005742), Xenopus laevis (Xl, NM_ ), Drosophila melanogaster (Dm, AY061349) and Caenorhabditis elegans (Ce, NM_076789). The sequences were aligned using the CLUSTALW program. White-on-black letters indicate amino acid residues conserved in more than 9 sequences.

6 Molecular mass (kda) Th Fe GST-PDIL2;3(ama'mb) PDIL2;3(ama'mb) PDIL2;3(AX5A) Time (min) Al Co Supplemental Figure 5. Size exclusion chromatography of GST- PDIL2;3(ama'mb), PDIL2;3(ama'mb), and PDIL2;3(AX5A). Thyroglobulin (Th, MW 669,000), ferritin (Fe, MW 440,000), aldolase (Al, MW 158,000), and conalbumin (Co, MW 75,000) were loaded on a Superdex 200 column and used as standards to estimate the molecular masses of the purified proteins. Calculated molecular masses of GST-PDIL2;3(ama'mb), PDIL2;3(ama'mb) and PDIL2;3(AX5A) are 72, Da, 45, Da, and 46, Da, respectively. The major peaks of GST-PDIL2;3(ama'mb), PDIL2;3(ama'mb), and PDIL2;3(AX5A) were detected at min, min, and min, respectively. Approximate molecular masses are estimated 316 kda for GST- PDIL2;3(ama'mb) and 208 kda for PDIL2;3(ama'mb) and PDIL2;3(AX5A).

7 Supplemental Data. Onda et al. (2011). Plant Cell /tpc spgfp-pdil1;1 spgfp-pdil2; Rhodamine B esp2 (pdil1;1) WT + OsTIP3-GFP Merged esp2 A spdsred-pdil1;1 T S P T S P T S P T S P pgt αgt Glb βgt PB-I PB-I PB-II PB-II Supplemental Figure 6. Complementation analysis of esp2, a null mutant of PDIL1;1. (A) Proteins extracted from mature seeds of the WT (lanes 1-3), esp2 (lanes 4-6) and esp2 expressing spdsred-pdil1;1 (lanes 7-9) or spgfp-pdil2;3 (lanes 10-12) under the control of rice TIP3 promoter were fractionated into the S and P fractions (see METHODS for detail). Total proteins (lanes T) were extracted under reducing condition. Proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. pgt, proglutelins; αgt, glutelin acidic subunit; Glb, α-globulin; βgt, glutelin basic subunit. Proglutelins were efficiently extracted from the wild-type seeds without a reducing agent (arrowhead), whereas extraction of proglutelins from the esp2 seeds required a reducing agent. Note that proglutelins were efficiently extracted without a reducing agent from the esp2 seeds expressing spgfp-pdil1;1. However, the solubility of proglutelins did not change in the esp2 seeds expressing spgfppdil2;3. (B) Confocal fluorescence images of the Rhodamine-stained esp2 endosperm expressing a PB-II membrane marker rice TIP3-GFP alone (upper panels) or expressing both spdsred-pdil1;1 and TIP3-GFP (lower panels). The fluorescence signals of DsRed-PDIL1;1 are not visible under the conditions for obtaining Rhodamine signals at the appropriate intensity. Bars = 5 µm.

8 Supplemental References : Ferrari DM, Soling HD. (1999) The protein disulphide-isomerase family: unravelling a string of folds. Biochem J. 339 : Karala AR, Lappi AK, Ruddock LW. (2010) Modulation of an active-site cysteine pka allows PDI to act as a catalyst of both disulfide bond formation and isomerization. J Mol Biol. 396 : Lappi AK, Lensink MF, Alanen HI, Salo KEH, Lobell M, Juffer AH, Ruddock LW. (2004) A conserved arginine plays a role in the catalytic cycle of the protein disulphide isomerases. J Mol Biol. 335 : Tamura K, Dudley J, Nei M, Kumar S. (2007) MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24 :