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1 R2A MASNSEKNPLL-SDEKPKSTEENKSS-KPESASGSSTSSAMP---GLNFNAFDFSNMASIL 56 R2B MASSSEKTPLIPSDEKNDTKEESKSTTKPESGSGAPPSPS-PTDPGLDFNAFDFSGMAGIL 60 R2A NDPSIREMAEQIAKDPAFNQLAEQLQRSIPNAGQEGGFPNFDPQQYVNTMQQVMHNPEFK 116 R2B NDPSIKELAEQIAKDPSFNQLAEQLQRSVPTGSHEGGLPNFDPQQYMQTMQQVMENPEFR 120 R2A TMAEKLGTALVQDPQMSPFLDAFSNPETAEHFTERMARMKEDPELKPILDEIDAGGPSAM 176 R2B TMAERLGNALVQDPQMSPFLEALGNPAASEQFAERMAQMKEDPELKPILAEIDAGGPSAM 180 R2A MKYWNDPEVLKKLGEAMGMPVAGLPDQTVSAEPEVAEEGEEEESIVHQTASLGDVEGLKA 236 R2B MKYWNDKDVLAKLGEAMGIAVGA--DQTVAAEPEEAEEGEEEESIVHQTASLGDVEGLKA 238 R2A ALASGGNKDEEDSEGRTALHFACGYGELKCAQVLIDAGASVNAVDKNKNTPLHYAAGYGR 296 R2B ALASGGNKDEEDSEGRTALHFACGYGEVRCAQVLLDAGANANAIDKNKNTPLHYAAGYGR 298 R2A KESVSLLLENGAAVTLQNLDEKTPIDVAKLNSQLEVVKLLEKDAFL 342 R2B KECVSLLLENGAAVTQQNMDNKNPIDVARLNNQLDVVKLLEKDAFL 344 Figure S1 Alignment of AKR2A and AKR2B amino acid sequences. The amino acid sequences of AKR2A (At4g35450) and AKR2B (At2g17390) were aligned. Gaps were introduced to maximize the alignment. Nonidentical amino acids are shown in red. 1

2 OEP7 MGKTSGAKQATVVVAAMALGWLAIEIAFKPFLDKFRSSIDKSDPTKDPDDFDTAATATTSKEGL OEP7(1-28)[7G] MGKTSGAKQATVVVAAMALGWLAIEIAFGGGGGGG OEP7[12I] MGKTSGAKIIIIIIIIIIIIWLAIEIAFKPFLDKFRSSIDKSDPTKDPDDFDTAATATTSKEGL OEP7[12F] MGKTSGAKFFFFFFFFFFFFWLAIEIAFKPFLDKFRSSIDKSDPTKDPDDFDTAATATTSKEGL coep64(1-29) MASQAANLWVLIGLGLAGILMLTKKLKKT coep64(1-29)[6g] MASQAANLWVLIGLGLAGILMLTGGGGGG Toc33 Toc34 KGKKLIPLIIGAQYLIVKMIQGAIRNDIKTSGKPL RGKKLIPLMFAFQYLLVMKPLVRAIKSDVSRESKLAWELRDSGLASRRS H + -ATPase( ) AKIRGIGWGWAGVIWLYSIVTYFPLDVFKFAIRYILSGK PMP22 NYKYVPLHFRVILHSLVAFFWGIFLTLRARSMTLALAKAK moep64 MSNTLSLIQSNASNPKVWVVIGVTVAGIVILAETRKRRIRA Tom20-2 NTEFTYDVCGWIILACGIVAWVGMA Figure S2 Amino acid sequences of the constructs used in in vitro pull-down assays. The numbers in parentheses denote amino acid residues. The amino acid residues that were replaced in the TMD of OEP7 or the CPRs of OEP7 and coep64 are underlined. To generate GST or GFP fusion proteins, these sequences were fused in-frame to the C-terminus of GST or GFP. 2

3 a GST GST:AKR2A AKR2A MBP MBP:OEP7 OEP7 b GST:AKR2 GST MBP:OEP7 MBP GST:OEP7 His:AKR2A (kd) c Input GST+ MBP GST:AKR2A+ MBP GST+ MBP:OEP7 GST:AKR2A + MBP:OEP7 64 kd Anti-GST Figure S3 AKR2A interacts with OEP7 in vitro. (a) Schemes of the constructs. (b) Purification of recombinant proteins used in the pull-down assays. His-, GST- and MBP-tagged recombinant proteins were purified from E. coli extracts by Ni + -NTA, glutathione agarose, or amylose resin affinity column chromatography, respectively. The purified proteins were separated by SDS/PAGE and the gels were stained with Coomassie blue. (c) Interaction of AKR2A with OEP7 in vitro. Protein pull-down experiments were performed using affinity-purified GST:AKR2A (5 µg) and MBP:OEP7 (5 µg) as prey and bait, respectively, in buffer containing 100 mm NaCl. The proteins were precipitated with amylose resin and the pelleted proteins were subjected to Western blot analysis using an anti- GST antibody. Input, 10% of total prey. 3

4 a WT akr2b LP + RP 900 bp LP + LB 410 bp b T-DNA LB ATG 500 bp TGA LP RP c WT akr2b Anti-AKR2A Anti-actin d AKR2A AKR2B 1029/1035 bp 18S rrna 18S rrna 180 bp Figure S4 Isolation of akr2b mutants. (a) A putative mutant with a T-DNA insertion in AKR2B was confirmed by PCR using T-DNA left border and genespecific primers. Total genomic DNA was isolated from wild type (WT) and akr2b plants and used as a template for PCR. The gene-specific fragment was amplified by PCR using gene-specific primers. LP, forward primer of AKR2B; RP, reverse primer of AKR2B; LB, left border primer of T-DNA. The gene-specific primers produced a 900 bp fragment from WT plants, but not from akr2b plants. Instead, LB and LP produced a 410 bp fragment from akr2b plants. (b) Schematic representation of the T-DNA insertion. Red boxes, coding regions; blue boxes, 5 and 3 untranslated regions. The PCR product obtained by LP and LB was sequenced to determine the orientation of T-DNA insertion and the insertion is indicated. (c) AKR2 protein levels in wild-type and akr2b plants were determined using anti- AKR2A antibody. Actin levels were used as loading control. (d) To compare the transcript levels of AKR2A and AKR2B, total RNA prepared from wild-type plants were used for semi-quantitative RT-PCR. 18S rrna was included as internal control. The primers used for PCR amplification were 5 5 -ATGGCTTCCAATTCGGAGAA-3 and 5 -TCAAAGGAAAGCATCCTTCTC- 3 for AKR2A and 5 -ATGGCTTCAAGCTCAGAGAA-3 and 5 - TCAGAGGAAAGCGTCCTTC-3 for AKR2B. 4

5 Wild-type Untransformed T7:AKR2A Wild-type HA:AKR2B T7:AKR2A HA:AKR2B akr2b 45 kd HA:AKR2B T7:AKR2A AKR2A 31 kd Control serum Anti-AKR2A Anti-T7 Anti-HA Anti-AKR2A Figure S5 Generation of an anti-akr2 antibody. Full length His:AKR2A purified from E. coli extracts by Ni-NTA agarose column chromatography was used to raise an antibody in rabbits. The antibody was affinitypurified from total serum using the antigen. To determine the specificity of the antibody, extracts of protoplasts transformed with T7:AKR2A and HA:AKR2B were analyzed by Western blotting using anti-akr2a, anti- T7 and anti-ha antibodies. The protein extracts obtained from akr2b plants were also tested. Control serum was included in the Western blot analysis. Note that the anti-akr2a antibody also cross-reacted with AKR2B weakly. 5

6 His:AKR2A : His:AKR2A-C : (µg) (µg) (kd) 45 Anti-His BSA : His:AKR2A-C : BSA (kd) 66 Anti-His 18 Figure S6 Competition for AKR2A binding to chloroplasts. Purified chloroplasts (equivalent to 20 µg chlorophyll) were incubated with the indicated amount of His:AKR2A and His:AKR2A-C (top panel) or His: AKR2A and BSA (bottom panel), and the chloroplast-bound proteins were analyzed as described in the Methods. BSA was detected by Coomassie blue staining. 6

7 R6 T7:AKR2A R6 T7:AKR2A : OEP7:GFP 37 kd Anti-GFP 38 kd Anti-T7 Coomassie (RbcL) 6 h 18 h Figure S7 The apparent modification of OEP7 proteins is decreased by coexpression of AKR2A in protoplasts. Protoplasts were transformed with the indicated constructs and extracts prepared at the indicated times after transformation were analyzed by Western blotting using anti-gfp and anti-t7 antibodies. The gel was stained with Coomassie blue to evaluate loading. R6, control vector without coding region. 7

8 Figure S8 Full scans 8

9 Figure S8 continued 9

10 Supplementary Table S1. Nucleotide sequences of primers Name of primers Sequence H-AKR2-F ( 5 -CTCGAGATGGCTTCCAATTCGGAG-3' H-AKR2-R 5 - TCAAAGGAAAGCATCCTT-3 H-AKR2N-R 5 -TCATGCTACCTCAGGTTCAGC-3 H-AKR2N1A-R 5 -TCACGCATTAACACTTGCTCC-3 RNAi-5B 5'-GGATCCCCCAAATCGACGGAGGAGAATA-3' RNAi-3C 5'-ATGCATCTCAGGGTTATGCATAACCTG-3' RNAi-5X 5'-CTCGAGCCCAAATCGACGGAGGAGAATA-3' RNAi-K 5'-GGTACCCTCAGGGTTATGCATAACCTG-3' AKR-RT 5 -GAAGAAGGTGAAGAAGAAGAG-3 AKR2B-5 5'-ATGGCTTCAAGCTCAGAGAA-3' AKR2B-3 5'-TCAGAGGAAAGCGTCCTTC-3' AKR2A2-F 5 -CTCGAGGAAGAAGGTGAAGAAGAAG-3 G-O7-F 5 -CCCGGGC GATGGGAAAAACTTCGG-3 G-O7-R 5 -TCAAAGGAAAGCATCCTT-3 G-O7(20-64)-F 5 -CCCGGGTAGGATGGTTAGCCATAG-3 G-O7(1-28)-R 5 -TTAGAAAGCGATCTCTATGGC -3 G-O7(1-35)-R 5 -TTAGAATTTATCGAGGAAAGG-3 R-AKR2A-F 5 -CCCGGGCAATGGCTTCCAATTCGGAG-3 R-AKR2A-R 5 -TCAAAGGAAAGCATCCTT-3 HATP-F 5'-CCCGGGCAAAGATTAGGGGTATT-3 HATP-R 5 -CTCGAGCTTTCCGCTCAAGATGTA-3 G-Toc33-5 5'-CCCGGGAAAGGAAAGAAACTCATC-3' G-Toc33-3 5'-CTCGAGTTAAAGTGGCTTTCCACT-3' G-Toc34-5 5'-CCCGGGAGAGGAAAAAAACTGATT-3' G-Toc34-3 5'-CTCGAGTCAAGACCTTCGACTTGC-3' G-cT TCTAGAATGGCGTCTCAAGCTGCG-3 G-cT GGATCCCGGTCTTCTTCAGCTTTT-3 G-PM-5 5'-AACTACAAGTATGTGCCACTGC-3' G-PM-3 5'-TTCTCTAACAAGGGTCCAGGGAGTTCT-3' G-mT64-5 5'-ATGTCGAATACGCTTTCTTTGAT-3' G-mT64-3 5'-GGCTCTAATCCGCCGCTTCCGGG-3' G-Tom-5 5'-CCCTCGAGTTATCTGGCAGGAGGTGG-3' G-Tom-3 5'-CCCCCGGGCTTATGATGTATGCGGTTG-3' Toc GCTCTAGATGGCTAGCATGACTGGTGGACAGCAAATGGTTA GATCCCCGCCTCTC-3 Toc TCCCCCGGGTTAGTACATGCTGTACTTGTCG-3 PM-5 5'-ATGGGATCTTCACCACCGAAG-3' PM-3 5'-TCACTTAGCCTTTGCCAAAGCT-3' H-O7-R1 5 -ATCTGGAACATCGTATGGGTAACCCTCTTTGGATGTGG-3 H-O7-R2 5 -CTCGAGTTAAGCGTAATCTGGAACATCGTATGG ATGATAACTCGACGGATCGC CCTCCAATGGATCCTCGTA-3 LB 5 -GCGTGGACCGCTTGCTGCAACT-3 LP 5 -ACGGTTGGCATGTTAAAAATC-3 RP 5 -GAAGGACTGCTTTGCATTTTG-3

11 Supplemental Materials Supplemental Methods Two-hybrid screening. The full-length coding region of OEP7 was cloned into the pas2-1 vector (Clontech) and used as bait. Screening was performed using an Arabidopsis cdna library. Transformed yeast cells were grown on SD/leu - /trp - agar plates supplemented with 2% glucose and streaked on SD/leu - /trp - agar plates supplemented with 2% galactose. To detect galactosidase expression, a Whatman filter soaked with lysis buffer (50 mm Tris-HCl, ph 8.0, 1.0 mm MgCl 2, 50 mm NaCl, 30 mm -mercaptoethanol) supplemented with 1 mm X-gal and 20 μg/ml zymolyase was placed on the plate. Construction of fusion proteins The construct used for yeast two hybrid screening was generated by sub-cloning the fulllength OEP7 coding region into the pas2-1 vector (Clontech) digested with EcoR I and BamH I. The constructs used for the in vitro pull-down assays were generated by subcloning PCR products into the Xho I and Hind III sites of the prset-a vector (Invitrogen) for His tagging, and the Xma I and Xho I sites of the pgex-5x-3 vector (Amersham Biosciences) for GST tagging. The PCR primers (see Table S1) are as follows: H-AKR2-F and H-AKR2-R for His:AKR2A(F); H-AKR2-F and H-AKR2N-R for His:AKR2A-N1; H-AKR2-F and H-AKR2N1A-R for His:AKR2A-N2; AKR2A2-F and H-AKR2-R for His:AKR2A-C; G-O7-F and G-O7-R for GST:OEP7(F); G-O7(20-

12 64)-F (5 -CCCGGGTAGGATGGTTAGCCATAG-3 ) and G-O7-R for GST:OEP7(20-64); G-O7-F and G-O7(1-28)-R for GST:OEP7(1-28); G-O7-F and G-O7(1-35)-R for GST:OEP7(1-35); R-AKR2A-F and R-AKR2A-R for RFP:AKR2A; HATP-F and HATP-R for GST:H + -ATPase (aa ). G-Toc33-5 and G-Toc33-3 for GST:Toc33, G-Toc34-5 and G-Toc34-3 for GST:Toc34, G-cT64-5 and G-cT64-3 for GST:cOEP64(1-29); G-PM-5 and G-PM-3 for GST:PMP22, G-mT64-5 and G-mT64-3 for GST:mOEP64 (moep64, formerly mitochondrial Toc64, At5g09420), G-Tom-5 and G-Tom-3 for GST:Tom20-2. MBP:OEP7 was generated by sub-cloning full length OEP7 into the pmal-c2 vector (New England BioLabs) digested with EcoR I, while Hind III T7-tagged AKR2A deletion mutants were generated by sub-cloning AKR2A fragments into the T7-tagging vector digested with BamH I and Hind III. To construct OEP7:HA, the OEP7 coding region was amplified by PCR using the primers G-O7-F, H-O7-R1, and H-O7-R2. The product was then ligated into pbluescriptii KS(+) (Stratagene). Constructs used in in vitro transcription and translation were generated by sub-cloning the Xba I and EcoR I fragments of GFP, OEP7:HA and T7:AKR2A into pbluescriptii KS(+) (Stratagene) digested with Xba I and EcoR I. To construct NLS:RFP:AKR2A, AKR2A was C- terminally fused to NLS:RFP (1). To construct HA:AKR2B, AKR2B (At2g17390) cdna was PCR amplified using gene-specific primers AKR2B-5 and AKR2B-3 and the resulting PCR product was ligated to a vector containg HA epitope. Expression of recombinant proteins.

13 Recombinant GST- and His-tagged AKR2A and MBP-fused OEP7 proteins were expressed in E. coli strain BL21(DE3)LysS. The GST-, His- and MBP-fusion proteins were purified by affinity column chromatography using glutathione-agarose beads (Amersham Pharmacia Biotech, Buckinghamshire, UK), Ni + -NTA (Qiagen, Hilden, Germany), or amylose resin (New England BioLabs), respectively, according to the manufacturers protocols. Isolation of the akr2b mutant A putative mutant with a T-DNA insertion in AKR2B was isolated from a SALK T-DNA insertion line obtained from the Arabidopsis Biological Resources Center (Ohio State University, Columbus, OH). The T-DNA insertion in the gene was confirmed by PCR using the T-DNA left border (LB) primer and the gene-specific primers LP and RP (2). As a control, an AKR2B-specific product was also amplified by PCR using the LP and RP gene-specific primers. PCR was performed by 50 cycles of denaturation at 94 o C for 30 sec, annealing at 50 o C for 30 sec and elongation at 72 o C for 30 sec. Protein fractionation and Western blot analysis Protein extracts were prepared from transformed protoplasts as described previously (3) and fractionated into soluble and pellet fractions by ultracentrifugation at 100,000 g for 30 min. The pellet fraction containing membrane proteins was resuspended in lysis buffer, treated with 0.1 M Na 2 CO 3 to remove peripheral proteins, and pelleted by ultracentrifugation. Western blots were developed using an ECL kit (Amersham Pharmacia Biotech).

14 Supplementary references 1. Lee, Y.J., Kim, D.H., Kim, Y.-W. & Hwang, I. Identification of a signal that distinguishes between the chloroplast outer envelope membrane and the endomembrane system in vivo. Plant Cell 13, (2001). 2. Alonso, J.M. et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, (2003). 3. Lee, Y.J., Kim, D.H., Kim, Y.-W. & Hwang, I. Identification of a signal that distinguishes between the chloroplast outer envelope membrane and the endomembrane system in vivo. Plant Cell 13, (2001).