AKR2A-mediated import of chloroplast outer membrane proteins is essential for chloroplast biogenesis

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AKr2A-mediated import of chloroplast outer membrane proteins is essential for chloroplast biogenesisWonsil Bae1,3, Yong Jik Lee1,2,3, Dae Heon Kim1, Junho Lee1, Soojin Kim1, Eun Ju Sohn1,2 and Inhwan Hwang1,4

In plant cells, chloroplasts have essential roles in many biochemical reactions and physiological responses1. Chloroplasts require numerous protein components, but only a fraction of these proteins are encoded by the chloroplast genome. Instead, most are encoded by the nuclear genome and imported into chloroplasts from the cytoplasm post-translationally2–5. Membrane proteins located in the chloroplast outer envelope membrane (OEM) have a critical function in the import of proteins into the chloroplast. However, the biogenesis of chloroplast OEM proteins remains poorly understood. Here, we report that an Arabidopsis ankyrin repeat protein, AKR2A, plays an essential role in the biogenesis of the chloroplast OEM proteins. AKR2A binds to chloroplast OEM protein targeting signals, as well as to chloroplasts. It also displays chaperone activity towards chloroplast OEM proteins, and facilitates the targeting of OEP7 to chloroplasts in vitro. AKR2A RNAi in plants with an akr2b knockout background showed greatly reduced levels of chloroplast proteins, including OEM proteins, and chloroplast biogenesis was also defective. Thus, AKR2A functions as a cytosolic mediator for sorting and targeting of nascent chloroplast OEM proteins to the chloroplast.

The targeting of nuclear-encoded proteins to chloroplasts is critical for plant survival6–8. Studies examining the mechanisms of protein targeting to chloroplasts have largely focused on how precursor proteins cross the chloroplast envelope membranes2–4,6,7. However, the mechanisms by which proteins are targeted to and inserted in the chloroplast OEM remain poorly understood9–13. Previously, a hydrophobic transmembrane domain (TMD) together with a flanking positively charged region was identified as a signal targeting proteins to the chloroplast OEM14,15. A cytosolic factor may rec-ognize the targeting signal to deliver OEM proteins to the chloroplast.

To investigate the molecular mechanisms of protein targeting to the chlo-roplast OEM, we screened for proteins that interact with the Arabidopsis OEM protein OEP7 (ref. 14) using a yeast two-hybrid screen and identi-fied an Arabidopsis protein, AKR2, which contains four ankyrin repeats (Fig. 1A). Previously, AKR2 was identified as a protein that participates

in anti-oxidation metabolism and disease resistance16. This protein was renamed AKR2A, as it is highly homologous to AKR2B of Arabidopsis (see Supplementary Information, Fig. S1a). The interaction between AKR2A and OEP7 was confirmed by in vitro pulldown experiments using His–AKR2A and GST–OEP7 or GST–AKR2A and MBP–OEP7 (Fig. 1B, C and see Supplementary Informaiton, Fig. S2). These experiments demonstrate that the AKR2A–OEP7 interaction is specific.

To delineate the OEP7 and AKR2A domains responsible for the interac-tion, deletion mutants were generated for both proteins (Fig. 1C, E) and expressed as GST- and His-fused proteins, respectively. His–AKR2AN2 and His–AKR2A, but not His–AKR2AN1 or His–AKR2AC, bound to GST–OEP7 (Fig. 1B), indicating that the amino-terminal region of AKR2A and the first two ankyrin repeats are necessary for its interaction with OEP7. For OEP7, both the TMD and carboxy-terminal-positive region (CPR) were required for AKR2A binding (Fig. 1D). The TMD and CPR of OEP7, and chloroplast OEP64 (cOEP64, formerly known as AtToc64), function as a targeting signal for chloroplast localization14,15. Thus, AKR2A may target TMD plus CPR-bearing proteins to the chloroplast OEM. To exam-ine this hypothesis, the relationship between chloroplast targeting and the interaction of AKR2A with OEP7 or cOEP64 was investigated using various OEP7 and cOEP64 mutants. In previous studies these mutants, OEP712I and cOEP641–29, were efficiently targeted to chloroplasts, whereas OEP71–28(7G), OEP712F and cOEP641–29(6G) were targeted to endomembrane compartments14,15. OEP712I and cOEP641–29 bound strongly to AKR2A, whereas OEP71–28(7G), OEP712F and cOEP641–29(6G) displayed only weak binding to AKR2A (Fig. 1F, G). Thus, the binding affinity of AKR2A for these OEP7 and cOEP64 mutants correlated positively with their targeting to chloroplasts. The binding of AKR2A to other membrane proteins was also examined. GST-fusion proteins were generated using the TMD plus a short flanking region from two chloroplast OEM proteins (Toc33 and Toc34), two mitochondrial proteins (Tom20-2 and mOEP64, formerly mitochondrial Toc64)17, a peroxisomal membrane protein (PMP22), and a plasma membrane protein (the last TMD of H+-ATPase; see Supplementary Information, Fig. S1b). His–AKR2A bound strongly to GST–Toc33 and GST–Toc34, but not to the other fusion proteins (Fig. 1G). These results indicate that AKR2A binds specifically to chloroplast proteins. In addition,

1Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Korea. 2Current address: Department of Cell Biology, University of California-Riverside, CA 92521, USA. 3These authors contributed equally to this work.4Correspondence should be addressed to I.H. (e-mail: [email protected])

Received 19 November 2007; accepted 12 December 2007; Published online 13 January 2008; DOI: 10.1038/ncb1683

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Figure 1 AKR2A interacts specifically with chloroplast OEM proteins. (A) Yeast two-hybrid assay. β-galactosidase activity was assayed as described in the Supplementary Information, Methods. (a) pGAL4(BD) plus pGAL4(AD). (b) pGAL4(BD)–OEP7 plus pGAL4(AD). (c) pGAL4(BD) plus pGAL4(AD)–AKR2A. (c) pGAL4(BD)–OEP7 plus pGAL4(AD)–AKR2A. (B–E) Identification of the interaction domains of AKR2A and OEP7. Protein pulldown assays were performed with the indicated combinations of proteins. In B, full-length or deletion mutants of AKR2A (all His-tagged; 5 µg) were used as prey and full-length OEP7 (GST-tagged; 5 µg) was used as bait. In D, full-length His-tagged AKR2A and various deletion mutants of OEP7 (all GST-tagged) were used as prey and bait, respectively. In F, full-length His-tagged AKR2A was used as prey and various substitution and/or deletion mutants of OEP7 were used as bait. The proteins were precipitated

with glutathione–agarose beads and the precipitates were then subjected to western blot analysis using an anti-His antibody. The blots were stained with Coomassie blue (Coomassie) to show that equal amounts of bait were used for the pulldown assays. N1, AKR2AN1; N2, AKR2AN2; C, AKR2AC; Fl, full-length AKR2A; Input, 5% of His–AKR2A proteins. Constructs used in the experiments are shown in C and E. CPR, C-terminal positively charged region; TMD, transmembrane domain. (G) Specificity of the interaction between AKR2A and various membrane proteins. Protein pulldown assays were performed with the indicated combination of His-tagged full length AKR2A (5 µg) as prey and GST alone or various organellar proteins (all GST-tagged; 5 µg) as bait. Toc33, aa 263–297; Toc34, aa 265–313; mOEP64, aa 1–41; PMP22, aa 151–190, ATPase (H+-ATPase), aa 810–848; Tom20-2, aa 176–200. Input, 10% of His-tagged full-length AKR2A.

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interactions between the closely related homologue AKR2B and these pro-teins followed the same pattern as AKR2A (data not shown), suggesting that both proteins perform the same function.

The interaction between AKR2A and OEP7 was examined in vivo. As reported previously, in protoplasts, GFP–OEP7 alone was not targeted to chloroplasts but instead produced a punctate staining pattern14. In

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Figure 2 AKR2A interacts with OEP7 in vivo and prevents OEP7 aggregation. (a) Effect of T7–AKR2A on the staining pattern of GFP–OEP7. Protoplasts were transformed with the indicated constructs and localization of GFP–OEP7 was examined. R6, expression vector without any coding region; Red fluorescence, chlorophyll. (b) Coimmunoprecipitation of T7–AKR2A and GFP–OEP7. Protein extracts from transformed protoplasts were subjected to immunoprecipitation with the anti-T7 antibody. Precipitates and total protein extracts were analysed by western blotting using the indicated antibodies. R6, control vector; T7–Fl, full-length T7–AKR2A. (c) Subcellular fractionation of GFP–OEP7. Protein extracts from transformed protoplasts were separated into soluble (S) and pellet (P) fractions by ultracentrifugation. The fractions were then subjected to western blot analysis using the indicated antibodies. R6, control vector. (d) Targeting of OEM proteins to the nucleus by NLS–RFP–AKR2A. Protoplasts were transformed with the indicated constructs and localization of GFP and RFP fusion proteins was examined. Chloroplasts (CH) are shown in blue. (e) Effect of T7–AKR2 on the localization of cOEP64N67–GFP. Protoplasts were transformed with the indicated constructs and the

localization of cOEP64N67–GFP was examined. OEP7–RFP was used as a marker for chloroplast OEM proteins. R6, control vector. Constructs used in this experiment are schematically presented at the bottom. Autofluorescence of chlorophyll is depicted in blue. (f) Targeting efficiency of cOEP64N67–GFP in the presence of AKR2 proteins. Chloroplasts were purified from lysates of protoplasts that had been transformed with the indicated constructs. Chloroplasts OEM proteins were prepared from purified chloroplasts at high pH and were analysed by western blotting using the indicated antibodies. Toc75 serves as control for protein loading. T, total extracts; C, protein extracts of purified chloroplasts. (g) To compare endogenous and transiently expressed AKR2 protein levels, protein extracts were prepared from protoplasts transformed with T7–AKR2A or HA–AKR2B (20 µg) and analysed by western blotting using anti-AKR2A antibody. As anti-AKR2A only detects AKR2B when overexpressed (see Supplementary Information, Fig. S4a), the AKR2B levels could not be quantified properly using anti-AKR2A antibody. C, untransformed control extracts; A, T7–AKR2A; B, HA–AKR2B. The scale bars represent 10 µm in a, d and e.



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contrast, in the presence of T7–AKR2A, GFP–OEP7 produced a diffuse pattern (Fig. 2a). Binding of T7–AKR2A to OEP7 may prevent aggrega-tion of GFP–OEP7. To confirm the interaction, coimmunoprecipitation experiments were performed. GFP–OEP7, but not GFP, was detected in anti-T7 precipitates (Fig. 2b), indicating that GFP–OEP7 interacts with AKR2A in vivo. Furthermore, GFP–OEP7, which was insoluble when expressed alone, was primarily detected in the soluble fraction when co-expressed with T7–AKR2A (Fig. 2c), suggesting that AKR2A binding to GFP–OEP7 prevents aggregation of the protein. These results suggest that AKR2 possesses chaperone activity, as observed with PEX19 and AIP for membrane proteins of peroxisomes and mitochondria, respec-tively18,19. However, despite its solubilization by AKR2A, GFP–OEP7 was not targeted to the chloroplast (Fig. 2a). We speculated that the large N-terminal GFP domain may block the integration of OEP7 into the chloroplast OEM.

To gain an insight into its role in vivo, we asked whether AKR2A bearing a nuclear localization signal (NLS) can divert chloroplast OEM proteins to the nucleus. In the presence of NLS–RFP–AKR2A (which consisted of an NLS, red fluorescent protein (RFP) and AKR2A, and localized to the nucleus), similarly to NLS–GFP14, coexpressed GFP–OEP7 localized to the nucleus (Fig. 2d). The negative control protein NLS–RFP did not induce nuclear localization of GFP–OEP7. These results demonstrate that AKR2A can deliver interacting proteins to a specific compartment.

Next, we examined whether AKR2 can increase the targeting efficiency of proteins to the chloroplast in vivo using several chimeric proteins. One of them, cOEP64N67–GFP, consisting of the N-terminal 67 amino acid segment of cOEP64 and GFP, seemed to be an ideal reporter pro-tein. Previously, it was demonstrated that the N-terminal 29 amino acid region of cOEP64 is sufficient to target GFP to chloroplasts15, and that cOEP64N67–GFP was similarly targeted to chloroplasts. However, at high expression levels, cOEP64N67–GFP aggregated around chloroplasts and only a minor fraction was imported into the chloroplasts (Fig. 2e), sug-gesting that the additional N-terminal segment increases the tendency to form aggregates. We tested whether high AKR2 levels supported target-ing of cOEP64N67–GFP to chloroplasts. cOEP64N67–GFP and OEP7–RFP were introduced into protoplasts together with T7–AKR2A or HA–AKR2B. In the presence of these AKR2 proteins, cOEP64N67–GFP closely overlapped OEP7–RFP at chloroplasts and only a minor fraction formed puncta (Fig. 2e). Furthermore, the amount of cOEP64N67–GFP that cop-urified with chloroplasts was 2–3 fold greater than the control (Fig. 2f). AKR2B was more efficient than AKR2A in targeting cOEP64N67–GFP to chloroplasts; the reason for this difference is not currently understood. Levels of transiently expressed AKR2 were several fold higher than the endogenous proteins (Fig. 2g), indicating that higher levels of AKR2 facilitated targeting of overexpressed cOEP64N67–GFP.

A genetic approach was used to examine the role of AKR2 in planta. An Arabidopsis mutant with a T-DNA insertion in AKR2B, but not AKR2A, was available (Fig. 3a and see Supplementary Information, Fig. S3). akr2b mutants seemed to be normal (Fig. 3b). In akr2b plants, AKR2A protein levels were similar to wild type (see Supplementary Information, Fig. S3c). In wild-type plants, AKR2B transcript levels were higher than AKR2A (see Supplementary Information, Fig. S3d). Transgenic plants harbouring an AKR2A RNAi construct under a dexamethasone (dex)-inducible promoter20,21 were generated in wild-type or akr2b backgrounds. Wild-type background RNAi plants did

not display any noticeable phenotype in the presence of dex (data not shown). In contrast, RNAi transgenic plants in the akr2b background, RNAi(AKR2A)–akr2b, displayed a yellow leaf or albino phenotype in the presence of dex (Fig. 3b). In RNAi(AKR2A)–akr2b plants, AKR2A transcripts were scarcely detectable (Fig. 3c) and the AKR2A protein level was reduced to 5–20% of control akr2b plants when examined using anti-AKR2A (Fig. 3d and see Supplementary Information, Fig. S4a). The levels of the chloroplast OEM proteins OEP7, Toc33, Toc34, Toc75 and Toc159 were examined in RNAi(AKR2A)–akr2b plants using specific antibodies (Fig. 3e). Compared to control akr2b plants, the transgenic plants had much lower levels of all chloroplast OEM proteins examined.

RbcS, Lhcb4 and PsaG, proteins that are imported into chloroplasts, were also greatly reduced in RNAi(AKR2A)–akr2b plants (Fig. 3e), which is likely to be due to the low levels of Toc proteins, as previously observed in ppi1 and ppi2 plants22–24. In contrast, porin and catalase in mitochondria and peroxisomes, respectively, were not affected in RNAi(AKR2A)–akr2b plants (Fig. 3e). Consistent with the western blot data, chloroplasts in cells of the RNAi(AKR2A)–akr2b plant leaves lacked thylakoid membranes and instead were filled with numerous small and large vacuoles (Fig. 3f). However, mitochondria and per-oxisomes showed normal morphology. These observations support the hypothesis that AKR2 has a pivotal function in targeting of chloroplast OEM proteins.

The above results demonstrate that AKR2A binds specifically to chloroplast OEM proteins. However, binding of AKR2A to substrate proteins may not be sufficient for targeting these proteins to chloroplasts. Therefore, we asked whether AKR2A binds to chloroplasts. Initially, we examined whether endogenous AKR2A copurifies with chloroplasts. AKR2A was detected in the purified chloroplast fraction, whereas the negative control proteins AALP and AtPEP12p were not (Fig. 4a). AKR2A binding to chloroplasts was examined in vitro using full-length and deletion mutants of His–AKR2A or His–GFP. His–AKR2A, His–AKR2AN2 and His–AKR2AC, but not His–AKR2AN1 or the control His–GFP, copelleted with chloroplasts, indicating that AKR2A binds to chloroplasts through its C-terminal ankyrin repeat domain (Fig. 4b). Furthermore, full-length AKR2A, but not bovine serum albumin, com-peted with His–AKR2AC for binding to chloroplasts (see Supplementary Information, Fig. S4b). Protoplasts transformed with T7–AKR2A or T7–AKR2AN1 were immunostained with anti-T7 and anti-Toc159 antibodies. T7–AKR2A staining overlapped endogenous Toc159 (Fig. 4c). In con-trast, T7–AKR2AN1 produced a weak diffuse pattern and did not overlap Toc159. These results confirm that AKR2A binds to chloroplasts.

To further confirm that AKR2A functions in protein targeting of chloroplast OEM proteins, in vitro import experiments were per-formed using purified chloroplasts, in vitro translated OEP7 and affinity-purified AKR2A25,26. Targeting of OEP7 alone to chloro-plasts was almost undetectable (Fig. 4d). OEP7 and AKR2A were then cotranslated in reticulocyte lysates, using a constant amount of OEP7–HA and increasing amounts of AKR2A. GFP was added to provide equivalent DNA levels. The in vitro-translated products were incubated immediately with purified chloroplasts, and proteins inserted into the chloroplast OEM were determined. In the presence of cotranslated AKR2A (0.5 µg), 20% of input OEP7–HA was inserted into chloroplasts (Fig. 4d, e). At 2 µg AKR2A, the import efficiency of OEP7–HA was 2.2-fold greater than at 0.5 µg AKR2A. These results



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demonstrate that AKR2A facilitates targeting of OEP7 to chloroplasts in vitro. In contrast, cotranslated GFP and AKR2A were not inserted into chloroplasts, confirming the specificity of AKR2A for chloroplast

OEM proteins. To confirm insertion of OEP7–HA into the chloroplast OEM, chloroplasts were purified from the import reaction mixture and treated with thermolysin25,26. Thermolysin-treatment generated

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Figure 3 RNAi(AKR2A)–akr2b plants have defects in OEM protein targeting and chloroplast development. (a) Lack of AKR2B transcripts. AKR2B and 18S rRNA transcript levels were examined by semi-quantitative RT–PCR using total RNA from wild-type (WT) and akr2b mutants. (b) Phenotypes of RNAi(AKR2A)–akr2b transgenic plants. The plants were grown on Murashige and Skoog (MS) plates containing 30 µM dex for 1 or 2 weeks. RNAi, RNAi(AKR2A)–akr2b. (c) AKR2A transcript levels in RNAi plants. Total RNA prepared from akr2b and RNAi(AKR2A)–akr2b (line 8) plants was subjected to semi-quantitative RT–PCR analysis of AKR2A and 18S rRNA transcript levels. (d) AKR2A protein levels. Protein extracts from akr2b and RNAi(AKR2A)–akr2b plants were analysed by

western blotting using anti-AKR2A, anti-actin and anti-PEP12p. Actin and PEP12p served as controls to normalize protein levels. (e) Western-blot analysis of various chloroplast proteins. Protein extracts from akr2b and RNAi(AKR2A)–akr2b (line 8) plants were analysed by western blotting using the indicated antibodies. (f) Ultrastructure of RNAi(AKR2A)–akr2b plants. Ultrathin sections prepared from leaf tissues of 1-week old akr2b and RNAi(AKR2A)–akr2b plants were examined by electron microscopy. In the main panels, the scale bars represent 2 µm and show whole cells. In the inserts, the scale bars in the top panels represent 2 µm and in the bottom panels represent 1 µm. CH, chloroplast; M, mitochondrion; N, nucleus; P, peroxisome; V, vacuole.



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a new band with a relative molecular mass, Mr, of 4,000–5,000, cor-responding to the N-terminal region plus TMD, with concomitant loss of OEP7–HA at Mr 10K (Fig. 4f), strongly suggesting that OEP7–HA is inserted into the chloroplast OEM.

For a protein to function as a cytosolic mediator of protein targeting to organelles, it should recognize both the targeting signal of organel-lar proteins and the target organelle. AKR2A has both activities. The

N- and C-terminal regions bind to the targeting signal of chloroplast OEM proteins14 and the chloroplast, respectively. These biochemi-cal properties suggest that AKR2A may bind to the targeting signal of chloroplast OEM proteins in the cytoplasm and deliver the OEM proteins to a receptor on the chloroplast surface. Consistent with this hypothesis, AKR2A facilitated the import of a substrate protein to chloroplasts in vitro and in vivo. AKR2A also displayed chaperone

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Figure 4 AKR2A binds to chloroplasts and enhances chloroplast targeting of OEP7. (a) Binding of AKR2A to chloroplasts in vivo. Proteins copurified with chloroplasts were analysed by immunoblotting with anti-AKR2A. Anti-AALP and anti-PEP12p served as controls for soluble and membrane proteins, respectively. T, total extracts; C, chloroplast extracts. (b) To examine binding of AKR2A to chloroplasts in vitro, purified chloroplasts were incubated with the indicated recombinant proteins (all His-tagged). Proteins pelleting with chloroplasts were detected with anti-His. Blots were stained with Coomassie blue to show that equal amounts of proteins were used for the pulldown assays. RbcL, the large subunit of the rubisco complex. (c) Localization of T7–AKR2A. Protoplasts transformed with T7–AKR2A or T7–AKR2AN1 were immunostained with anti-T7 and anti-Toc159 antibodies. Soluble proteins in the cytosol could not be well fixed in these conditions and, thus, were barely detectable. The scale bar represents 10 µm. (d) In vitro targeting of OEP7–HA to the chloroplast OEM. The indicated amounts of plasmids were used for in vitro translation in rabbit reticulocyte lysates. A GFP plasmid was included to adjust the total

amount of plasmid in the reaction mixture. The input (10%; top panel) and the membrane fraction (pellet) from the import assay mixtures (bottom panel) were analysed by fluorography. OEP7–HA produced two bands in both reticulocyte lysates and protoplasts (see Supplementary Information, Fig. S5). The upper band may correspond to the modified form. The lower band was more intense in the presence of AKR2A (see Supplementary Information, Fig. S5) and predominantly targeted to chloroplasts during in vitro import. (e) Quantification of OEP7 proteins inserted into chloroplasts. The OEP7 band intensity was measured using an image analysis program. The import efficiency was expressed relative to that obtained with 0.5 µg AKR2A DNA (n = 3). The error bars represent s.d. (f) Insertion of OEP7 into chloroplast membranes. At the end of the import experiment, chloroplasts were re-isolated and 10% of re-isolated chloroplasts was saved as a control (– Thermo). The remaining 90% of re-isolated chloroplasts were treated with thermolysin (+ Thermo). After thermolysin treatment, chloroplast membrane proteins were analysed by SDS–PAGE. Translation, translation products; OEP7 fragments, degradation product of OEP7–HA.



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activity towards chloroplast OEM proteins, which is another criti-cal activity needed by a cytoplasmic factor(s) that targets membrane proteins to specific organelles. Consistent with the proposed role of AKR2A, RNAi(AKR2A)–akr2b plants, which displayed low levels of AKR2A, showed greatly reduced levels of chloroplast OEM proteins and significantly impaired chloroplast biogenesis. The biological role of AKR2A proposed in this study is different from that hypothesized in a previous study16. We cannot exclude the possibility that AKR2A also may play a role in anti-oxidation metabolism and disease resist-ance either directly or indirectly.

METHODSGrowth of plants. Arabidopsis thaliana (ecotype Columbia) was grown on Murashige and Skoog plates at 22 °C in a culture room or in a greenhouse under conditions of 70% relative humidity, 20 °C and a 16 h light, 8 h dark cycle.

Generation of AKR2A RNAi transgenic plants and semi-quantitative RT–PCR analysis. To generate the AKR2A RNAi construct, a 292 base pair (bp) AKR2A fragment was amplified by PCR. The primers used were RNAi-5B and RNAi-3C for AKR2A-B/C, and RNAi-5X and RNAi-K for AKR2A-X/K (see Supplementary Information, Table S1 for primer sequences). The AKR2A-B/C and AKR2A-X/K PCR products were ligated into the BamHI–ClaI sites and XhoI–KpnI sites of the Kannibal vector, respectively27. The resulting construct was ligated into pTA7002. The RNAi construct was introduced into Agrobacterium and then into akr2b mutant plants using the floral dip method28.

For RT–PCR analysis, total RNA was isolated from the leaf tissues of akr2b and RNAi(AKR2A) –akr2b plants by standard protocols. Reverse transcription was performed with 10 µg of total RNA using Superscript RT (Gibco BRL) and 10 ng of a random primer in a 100 µl reaction volume. PCR was performed using 5 µl reverse transcriptase at 94 °C for 30 s, 50 °C for 30 s and 72 °C for 30 s for 50 cycles using the AKR–RT primer and a random primer. As internal controls, 18S rRNA was amplified under the same conditions for 20 cycles using the 18-5 and 18-3 primers.

In vitro protein pulldown assays. Prey proteins were pre-cleared by incubating 10 µg prey protein in protein pulldown buffer with 20 µl of a 50% glutathione–aga-rose bead solution for 4 h at 4 °C, and then adding an additional 20 µl of the 50% glutathione–agarose bead solution and incubating for 1 h at 4 °C. Purified bait and pre-cleared prey proteins were incubated in 10 mM Tris–HCl at pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5% NP40, 1% Triton X-100, 150 mM NaCl, 1 mM DTT) for 4 h at 4 C. The unbound proteins were removed by washing, and the bound proteins were pelleted and analysed by immunoblotting using appropriate antibodies.

In vivo targeting and immunohistochemistry. Plasmids were purified using Qiagen columns according to the manufacturer’s protocol. Constructs were intro-duced into Arabidopsis protoplasts prepared from leaf tissues by polyethylene glycol-mediated transformation, as described previously14,29. GFP and RFP images were directly captured with a cooled charged-coupled device camera using a Zeiss Axioplan fluorescence microscope. The filter sets used were XF116 (exciter, 474AF20; dichroic, 500DRLP; emitter, 510AF23), XF33/E (exciter, 535DF35; dichroic, 570DRLP; emitter, 605DF50), and XF137 (exciter, 540AF30; dichroic, 570DRLP; emitter 585ALP; Omega), to detect GFP, RFP and chlorophyll autofluo-rescence, respectively. The data were processed using Photoshop software and the images were rendered in pseudocolour.

For immunostaining, protoplasts were fixed in 4% paraformaldehyde for 30 min and permeabilized in buffer containing 10 mM Tris–HCl at pH 7.4, 0.9% NaCl, 0.25% gelatin, 0.02% SDS and 0.1% Triton X-100 for 10 min. Fixed pro-toplasts were incubated with anti-HA and anti-Toc159 antibodies followed by TRITC-conjugated goat anti-mouse IgG (Zymed) and FITC- conjugated anti-rab-bit IgG, respectively, as secondary antibodies14. The immunostained protoplasts were mounted in 120 mM Tris at pH 8.4 and 30% glycerol containing Mowiol4-88 (Calbiochem).

Coimmunoprecipitation. Protein extracts from transformed protoplasts were incubated with 1 µg of an anti-T7 monoclonal antibody (Novagen) for 4 h at 4 °C

and, subsequently, 20 µl of a 50% protein A–Sepharose bead solution (Pharmacia Biotech) was added and incubated for 1 h at 4 °C. The beads were washed three times and proteins were eluted by boiling for 5 min in 20 µl sample buffer. The eluates were separated by SDS–PAGE and analysed by immunoblotting using appropriate antibodies.

In vitro protein import into chloroplasts. Plasmid DNAs were linearized with EcoRI and the template DNAs were transcribed and translated using the rabbit reticulocyte TNT Quick Coupled transcription/translation System according to the manufacturer’s protocol (Promega). After incubation at 30 °C for 60 min, the reaction was terminated by incubation with 1.25 mM cycloheximide on ice for 15 min. Chloroplasts were isolated from protoplasts by standard procedures on Percoll gradients25,26. Protein import into chloroplasts (equivalent to 20 µg chlorophyll) was assessed in 100 µl import buffer (50 mM Hepes–KOH at pH 7.6, 3 mM MgSO4, 330 mM sorbitol) containing 10% in vitro-translated 35S-labelled

proteins. Import was initiated by adding chloroplasts and stopped after 30 min. Intact chloroplasts were then re-isolated by centrifugation at 1,000g for 5 min at 4 °C and resuspended in buffer (50 mM Hepes–KOH at pH 7.6, 3 mM MgSO4, 150 mM NaCl). Lysates were treated with 0.1 M Na2CO3 and fractionated into soluble and membrane fractions by ultracentrifugation. Thermolysin treatment was performed as described previously25,26. Briefly, re-isolated chloroplasts were incubated with thermolysin (10 µg ml–1) in import buffer at 4 °C for 30 min and chloroplasts were then pelleted by centrifugation at 1,000g for 30 min at 4 °C. Chloroplast membrane proteins were solubilized with 1% Triton X-100 in import buffer. The soluble fraction was obtained by centrifugation at 10,000g for 30 min and proteins were precipitated with 1% cold trichloroacetic acid. The pellet was washed with a mixture of ethanol:ethylether (1:1) to remove chlorophyll. Proteins were separated by 15% SDS–PAGE and visualized by fluorography using Amplify (Amersham Biosciences) according to the manufacturer’s instructions.

Electron microscopy. Plant leaf tissues were fixed for 4 h with 2% paraformal-dehyde and 2% glutaraldehyde in 50 mM cacodylate buffer (pH 7.2), rinsed with the same buffer, and post-fixed with 1% osmium tetroxide in cacodylate buffer for 2 h. After dehydration, the specimens were embedded in LR White (London Resin Co.). Ultra-thin sections (60–80 nm thick) were collected on uncoated copper grids (150 mesh), stained with 3% uranyl acetate and Reynolds’ lead citrate, and examined using a transmission electron microscope (Jeol JEM-1011) at 80 kV.

AKR2A binding to chloroplasts. Purified His-tagged proteins (10 µg) were incu-bated with intact chloroplasts (equivalent to 20 µg chlorophyll) in 1 ml import buffer on ice for 30 min. Subsequently, chloroplasts were precipitated by low speed cen-trifugation (500g) for 5 min at 4 °C. Proteins pelleting with chloroplasts were boiled in SDS–PAGE buffer and analysed by immunoblotting with an anti-His antibody.

Note: Supplementary Information is available on the Nature Cell Biology website.

ACKNOWLEDGMENTSWe thank H.-M. Li (Institute of Molecular Biology, Academia Sinica, Taiwan) for the anti-OEP7, anti-Toc33, anti-Toc34, anti-Toc75 and anti-Toc159 antibodies. The akr2b mutant (SALK T-DNA insertion line) was obtained from the Arabidopsis Biological Resources Center. This work was supported in part by a grant from Crop Functional Genomics Research Center (M107KG010003-07K0701-00330), Systems Biodynamics-National Core Research Center (R15-2004-033-05002-0) and the National Creative Research Initiatives program funded by the Ministry of Science and Technology, Korea and Core research fund of POSTECH.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

Published online at http://www.nature.com/naturecellbiology/ reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions

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S U P P L E M E N TA RY I N F O R M AT I O N

WWW.NATURE.COM/NATURECELLBIOLOGY 1

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. Non-identical amino acids are shown in red.

R2A MASNSEKNPLL-SDEKPKSTEENKSS-KPESASGSSTSSAMP---GLNFNAFDFSNMASIL 56R2B MASSSEKTPLIPSDEKNDTKEESKSTTKPESGSGAPPSPS-PTDPGLDFNAFDFSGMAGIL 60

R2A NDPSIREMAEQIAKDPAFNQLAEQLQRSIPNAGQEGGFPNFDPQQYVNTMQQVMHNPEFK 116R2B NDPSIKELAEQIAKDPSFNQLAEQLQRSVPTGSHEGGLPNFDPQQYMQTMQQVMENPEFR 120

R2A TMAEKLGTALVQDPQMSPFLDAFSNPETAEHFTERMARMKEDPELKPILDEIDAGGPSAM 176R2B TMAERLGNALVQDPQMSPFLEALGNPAASEQFAERMAQMKEDPELKPILAEIDAGGPSAM 180

R2A MKYWNDPEVLKKLGEAMGMPVAGLPDQTVSAEPEVAEEGEEEESIVHQTASLGDVEGLKA 236R2B MKYWNDKDVLAKLGEAMGIAVGA--DQTVAAEPEEAEEGEEEESIVHQTASLGDVEGLKA 238

R2A ALASGGNKDEEDSEGRTALHFACGYGELKCAQVLIDAGASVNAVDKNKNTPLHYAAGYGR 296R2B ALASGGNKDEEDSEGRTALHFACGYGEVRCAQVLLDAGANANAIDKNKNTPLHYAAGYGR 298

R2A KESVSLLLENGAAVTLQNLDEKTPIDVAKLNSQLEVVKLLEKDAFL 342R2B KECVSLLLENGAAVTQQNMDNKNPIDVARLNNQLDVVKLLEKDAFL 344



2 WWW.NATURE.COM/NATURECELLBIOLOGY

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.

OEP7OEP7(1-28)[7G]

OEP7[12I]OEP7[12F]

cOEP64(1-29)cOEP64(1-29)[6G]

H+-ATPase(810-848)

MSNTLSLIQSNASNPKVWVVIGVTVAGIVILAETRKRRIRANYKYVPLHFRVILHSLVAFFWGIFLTLRARSMTLALAKAK

MGKTSGAKQATVVVAAMALGWLAIEIAFKPFLDKFRSSIDKSDPTKDPDDFDTAATATTSKEGLMGKTSGAKQATVVVAAMALGWLAIEIAFGGGGGGGMGKTSGAKIIIIIIIIIIIIWLAIEIAFKPFLDKFRSSIDKSDPTKDPDDFDTAATATTSKEGLMGKTSGAKFFFFFFFFFFFFWLAIEIAFKPFLDKFRSSIDKSDPTKDPDDFDTAATATTSKEGLMASQAANLWVLIGLGLAGILMLTKKLKKTMASQAANLWVLIGLGLAGILMLTGGGGGG

AKIRGIGWGWAGVIWLYSIVTYFPLDVFKFAIRYILSGK

mOEP64

Toc33Toc34

KGKKLIPLIIGAQYLIVKMIQGAIRNDIKTSGKPLRGKKLIPLMFAFQYLLVMKPLVRAIKSDVSRESKLAWELRDSGLASRRS

Tom20-2 NTEFTYDVCGWIILACGIVAWVGMA

PMP22




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.

GST:AKR2

GSTM

BP:OEP7

MBP

GST:OEP7

His:AKR2A

66

45

(kD)

30

64 kD

GST+ M

BP

GST:AKR2A

+ M

BP

GST+ M

BP:OEP7

GST:AKR2A

+

MBP:O

EP7

Inpu

t

Anti-GST

b

c

a

GST

GST:AKR2A

MBP

MBP:OEP7

AKR2A

OEP7




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 gene-specific 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.

LP + LB

LP + RP 900 bp

410 bp

WT

a

akr2b

T-DNA

LB

b

c

ATG TGA

WT akr2b

Anti-AKR2A

Anti-actin

AKR2A AKR2B

18S rRNA 18S rRNA

1029/1035 bp

180 bp

d

500 bp

LP RP




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 affinity-purified 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.

Controlserum

Wild-typeWild

-type

akr2

bT7:

AKR2AT7:

AKR2A

HA:AKR2B

HA:AKR2B

Anti-AKR2A Anti-AKR2AAnti-T7 Anti-HA

Untra

nsfo

rmed

45 kD

31 kD

T7:AKR2A

AKR2A

HA:AKR2B




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.

Anti-His

Anti-His

BSA

5 5 10 200 5 5 5

5 0 5 10 20 (µg)

0 5 5 5 5 (µg)

BSA :His:AKR2A-C :

His:AKR2A :

His:AKR2A-C :

66

18

45

31

21.5

(kD)

(kD)




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.

38 kD

37 kD

: OEP7:GFP

R6 T7:AKR2A

R6T7:

AKR2A

6 h 18 h

Anti-GFP

Anti-T7

Coomassie(RbcL)

+ + + +




Figure S8 Full scans




Figure S8 continued


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-cT64-5 5’-TCTAGAATGGCGTCTCAAGCTGCG-3’ G-cT64-3 5’-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' Toc159-5 5’-

GCTCTAGATGGCTAGCATGACTGGTGGACAGCAAATGGTTAGATCCCCGCCTCTC-3’

Toc159-3 5’-TCCCCCGGGTTAGTACATGCTGTACTTGTCG-3’ PM-5 5'-ATGGGATCTTCACCACCGAAG-3' PM-3 5'-TCACTTAGCCTTTGCCAAAGCT-3' H-O7-R1 5’-ATCTGGAACATCGTATGGGTAACCCTCTTTGGATGTGG-3’ H-O7-R2 5’-CTCGAGTTAAGCGTAATCTGGAACATCGTATGG-3’ 18-5 5’-ATGATAACTCGACGGATCGC-3’ 18-3 5’-CCTCCAATGGATCCTCGTA-3’ LB 5’-GCGTGGACCGCTTGCTGCAACT-3’ LP 5’-ACGGTTGGCATGTTAAAAATC-3’ RP 5’-GAAGGACTGCTTTGCATTTTG-3’


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 MgCl2, 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 full-

length 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 sub-

cloning 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-


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 810-848). 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.


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 oC for 30

sec, annealing at 50 oC for 30 sec and elongation at 72 oC 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 Na2CO3 to remove peripheral proteins, and pelleted by

ultracentrifugation. Western blots were developed using an ECL kit (Amersham

Pharmacia Biotech).


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, 2175-2190 (2001).

2. Alonso, J.M. et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana.

Science 301, 653-657 (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, 2175-2190 (2001).


AKR2A-mediated import of chloroplast outer membrane proteins is essential for chloroplast biogenesis

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