A protein oxidase catalysing disulfide bond formation is localized to the chloroplast thylakoids

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1 A protein oxidase catalysing disulfide bond formation is localized to the chloroplast thylakoids Wei-Ke Feng 1, *, Liang Wang 1, *, Ying Lu 1 and Xiao-Yun Wang 1,2 1 College of Life Science, Shandong Agricultural University, Shandong Taian, China 2 State Key Laboratory of Crop Biology, Shandong Agricultural University, Shandong Taian, China Keywords chloroplast; disulfide bond formation; site-directed mutagenesis; topology; TP Correspondence X.-Y. Wang, College of Life Science, Shandong Agricultural University, Shandong Taian , China Fax: Tel: xyunwang@sdau.edu.cn *These authors contributed equally to this work (Received 30 March 2011, revised 8 July 2011, accepted 19 July 2011) doi: /j x In chloroplasts, thiol disulfide-redox-regulated proteins have been linked to numerous metabolic pathways. However, the biochemical system for disulfide bond formation in chloroplasts remains undetermined. In the present study, we characterized an oxidoreductase, AtVKOR-DsbA, encoded by the gene At4g35760 as a potential disulfide bond oxidant in Arabidopsis. The gene product contains two distinct domains: an integral membrane domain homologous to the catalytic subunit of mammalian vitamin K epoxide reductase (VKOR) and a soluble DsbA-like domain. Transient expression of green fluorescent protein fusion in Arabidopsis protoplasts indicated that AtVKOR-DsbA is located in the chloroplast. The first 45 amino acids from the N-terminus were found to act as a transit peptide targeting the protein to the chloroplast. An immunoblot assay of chloroplast fractions revealed that AtVKOR-DsbA was localized in the thylakoid. A motility complementation assay showed that the full-length of AtV- KOR-DsbA, if lacking its transit peptide, could catalyze the formation of disulfide bonds. Among the 10 cysteine residues present in the mature protein, eight cysteines (four in the AtVKOR domain and four in the AtDsbA domain) were found to be essential for promoting disulfide bond formation. The topological arrangement of AtVKOR-DsbA was assayed using alkaline phosphatase sandwich fusions. From these results, we developed a possible topology model of AtVKOR-DsbA in chloroplasts. We propose that the integral membrane domain of AtVKOR-DsbA contains four transmembrane helices, and that both termini and the cysteines involved in catalyzing the formation of disulfide bonds face the oxidative thylakoid lumen. These studies may help to resolve some of the issues surrounding the structure and function of AtVKOR-DsbA in Arabidopsis chloroplasts. Structured digital abstract l AtVKOR-DsbA and D1 colocalize by cosedimentation in solution (View interaction) Introduction It has long been postulated that disulfide bond formation may regulate protein activity. This idea comes from studies in which enzymes and transcription factors have been shown to lose activity after oxidation of cysteine residues in vitro and to regain it when exposed to the disulfide reductant thioredoxin (Trx) Abbreviations AP, alkaline phosphatase; GFP, green fluorescence protein; IPTG, isopropyl thio-b-d-galactoside; TM, transmembrane; TP, transit peptide; Trx, thioredoxin; VKOR, vitamin K epoxide reductase; XP, 5-bromo-4-chloro-3-indolyl phosphate. FEBS Journal 278 (2011) ª 2011 The Authors Journal compilation ª 2011 FEBS 3419

2 [1]. In chloroplasts, Trx-regulated Calvin enzymes are inactivated in the dark by the formation of regulatory disulfides [2]. Some studies suggest that the gain of function caused by disulfide bond formation may be a common way of responding to cellular stress [1]. The formation of disulfide bonds is mainly localized in subcellular organelles involved in secretory pathways, such as the periplasm in bacteria and the endoplasmic reticulum in eukaryotes [3]. Electron transfer pathways that promote the formation of protein disulfide bonds differ little between organisms. In prokaryotes, the formation of disulfide bonds in substrate proteins is catalyzed by Dsb proteins: DsbA and DsbB [4,5]. DsbA, a soluble periplasmic protein with a canonical CXXC motif in its active site, rapidly oxidizes cysteine residues in protein substrates, becoming reduced in the process [4]. Then the integral membrane protein DsbB reoxidizes DsbA and shuttles electrons received from DsbA to the electron transport chain via membrane-bound quinones [5]. In eukaryotes, this process is catalyzed in a similar way by protein-disulfide isomerase [6]. However, in many widely divergent aerobic bacteria, including all cyanobacteria and some actinobacteria, delta and epsilon proteobacteria, and spirochetes, a different protein replaces DsbB [7]. The bacterial homolog of the eukaryotic enzyme vitamin K epoxide reductase (VKOR) reoxidizes its DsbA-like partner and reduces quinones [7]. In mammals, VKOR is an integral membrane enzyme of the endoplasmic reticulum. Its catalytic subunit, VKOR complex subunit 1, recycles reduced vitamin K, which is then used as a co-factor in the c-carboxylation of glutamic acid residues in blood coagulation enzymes [8]. VKOR is a member of a large family of predicted enzymes that are present in vertebrates, Drosophila, plants, bacteria and archaea, including the cell envelope-located proteins in Mycobacterium tuberculosis, Synechocystis sp. PCC 6803, Synechococcus sp., and predicted plastidtargeted proteins in green algae and Arabidopsis [8 13]. In plants, the formation of disulfide bonds is also an important step in the maturation of storage and secretory proteins. The disulfide bonds in regulatory proteins serve as signaling elements in chloroplasts. Regulatory disulfides are preferentially reduced by the dithiol reductant Trx, although their oxidant counterpart has yet to be identified [2]. Because cyanobacterial VKOR, with its Trx-like domain, is essential for the formation of disulfide bonds, and because plant VKOR homologs have a similar structure with two fused domains, we hypothesized that the VKOR homologs in higher plants might act as oxidants to catalyze the formation of regulatory disulfide bonds [10,11]. A recent study characterized the At4g35760 gene product from Arabidopsis. Transient expression of the full-length coding region fused with green fluorescent protein (GFP) suggested that the protein was localized in the chloroplasts. A segment of 70 amino acids at the N-terminus was presumed to be a transit peptide (TP). As a bimodular oxidoreductase, the VKOR domain of this protein can accept electrons from reduced Trx-like (DsbA-like) domains and donate electrons to naphthoquinone. Because members of the Trx superfamily involved in redox reactions can act either as reductants or oxidants, the function of Arabidopsis VKOR with its Trx-like domain requires some clarification [14]. It should also be determined whether it promotes the formation of disulfide bonds by electron transfer. In the present study, we report the subcellular localization of the Arabidopsis thaliana VKOR homolog and determine the length of its TP with direct experimental evidence [13]. The protein was found to be located in the thylakoid. We also clarify its function in disulfide bond formation, identify the cysteines essential for electron transfer and propose a likely membrane topology of AtVKOR-DsbA. Results Localization of AtVKOR-DsbA and position of TP The putative subcellular localization and TP of AtV- KOR-DsbA were predicted online with TARGETP and PREDSL software (Table 1). Use of the software showed that AtVKOR-DsbA was most likely localized in the chloroplasts and that the first 45 amino acids from the N-terminus were probably the TP. To obtain direct experimental evidence, we fused the presumed TP of AtVKOR-DsbA inframe to the N-terminus of GFP and transiently expressed the fusion protein in Table 1. Prediction of subcellular localization and the TP of AtV- KOR-DsbA using different software programs. The subcellular localization and the TP of AtVKOR-DsbA were predicted by PREDSL and TARGETP. Numbers given under the ctp, mtp and SP scores represent the probabilities that AtVKOR-DsbA will be localized in different subcellular regions. Numbers under the TP length represent the amino acid residues of AtVKOR-DsbA. ctp, chloroplast transit peptide; mtp, mitochondrial targeting peptide; SP, secretory pathway signal peptide. PREDSL: TARGETP: Software ctp score mtp score SP score Prediction PREDSL Chloroplast 45 TARGETP Chloroplast 45 TP length 3420 FEBS Journal 278 (2011) ª 2011 The Authors Journal compilation ª 2011 FEBS

3 A GFP B Chlorophyll E GFP F Chlorophyll TP-GFP C D GFP alone G H Bright field Merged Bright field Merged Fig. 1. Subcellular localization of AtVKOR-DsbA. The fusion construct of the TP of AtVKOR-DsbA with pjit163-hgfp and pjit163-hgfp alone was introduced into Arabidopsis protoplasts. The protoplasts were incubated overnight at 23 C and then analyzed by confocal microscopy. (A D) Transient expression of GFP fused to the TP of AtVKOR-DsbA in Arabidopsis protoplast. (E H) GFP alone. (A, E) GFP fluorescence in the transformed protoplasts. (B, F) Chlorophyll fluorescence of the same protoplast as in (A) and (E). (C, G) Bright field images of protoplasts. (D, H) Merged images of (A) and (B), as well as (E) and (F). AtVKOR-DsbA-GFP D1 Rubisco activase Chr Str Fig. 2. Suborganellar localization of AtVKOR-DsbA. Chloroplasts, stroma and thylakoids were isolated from transgenic plants expressing AtVKOR-DsbA, fused with GFP-tags, fractionated by SDS PAGE, transferred to poly(vinylidene difluoride) membrane, and visualized using anti-gfp, anti-rubisco activase (as a control for stromal proteins) or anti-d1 (as a control for thylakoid proteins). Arabidopsis protoplasts under the control of the constitutive 35S promoter. The subcellular location of the fusion protein was visualized by confocal microscopy (Fig. 1). TP-GFP showed co-localization of the green GFP fluorescence with the red chlorophyll signal, clearly indicating localization in the chloroplast (Fig. 1A D), whereas the GFP-alone control was found in the cytoplasm (Fig. 1E H). These results confirmed that the first 45 amino acids from the N-terminal were capable of targeting AtVKOR-DsbA to the chloroplast. This is in contrast with the findings of Furt et al. [13], who suggested that the At4g35760 gene product was localized in the chloroplast by transient expression of the full-length At4g35760 gene fused with GFP in tobacco mesophyll cells and that the TP was likely to be the first 70 amino acids, and not the first 45. The results obtained in the present study clearly show that the first 45 amino acids alone could act as a TP and target the protein to the chloroplast. To determine the suborganelle localization of AtV- KOR-DsbA within the chloroplast, the full-length At4g35760 gene fused with a GFP-tag at its C terminus was introduced into Agrobacterium tumefaciens and transformed to Arabidopsis. Intact Arabidopsis chloroplasts overexpressing GFP-tagged AtVKOR-DsbA tag were fractionated into stroma and thylakoid fractions. Both fractions were subjected to immunoblot analysis, and the purity of the fractions was assessed by monitoring the stromal rubisco activase and thylakoid D1 proteins (Fig. 2) [15]. As shown in Fig. 2, the AtVKOR- DsbA protein was exclusively detected in the thylakoid fraction using anti-gfp. The results demonstrate that AtVKOR-DsbA was localized in the thylakoids. Effects of AtVKOR-DsbA on the formation of disulfide bonds To assess the role of AtVKOR-DsbA in disulfide bond formation, we first performed functional complementation Thy FEBS Journal 278 (2011) ª 2011 The Authors Journal compilation ª 2011 FEBS 3421

4 assays in Escherichia coli Dsb null strains. In prokaryotes, the formation of disulfide bonds in substrate proteins is catalyzed by the Dsb proteins, DsbA and DsbB [4,5]. The soluble periplasmic protein DsbA, which has a canonical CXXC motif in its active site, rapidly oxidizes cysteine residues in protein substrates [4]. Then the integral membrane protein DsbB reoxidizes DsbA and shuttles electrons received from DsbA to the electron transport chain [5]. When DsbA or DsbB, or both, are deleted, these E. coli strains become non-motile as a result of their inability to form the disulfide bonds critical for proper folding of the flagellar motor protein FlgI [16]. In Synechococcus sp., a VKOR homolog, SynDsbAB, which resembles a fused DsbB-DsbA protein, was found to complement motility defects in Ddsb strains, so the protein was confirmed to be capable of catalyzing the formation of disulfide bonds [10]. Similar to SynDsbAB, AtVKOR-DsbA consists of two distinct domains: an N-terminal membrane domain (AtVKOR) and a C-terminal soluble Trx-like domain (AtDsbA). The DdsbB, DdsbA and DdsbAB strains of E. coli were used as hosts for AtVKOR- DsbA complementation assays on M63 minimal media. Wild-type strains with the ability to form disulfide bonds in FlgI were motile after 72 h of incubation on M63 minimal plates, whereas Ddsb strains with empty s remained non-motile. We first tested the ability of full-length AtVKOR-DsbA with TP in plasmid pwl1 to complement the DdsbA and DdsbB strains (Fig. 3A, B). However, neither Ddsb strain transformed with plasmid pwl1 regained motility. We assumed that the TP might have been cleaved, leading to native folding of AtVKOR-DsbA in the targeted organelle. A plasmid (pwl2) containing AtV- KOR-DsbA without TP was then constructed and transformed to Ddsb strains. Consistent with our expectations, AtVKOR-DsbA without TP was able to restore motility in DdsbB, DdsbA and DdsbAB strains (Fig. 3). Then a His-tag, which was considered as not being likely to affect protein folding or weaken protein expression, was introduced to the N-terminus of AtV- KOR-DsbA in pwl2, yielding pwl3 [9]. As shown in Fig. 3, pwl3 was better able to complement motility defects in Ddsb strains than pwl2 was. The results indicate that AtVKOR-DsbA without TP could complement the defect of disulfide bond formation in Dsb null strains. Next, we tested the ability of individual domains of AtVKOR-DsbA to complement the DdsbA and DdsbB strains (Fig. 3A, B). Because a linker of the last transmembrane (TM) segment connected VKOR to the Trx-like domain in Synechococcus sp., we separated the AtVKOR and AtDsbA domains before the last TM segment at position 204 [10]. However, pwl4 with His-tagged AtVKOR could not complement motility defects in DdsbB and pwl5 with His-tagged AtDsbA could not complement defects in DdsbA. We also attempted to divide the two domains at a different site, position 218, and performed the complementation assay again, although the Ddsb strains remained nonmotile (data not shown). AtVKOR and AtDsbA alone were unable to complement motility defects in DdsbAB (Fig. 3C). When the two individual domains were A B C pwl3 pwl3 pwl3 pwl1 pwl4 pwl4 pwl4 pwl5 pwl5 WT WT WT pwl2 pwl2 pwl2 Fig. 3. Complementation assay of Ddsb strains. pwl1, ptrc99a with wild-type AtVKOR-DsbA; pwl2, ptrc99a containing AtVKOR-DsbA without TP; pwl3, ptrc99a containing His-AtVKOR-DsbA without TP; pwl4, ptrc99a containing His-AtVKOR without TP; pwl5, ptrc99a containing His-AtDsbA; empty, ptrc99a alone. (A) DdsbB (HK325) transformed with empty s, pwl1, pwl2, pwl3 and pwl4. (B) DdsbA (HK361) transformed with empty s, pwl1, pwl2, pwl3 and pwl5; (C) DdsbAB (HK329) transformed with empty s, pwl2, pwl3, pwl4 and pwl5. E. coli cells transformed with different plasmids were stabbed at multiple locations on M63 medium containing 0.3% agar and 0.2% glucose as carbon source. The spots representing different strains after 72 h of incubation at 30 C are shown FEBS Journal 278 (2011) ª 2011 The Authors Journal compilation ª 2011 FEBS

5 transformed together into the DdsbAB strain, the motility defect remained uncomplemented (data not shown). One possible explanation for the fact that the separate protein domains failed to function in E. coli is that the position at which the two domains are cleaved may be important for protein activity. Another explanation is that separate domains may have weaker affinity for each other than the whole protein. Because no complement was gained by using two different dividing sites, it is quite possible that the fusion of the two domains does not require high affinity, possibly because of high local concentrations. Once cleaved, it would be difficult for the separate domains to interact with each other smoothly as a result of low affinity. These results suggest a high specificity: AtVKOR may oxidize fused AtDsbA, which then oxidizes FlgI further, although individual AtVKOR domains do not appear to be able to replace the function of DsbB. AtDsbA domains cannot act as substitutes for DsbA. From these results, we could conclude that AtVKOR- DsbA may have the ability to catalyze disulfide bond formation in E. coli. As a homolog of SynDsbAB in Synechococcus sp., AtVKOR-DsbA probably performs this function in chloroplasts as well. This should be confirmed by further experimental evidence. Cysteines essential for disulfide bond formation Similar to DsbB, the bacterial VKOR homolog MtbVKOR (M. tuberculosis) contains four conserved cysteines: one pair of separated cysteines and another pair in a canonical CXXC motif. In E. coli, DsbA catalyzes disulfide bond formation by oxidizing the cysteine residues of protein substrates. Then MtbVKOR accepts electrons from DsbA via one of the separated cysteines and transfers the electrons to the electron transport chain through the cysteines in the CXXC motif. However, in addition to the AtVKOR domain, the Arabidopsis VKOR homolog AtVKOR-DsbA contains another DsbA-like domain with four conserved cysteines [10]. Unlike DsbA, which has only one pair of cysteines in a CXXC motif, there are another two separated cysteines in the AtDsbA domain. AtVKOR- DsbA also contains two nonconserved cysteines. To assess the roles of these 10 cysteines in disulfide bond formation, site-directed mutagenesis was used to confirm motility complementation and b-galactosidase activity. A DdsbAB strain (MER600) containing a fusion of b-galactosidase to a large periplasmic domain of the cytoplasmic membrane protein MalF (a membrane protein involved in maltose transport) in the genome was used as the host cell in the b-galactosidase activity assay [17]. If complementation can promote the formation of disulfide bonds in the exported portion of b-galactosidase, it would render the enzyme inactive, as indicated by a white color. Otherwise, b-galactosidase would remain enzymatically active, as indicated by a blue color [18]. As shown in Fig. 4(A C), when we mutated AtVKOR-DsbA to change each of the eight conserved cysteines to alanines or mutated each pair of conserved cysteines located in the same domain, motility complementation was abolished. Altering the two nonconserved cysteines had little effect on motility. In the b-galactosidase activity assay, the mutants of conserved cysteines showed high b-galactosidase activity, suggesting a lack of disulfide bond formation. The two mutants of nonconserved cysteines displayed very low activities, indicating the formation of disulfide bonds (Fig. 4D F). These results indicate that all of the conserved cysteines in AtVKOR-DsbA are essential for disulfide bond formation in E. coli and that the two nonconserved cysteines are not involved. Analysis of AtVKOR-DsbA topology in E. coli using alkaline phosphatase (AP) To understand the structure function relationships relative to the activity of AtVKOR-DsbA in the formation of disulfide bonds, it is necessary to understand its membrane topology (i.e. the specific number of TM segments and their orientation in the membrane, including the locations of essential cysteines). The membrane topology of AtVKOR-DsbA without TP was first predicted using nine different topology prediction software programs with the default parameters. Three of the nine software programs predicted four TM helices in AtVKOR-DsbA, and the others predicted five (Table 2). All of the software except SOSUI predicted that the C-terminus of AtVKOR-DsbA would be located in the periplasm. It has been reported that the reliability of topology predictions is greatly increased if different topology prediction methods give the same prediction [19,20]. Therefore, it is likely that AtVKOR-DsbA has four TM helices and a C-terminus located in the periplasm. Figure 5 shows the membrane topology prediction and the probability profile of AtVKOR-DsbA as determined by the most commonly used software, TMHMM. It predicts that there are four TM helices in AtVKOR-DsbA: , , and However, TMHMM gives the candidate TM helix (36 56) predicted by the six other software programs listed in Table 2 only a 50% chance of truly existing. To obtain direct experimental evidence regarding the topology of AtVKOR-DsbA, we used the AP fusion FEBS Journal 278 (2011) ª 2011 The Authors Journal compilation ª 2011 FEBS 3423

6 A B C C46A C230A C109A C116A C109A/C116A C195A C293A C296A C293A/C296A C316A C331A AtVKOR-DsbA (WT) AtVKOR-DsbA (WT) C198A C195A/C198A AtVKOR-DsbA C316A/C331A (WT) D E F C46A C109A C116A C293A C296A C230A AtVKOR- DsbA(WT) C109A C116A AtVKOR- DsbA(WT) C293A C296A C195A C316A AtVKOR- DsbA(WT) C195A C198A C198A C316A C331A C331A Fig. 4. Role of cysteine residues in the function of AtVKOR-DsbA in DdsbAB strains. AtVKOR-DsbA was mutated in such a way that would change any one of its 10 cysteines to alanine. Each pair of conserved cysteines was located in the same domain. (A C). DdsbAB(HK329) transformed with mutant plasmids was stabbed at multiple locations on M63 medium containing 0.3% agar and 0.2% glucose as a carbon source. The spots represent different strains after 72 h of incubation at 30 C. (D F) b-galactosidase activity assay of DdsbAB (MER600) transformed with corresponding mutant plasmids in (A) to (C). E. coli cells were streaked on M63 minimal medium containing 1.2% agarose, 20 lgæml )1 5-bromo-4-chloro-3-indolyl b-d-galactoside and 1 mm IPTG and incubated at 37 C for 2 days. The strains with defective disulfide bond formation turned blue in color. Table 2. Prediction of membrane topology of AtVKOR-DsbA. Membrane topology of AtVKOR-DsbA, excluding the TP, was predicted using different software programs. The predicted TM helices are listed. Numbers under the TM represent the amino acid residues of AtVKOR- DsbA. TMHMM: SOSUI: THUMBUP: sparks.informatics.iupui.edu/index.php?pageloc=services; TOPCONS: PHILIUS: philius/runphilius.jsp; HMMTOP: TMPRED: POLYPHOBIUS: su.se/poly.html; HMM-TM: Software TM number C-terminus TM1 TM2 TM3 TM4 TM5 TMHMM 4 Outside a SOSUI THUMBUP 4 Outside TOPCONS 5 Outside PHILIUS 5 Outside HMMTOP 5 Outside TMPRED 5 Outside POLYPHOBIUS 5 Outside HMM TM 5 Outside a Outside: periplasm location of E. coli FEBS Journal 278 (2011) ª 2011 The Authors Journal compilation ª 2011 FEBS

7 1.2 1 Fig. 5. TMHMM topology prediction and probability profile for AtVKOR-DsbA. The top line shows the predicted topology with the predicted TM helices. The blue and pink curves show the posterior probabilities for the inside and outside loops, respectively. The striped profile shows the probability of TM helix. Probability Transmembrane Inside Outside approach in E. coli. This approach is based on the fact that AP is enzymatically active when expressed in the periplasm but inactive when expressed in the cytoplasm [21,22]. As a result, by determining the AP activity of fusions to different hydrophilic domains of AtVKOR-DsbA, it is possible to distinguish cytoplasmic from periplasmic domains and, in this way, to determine membrane topology. It is likely that a thylakoid protein, which has regions that are lumenal in the chloroplast, will act as periplasmic in E. coli. Reciprocally, stromal domains are predicted to be exposed in the cytoplasm [15]. We first constructed five AP fusions without TP at positions 79, 121, 145, 176 and 265 in an expression plasmid, pdhb7744. To preserve the likely topology determinants of the protein, the fusion joints were placed at or near the predicted C-terminal end of each hydrophilic region [23]. However, except for the distinctly higher AP activity of fusion 265, the other four fusions did not differ significantly (data not shown). Taking this into account, we only could reason that the C-terminal of AtVKOR- DsbA might be located in oxidative surroundings. Although assays of series of such fusions have provided accurate information on the topological structure of some proteins, this approach is still potentially limited [23 26]. Because the fusions are generated in such a way that AP replaces a C-terminal portion of the membrane protein, which is required for proper assembly, the fusion approach may give an unfaithful model [27,28]. To overcome the limitations described above, we constructed sandwich fusions in which AP was inserted into the intact AtVKOR-DsbA protein, rather than replacing the C-terminus of the membrane protein AtVKOR-DsbA with AP [29]. The AP activity of each sandwich fusion was determined on 5-bromo-4-chloro-3-indolyl phosphate (XP) plates (Fig. 6). Sandwich fusions S145 and S265 showed higher activity than fusions S121 and S176. This is consistent with the prediction of periplasmic localization of AP in S145 and S265 and cytoplasmic localization of AP in S121 and S176. The fusion S6 S176 S145 S265 S121 S79 S62 Fig. 6. AP activity of sandwich fusions in FA113 as determined on checking plates S6, S145 and S265 showed higher levels of enzymatic activity than S62, S79, S121 or S176 on XP plates. On LB medium containing 0.4 mgæml )1 XP and 1 mm IPTG, FA113 strains transformed with different sandwich fusion plasmids were streaked and incubated at 37 C for 2 days. A blue color indicates AP activity. showed activity approximating that of S145, indicating that the N-terminus is probably located in oxidative surroundings and that the first candidate TM helix shown in Fig. 5 is not likely to exist. However, both S62 and S79 showed very low activity, which is in contrast with the prediction of periplasmic localization of AP in S62 and S79. Given the first lower striped domain shown in Fig. 5, we presumed that the sequence of amino acids before the TM helix (80 102) may have been partially inserted into the membrane and may have affected the activity of AP fused to this segment. The question of whether the fusion positions of S62 and S79 are too close to the membrane to affect AP activity or whether they become degraded merits further study. We present a model of AtVKOR-DsbA topology in Fig. 7 based on most of the fusion results and on the ability of the protein to function in E. coli. However, two of the results are inconsistent with the proposed model. In this model, there are four TM helices and a S6 FEBS Journal 278 (2011) ª 2011 The Authors Journal compilation ª 2011 FEBS 3425

8 6aa partially inserted segment ahead in AtVKOR-DsbA. Both the N-terminus and C-terminus of AtVKOR- DsbA are located in the thylakoid lumen. In this way, the protein is quite different from its mammalian counterpart, VKOR, which has three TM helices, with its N-terminus in the lumen of the endoplasmic reticulum and its C-terminus in the cytoplasm [30]. The four pairs of conserved cysteines are positioned in different regions. Two pairs in canonical CXXC motif Cys195- Cys198 (C151 C154) and Cys293-Cys296 (C249 C252) (Fig. 7) are located in the third TM helix and third hydrophilic lumenal domain, respectively. The other two pairs, Cys109-Cys116 (C65 C72) and Cys316- Cys331 (C272 C287) (Fig. 7), are located in the first and third hydrophilic lumenal domains. Discussion 62aa C65 C72 79aa 121aa C252 C aa C151 C aa 265aa C272 C287 Lumen Stroma Fig. 7. Proposed membrane topology of AtVKOR-DsbA without TP in chloroplasts. The gray circles represent the positions of the AP fusions and the white circles represent conserved cysteines. The structure and function of VKOR, an integral membrane enzyme of the endoplasmic reticulum, has been widely studied in mammals. In recent years, reports of VKOR homologs have focused on Mycobacterium, cyanobacteria, Arabidopsis and a few others. Notably, homologs of VKOR among Mycobacterium and cyanobacteria were shown to catalyze the formation of disulfide bonds [9,10]. In both prokaryotes and eukaryotes, disulfide bond formation involves two proteins: a membrane-associated oxidoreductase that generates disulfide bonds and a soluble oxidoreductase that carries oxidizing equivalents from the membraneassociated oxidoreductase to substrates. In the present study, we report a novel protein, AtVKOR-DsbA, which exhibits the properties of both enzymes. The present study supports the conclusion that the Arabidopsis homolog is localized in the chloroplast and confirms that its TP is likely to be the first 45 amino acids from the N-terminus via a GFP fusion experiment in Arabidopsis protoplasts [13]. For suborganelle localization, we transformed the full-length At4g35760 gene fused with GFP into Arabidopsis and performed an immunoblot assay, which showed that AtVKOR-DsbA was localized in the thylakoid. After the protein entered the chloroplast, its TP was removed and the remaining protein was found to integrate into the thylakoid directly, as do most thylakoid membrane proteins with multiple TM helices [31]. Because E. coli supplies both an impressive array of general genetic tools and several approaches specific to disulfide bond formation, it makes a convenient surrogate host for the analysis of aspects of the structure and function of proteins involved in disulfide bond formation in other organisms. We used E. coli as a host to assess the topology of AtVKOR-DsbA, confirm its function in disulfide bond formation, and identify its essential cysteines. The results obtained suggest a possible topological model for AtVKOR-DsbA in which both the N-terminus and C-terminus exist in the periplasm. Between the two termini of the protein are four TM segments. This organization predicts that the first pair of conserved cysteines in AtVKOR-DsbA (Cys57, Cys65) will be located within the first hydrophilic periplasmic domain, that the second conserved pair (Cys139, Cys142) will be located at the periplasmic end of the third TM segment, and that the last two pairs will both be located in the third hydrophilic periplasmic domain. Because the topological analogy between bacteria (periplasm cytoplasm) and plastids (lumen stroma) is justified by the previous establishment of its reliability in the analysis of the thylakoid membrane polytopic protein CcsA, we assumed that AtVKOR-DsbA would be a thylakoid membrane protein whose N-terminus and C-terminus were both on the lumen side [32]. We also confirmed that all eight conserved cysteines are essential for disulfide bond formation. In E. coli, DsbB has its pairs of redox-active cysteines located in or close to the periplasm [16,33,34]. The redox-active cysteines in both the AtVKOR domain and DsbB are in (or close to) the two periplasmic domains. This proposed topology is consistent with the role of DsbB in oxidizing periplasmic DsbA in E. coli and suggests a similar role for the AtVKOR domain in oxidizing the DsbA-like domain (AtDsbA). The AtVKOR domain and DsbB would receive electrons from the AtDsbA domain or DsbA in one of their periplasmic domains and transfer those electrons to the CXXC motifs that are accessible in the periplasm. Topological analysis also revealed differences between DsbB-DsbA and AtVKOR-DsbA. First, the pairs of conserved cysteines in the AtVKOR domain (one separated pair and one 3426 FEBS Journal 278 (2011) ª 2011 The Authors Journal compilation ª 2011 FEBS

9 CXXC pair) appear in the amino acid sequence of the protein in an order opposite to that observed in DsbB. Specifically, the canonical CXXC in the AtVKOR domain is present between the second and third TM segments and the one in DsbB is located between the first and second TM segments. The second pair of cysteines is present in the first hydrophilic periplasmic loop in AtVKOR domain but in large periplasmic loops connecting the second and third TM segments in DsbB. Furthermore, there is another pair of conserved cysteines after the canonical CXXC in the AtDsbA domain but not in DsbA. These differences may prevent the individual domain AtVKOR-DsbA from acting as a complement to DsbB and DsbA. In the present study, expression of the full-length AtVKOR-DsbA gene without TP in Ddsb strains of E. coli restored the ability if the organism ability to form disulfide bonds in the periplasm. We confirmed that all eight conserved cysteines are essential for the function of AtVKOR-DsbA. This suggests that AtV- KOR-DsbA could catalyze the formation of disulfide bonds in the E. coli periplasm and that it may also play an important role in disulfide bond formation in chloroplasts. One clue to the potential function of AtVKOR-DsbA in chloroplasts originates from an analysis of its secondary structure. The AtVKOR domain shares significant structural similarity to DsbB, whereas the AtDsbA domain is similar to DsbA [10]. The results of the present study show that AtVKOR- DsbA was able to complement the function of DsbB or DsbA, or both, in E. coli Ddsb strains. However, when each individual domain was expressed separately, we found that the AtVKOR domain alone was not able to complement the function of DsbB and that the AtDsbA domain alone could not complement the function of DsbA. These results suggest that E. coli DsbA is unable to accept oxidizing equivalents from the AtVKOR domain, that E. coli DsbB cannot transfer oxidizing equivalents to the AtDsbA domain, and that only the two fused domains can complement the Dsb system. These results are not fully consistent with those of SynDsbAB in cyanobacteria. AtVKOR-DsbA and SynDsbAB share significant secondary structural similarity, although SynDsbA is able to complement the function of DsbA [10]. Because the SynDsbB domain of SynDsbAB cannot functionally interact with DsbA and because E. coli DsbB has no ability to accept electrons from the DsbA homolog in M. tuberculosis, we presumed that SynDsbB, DsbB and AtV- KOR would have relatively strict substrate specificities. It is reasonable to assume then that the individual domain of AtVKOR-DsbA could not complement the function of DsbB or DsbA. The results of the present study also show that Ddsb strains transformed with the full-length AtVKOR-DsbA gene including its TP had no disulfide bond formation activity. After removal of the TP, however, the protein regained function, suggesting that TP only serves to lead this protein to chloroplast, but does not play a role in disulfide bond formation. In the present study, AtVKOR-DsbA was found to be capable of complementing the functions of DsbA and DsbB in E. coli Dsb null strains, and so may comprise a potential oxidant for disulfide bond formation in Arabidopsis chloroplast proteins. Further studies will be needed to identify the substrates involved in the functional interval of AtVKOR-DsbA and the electron transport chain relevant to disulfide bond formation in chloroplasts. We consider that a better understanding of the oxidative side of chloroplast signaling will further our comprehension of the intricate regulatory networks in chloroplasts. The identification of VKOR homologs as possible universal oxidant counterparts of redox regulation in higher plants may have profound implications for our understanding of cellular signaling in general. Materials and methods Materials A. thaliana (ecotype Columbia) was grown in soil in a greenhouse under a 16 : 8 h light dark cycle (100 leæm )2 Æs )1 )at 22 C. Bacterial strains and their relevant genotypes are described in Tables 1 and S1. Cultures were generally grown in LB medium or M63 minimal medium supplemented with glucose and appropriate antibiotics [35]. Bioinformatic analysis The putative target location and TP of AtVKOR-DsbA was predicted online using TARGETP and PREDSL software. Online software, including TMHMM, HMM-TM, TMPRED, TOP- CONS, SOSUI, PHILIUS, POLYPHOBIUS, HMMTOP and THUMUP, was used to predict the membrane topology of AtVKOR- DsbA. Construction of s The At4g35760 cdna was amplified by PCR from an A. thaliana cdna library using gene-specific primers to construct plasmid pwl1. According to bioinformatic analysis, the gene product contained a TP of the first 45 amino acids. Plasmids pwl2 and pwl3 containing the AtVKOR- DsbA coding region, but not TP, were constructed using pwl1 as the template (pwl3 was His-tagged at the N-terminus and pwl2 was not His-tagged). Plasmids pwl4 and pwl5 contained regions encoding AtVKOR (amino acids FEBS Journal 278 (2011) ª 2011 The Authors Journal compilation ª 2011 FEBS 3427

10 45 214) and AtDsbA (amino acids ), respectively. All PCR products were cloned into plasmid ptrc99a. For topological analysis, we constructed AtVKOR-DsbAphoA fusions in which the phoa gene for AP, missing its signal sequence, was fused to certain positions in AtVKOR-DsbA [23]. The plasmid pdhb7744, an expression, contained phoa fused to the malf gene, which encoded a membrane protein involved in maltose transport. Different segments of AtVKOR-DsbA without TP were cloned to this in place of the malf gene. To make this possible, the was digested with BspEI and NcoI to remove the malf sequence. The AtVKOR-DsbA sequence was amplified by PCR with appropriate restriction sites encoded in the primers. The reading frame was adjusted by adding a glycine codon to the 3 primers of each of the seven fusion oligonucleotides between the last codon of each segment of AtVKOR-DsbA and the beginning codon of the phoa gene. The PCR products were cloned into pdhb7744, producing phoa fusions. Then all fusions were used to construct sandwich fusions in accordance with the method described by Ehrmann et al. [36]. In each fusion, the stop codon in the phoa gene was deleted, and the remaining sequences of AtVKOR-DsbA after the fusion positions were fused to the C-terminus of phoa, producing sandwich fusions. Then these sandwich fusions were transformed to E. coli strain FA113 [9]. The primers for these constructions are listed in Table S2. All constructs described in the present study were confirmed by sequencing. AP activity assay Strains of FA113 transformed with different sandwich fusion plasmids were streaked on LB medium containing 0.4 mgæml )1 XP and 1 mm isopropyl thio-b-d-galactoside (IPTG) and incubated at 37 C for 2 days. A blue color indicates AP activity. The protoplasts were resuspended in 100 ll of W5 solution and added to 1 ml of W5 (six-well plates). The protoplasts were then incubated for h in the dark at 23 C and fluorescence images were captured with confocal laser scanning microscopy, as described previously [38]. Antibodies Monoclonal anti-gfp was purchased from Tiangen (Beijing, China). Polyclonal anti-d1 and anti-rubisco activase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for marker proteins of suborganellar compartments were selected according to their specificity for proteins from Arabidopsis. Fractionation of suborganelle compartments in chloroplasts from Arabidopsis Intact Arabidopsis chloroplasts were isolated and purified from the leaves of 4 5-week-old plants overexpressing AtV- KOR-DsbA-GFP (oeatvkor-dsba-gfp), as described previously [39]. Plants overexpressing AtVKOR-DsbA-GFP (oeatvkor-dsba-gfp) were generated by introducing the AtVKOR-DsbA coding region minus its stop codon into the plant expression pbi121-gfp (Table S2). This produced a fusion protein upstream and in frame to GFP ORF under the control of the 35S promoter of cauliflower mosaic virus. We then transformed the flowers of wild-type plants with the AtVKOR-DsbA-GFP overexpression construct, as described previously [40]. Intact chloroplasts were ruptured by mixing with 10 volumes of lysis buffer (20 mm Hepes KOH, ph 7.5, and 10 mm EDTA) and incubated on ice for 30 min. To separate thylakoid and stroma phases, ruptured chloroplasts were centrifuged ( g for 30 min at 4 C). Stroma and thylakoid fractions were retrieved from the supernatant and the pellet, respectively [41]. Transient expression of GFP fusion constructs in Arabidopsis protoplasts The ORF of AtVKOR-DsbA TP was amplified from a cdna clone (Table S2). The PCR product was cloned into pjit163-gfp, producing a fusion protein under the control of the cauliflower mosaic virus 35S promoter. The constructed was then introduced into E. coli. Protoplast preparation took place in accordance with the method of Sheen [37]. For the transformation, 100 ll of protoplast suspension was carefully mixed with 10 lg of column-purified plasmid DNA and 110 ll of poly(ethylene glycol) Ca 2+ solution [4 g of poly(ethylene glycol) 4000, 3 ml of H 2 O, 2.5 ml of 0.8 M mannitol and 1 ml of 1 M CaCl 2 ] and incubated for 20 min at 23 C. Then the mixture was diluted with 0.44 ml of W5 (154 mm NaCl, 125 mm CaCl 2, 5 mm KCl and 2 mm MES KOH, ph 5.7) solution and spun at 100 g for 1 min to remove poly(ethylene glycol). Directed mutations of the cysteine residues There are 10 cysteines in the AtVKOR-DsbA coding region, at positions 46, 109, 116, 193, 195, 230, 293, 296, 316 and 331. To investigate the function of each cysteine in the formation of disulfide bonds, single and double mutants were produced using a QuickChangeÔ mutagenesis kit obtained from Stratagene (La Jolla, CA, USA). All mutations and corresponding primers are listed in Tables S1 and S2. Disulfide bond formation assay Motility complementation assay M63 minimal media was used for the motility confirmation [16]. E. coli cells transformed with different plasmids were stabbed on M63 medium. The strains were incubated at 30 C for 3 days FEBS Journal 278 (2011) ª 2011 The Authors Journal compilation ª 2011 FEBS

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