Identifying Interacting Proteins of Arabidopsis Cyclophilin38 (AtCYP38), a Key Factor for PSII Assembly/Repair, via Multiple Screening Approaches

inoculated onto SD/-Leu/Trp and SD/-Leu/Trp/His plates. A shows the conrmation of interaction between CYP38F domain and its screening results. B. Conrmation of interaction between CYP38N domain and its screening results. of interaction between CYP38C domain and its screening results.


Abstract Background
AtCYP38, a thylakoid lumen localized immunophilin, is essential for photosystem II (PSII) assembly and maintenance, but how AtCYP38 functions in chloroplast remains unknown. Based on previous functional studies and its crystal structure, we hypothesize that AtCYP38 should function via binding its targets or cofactors in the thylakoid lumen to in uence PSII performance. Therefore, identifying its target proteins and cofactors would be a key step to understand the working mechanism of AtCYP38.

Results
To identify potential interacting proteins of AtCYP38, we rst adopted two web-based tools, ATTED-II and STRING, and found 15 proteins functionally related to AtCYP38. We then screened a yeast two-hybrid library including an Arabidopsis genome wide cDNA with the N-terminal domain, the C-terminal domain, and the full-length mature protein of AtCYP38. 25 positive targets were identi ed, but a very limited number of target proteins were localized in the thylakoid lumen. In order to speci cally search interacting proteins of AtCYP38 in the thylakoid lumen, we created a yeast two-hybrid mini library including the thylakoid lumenal proteins and lumen fractions of thylakoid membrane proteins. After screening the mini library with 3 different forms of AtCYP38, we obtained 6 thylakoid membrane proteins and 9 thylakoid lumenal proteins as interacting proteins of AtCYP38. We further con rmed the localization of several identi ed proteins and their interaction between AtCYP38.

Conclusions
After analysis with two web-based tools and yeast two-hybrid screenings against two different libraries, we identi ed a couple of potential interacting proteins, which could be functionally related to AtCYP38.
We believe that the results will lay a foundation for unveiling the working mechanism of AtCYP38 in photosynthesis.

Background
In photosynthetic organisms, photosystem II (PSII) is a large multiprotein complex, which is composed of more than 20 protein subunits, many cofactors and pigment molecules on the thylakoid membrane [1,2].
It catalyzes the light-driven oxidoreduction of water and plastoquinone, and pumps electrons to the photosynthetic electron transfer chain [3,4]. Proper biogenesis and maintenance of PSII is essential for photosynthesis in chloroplasts. A large number of auxiliary proteins, including immunophilins in the thylakoid lumen, have been demonstrated to be required for the assembly and/or repair of PSII [5,6,7].
Immunophilins include two groups of proteins without signi cant sequence homology: cyclophilins (CYPs) and FK506-binding proteins (FKBPs), which are cellular receptors for potent immunosuppressive drugs: cyclosporin A and FK506, respectively [8,9]. CYPs and FKBPs share a common feature of peptidyl-prolyl cis/trans isomerase (PPIase) activity, which is essential for tra cking and protein folding in cells.
These proteins are widely present in prokaryotic and eukaryotic organisms with diverse cellular functions, including mediating protein folding, chaperone activity, scaffold facilitating [10,11]. In Arabidopsis, a large fraction of immunophilins (17 out of 52 members) are localized in chloroplasts, and interestingly, 16 of the 17 chloroplast immunophilins are presumably present in the thylakoid lumen [11,12,13]. Given a small number (~100) of proteins in the thylakoid lumen proteome and relatively a large number (16) of immunophilins in this compartment [12,13,14], it is reasonable to assume that immunophilins could be functionally related to the assembly and/or maintenance of thylakoid protein complexes. The recent ndings that AtCYP38 and AtFKBP20-2 play key roles in PSII assembly and repair con rm the critical physiological roles of immunophilins in the thylakoid lumen [15,16,17].
Arabidopsis cyclophilin38 (AtCYP38) is a greatly divergent immunophilin located in the thylakoid lumen of eukaryotic photosynthetic organisms [18]. Its ortholog in spinach, also named as TLP40, was originally identi ed as a cofactor of a chloroplast phosphatase [19], and was proposed to function as a negative regulator of a putative thylakoid protein phosphatase [20,21]. In Arabidopsis, AtCYP38 is indispensable for the correct assembly and rapid repair of PSII [16,17]. Arabidopsis mutant plants with a T-DNA disrupted AtCYP38 gene show a stunt growth phenotype and are very sensitive to high light stress [16,17]. This protein may be required for the proper folding of PSII core proteins D1 and CP43, and the successful assembly of PSII oxygen evolving complex (OEC) [17]. Recently, a study showed that AtCYP38 may negatively regulate PsbO2 activity, and subsequently facilitate the degradation of photo-damaged D1 protein [22]. By far, how AtCYP38 plays its roles in PSII assembly and maintenance is not clearly understood.
Crystal structure analysis demonstrated that AtCYP38 contains two distinct major domains: an Nterminal α-helical bundle and a C-terminal cyclophilin β-sheet barrel, with a short acidic loop as a linker in the middle [23]. The two domains are packed together to form a strong intramolecular interaction. This particular structure of AtCYP38 indicates a possible autoinhibition mechanism for its function via the Nand C-terminal interaction. Enzyme activity assay and structure analysis showed that AtCYP38 is not an active PPIase [23]. It is consistent with previous reports that PPIase activity in the thylakoid lumen was strictly attributed to only two immunophilins, AtCYP20-2 and AtFKBP13 [24,25,26]. It is likely that AtCYP38 plays important roles in maintaining normal con guration of its substrates with a chaperonelike activity via protein-protein interaction. According to the above comprehensive analysis, searching for partner proteins of AtCYP38 would be of great signi cance for understanding its working mechanism.
To identify its substrate proteins and potential cofactors, we rst carried out the co-expression network ATTED-II [27] and protein-protein interaction network STRING [28] analysis to nd potential partner proteins of AtCYP38. Then we performed yeast two-hybrid (YTH) assays to screen against an Arabidopsis genome wide cDNA library with three different forms of AtCYP38. Since AtCYP38 protein is located in the thylakoid lumen, we further constructed an YTH mini library including previously identi ed lumenal proteins and membrane proteins with lumenal fractions [12,13,14], and screened it with three different forms of AtCYP38. At the end, we con rmed their chloroplast localization of several identi ed proteins and their interactions with AtCYP38. We believe that we obtained certain interacting proteins functionally related to AtCYP38, and it will lay a foundation to understand the possible mechanism on how AtCYP38 plays its roles in the thylakoid lumen.

Results
Functionally related proteins of AtCYP38 revealed by web-based tools In order to search potential interacting proteins of AtCYP38, we rst adopted the co-expression network program ATTED-II to search for proteins whose expression pattern is related to AtCYP38. The program revealed 20 proteins are tightly co-expressed with AtCPY38 ( Fig. 1A and Table 1). Among these proteins, 8 proteins are localized in the thylakoid lumen, including FKBP18 (At1g20810), PPL (At1g76450), FKPB16-3 (At2g43560), PRP ( At2g44920), PPD6 (At3g56650), TAPX4 (At4g09010), TLP15 (At5g52970), and TLP17.4 (At5g53490); and 1 protein, HHL1 (At1g67700), resides on the thylakoid membrane. Considering the thylakoid lumen localization of AtCYP38, we assumed that these 9 proteins might be functionally related to AtCYP38. Meanwhile, the other 10 co-expression proteins, which are localized in the chloroplast stroma ( Fig. 1A and Table 1), are not likely to physically interact with AtCYP38.
In order to evaluate the ATTED-II co-expression results, we also applied the functional protein association tool, STRING, to screen potential proteins related to AtCYP38. The results showed that there are 10 chloroplast proteins functionally related to AtCYP38 at the highest con dence value. Out of these proteins, 4 are localized in the chloroplast thylakoid lumen, including TLP18.3 (At1g54780), PRXQ (At3g26060), MPH2 (At4g02530), and FKBP13 (At5g45680), and 2 proteins, LQY1 (At1g75690) and ATPD (At4g09650), reside on the thylakoid membrane ( Fig. 1B and Table 2). All the other proteins are not localized in the chloroplast thylakoid lumen. They might not physically interact with AtCYP38, though they could be functionally related.
After network analysis with these 2 different programs, we found that no protein was co-detected by both software. We assumed that the different results from these two web-based detections could be partially caused by their unique algorithms. Considering their localization and their functions in photosynthesis, we presumed that all these 15 lumen or membrane localized proteins might be functionally related to AtCYP38.
Target proteins screening against a library containing genome wide cDNA Yeast two-hybrid (YTH) screening is a powerful and well-established tool to identify interested proteins which could be physically associated with a speci c target protein. To identify potential targets and cofactors of AtCYP38, we launched a yeast two-hybrid screening with AtCYP38 as the bait protein. X-ray crystal structural analysis showed that the mature full-length AtCYP38 is composed of an N-terminal αhelical domain and a C-terminal cyclophilin β-sheet domain, and these two domains could possess different functions [23]. Therefore, we utilized three baits representing different forms of AtCYP38, including the mature full-length protein (CYP38F, without signal peptide), the N-terminal α-helical domain (CYP38N), and the C-terminal β-sheet domain (CYP38C) to perform yeast two-hybrid screening against an Arabidopsis genome wide cDNA library ( Fig. 2A).
We screened approximately a total of 2.0×10 7 yeast transformants with these 3 bait proteins, respectively. When CYP38F is used as the bait, we obtained about 70 con rmed positive colonies. After data ltering and interaction con rmation, we found that 8 chloroplast proteins could be interacting partners of AtCYP38 (Fig. 2B, 3A and Table 3). Only 3 proteins were localized in the chloroplast thylakoid lumen, including PSBP-1 (At1g06880), TSP9 (At3g47070) and DEG8 (At5g39830). After screening with CYP38N as the bait, we obtained approximately 50 con rmed positive clones, and further narrowed down to 4 candidate proteins according to their sequence results. However, none of 4 candidate protein was localized in chloroplast (Fig. 2B, 3B and Table 4). Notably, there are a very limited number of positive colonies obtained from screening with CYP38F and CYP38N as baits, indicating that those two baits display a low activity in the YTH screening assay.
When screening the same library with the CYP38C domain, we then obtained about 200 con rmed positive colonies, which were much more than those obtained from screening with CYP38F or CYP38N.
After data cleaning up and re-validation, we obtained 15 chloroplast proteins to be interacting candidates of AtCYP38 (Fig. 2B, 3C and Table 5). Interestingly, several chlorophyll binding proteins were identi ed in this screening, including LHCB1.1, LHCB1.2, LHCB1.5 and LHCB4.1. Other 3 thylakoid membrane proteins, the PSI subunit PASG, the Cytb6f complex subunit PETC, and ROD1 [29], an enzyme involved in lipid metabolism, were also found to interact with AtCYP38. Three interacting proteins are thylakoid lumen localized proteins, including PSBP-1, PETE2, and AtCYP38. Notably, PSBP-1 is also a partner of CYP38F. In addition, we also found some proteins are not localized in the thylakoid lumen but still can be identi ed by yeast two-hybrid assay (Table 5). However, it is unlikely that these proteins physically interact with AtCYP38, considering the different compartmental locations of these proteins and AtCYP38.
It was suggested that the C-terminal domain plays its function to bind other proteins, and the N-terminal bundle domain acts as the regulatory domain, which suppressing AtCYP38 to bind its targets [23]. To test this hypothesis, we cross examined the identi ed proteins via cDNA library screening with different baits individually. We found that all target proteins screened by CYP38F and CYP38N could interact with CYP38C, but proteins identi ed from screening with CYP38C could not interact with CYP38F or CYP38N (Fig. 4), which is in agreement with the above assumption.
Altogether, after screening 2.0×10 7 colonies with three different forms of AtCYP38, we obtained a very limited number of potential interacting partners of AtCYP38. Only a few thylakoid lumen proteins were identi ed in the YTH screening, including PSBP-1, TSP9, DEG8, PETE2 and AtCYP38. We initially expected that a larger number of interacting proteins of AtCYP38 could be identi ed, given its the chaperone-like activity. The unexpected YTH screening results prompted us to think that screening against a genome wide cDNA library may not be an e cient way to identify the interested interacting proteins. Because proteins in the thylakoid lumen only occupy a very small fraction of the plant cell proteome, it is not surprising that cDNA for thylakoid lumen proteins are relatively rare in this library. Therefore, there exist too much noise background due to an overwhelming amount of unrelated proteins in this screening, and consequently, the chance would be very low for AtCYP38 to sh out target proteins which are located in the chloroplast thylakoid lumen.
Yeast two-hybrid screening against a thylakoid lumen protein mini library Given that screening the genome wide cDNA library was not a very successful experience, we decided to launch an alternative screening strategy. Since AtCYP38 is a thylakoid lumenal protein, it would be a better choice to screen a library only harboring thylakoid lumen proteins and thylakoid membrane protein with lumenal domains, therefore massive unrelated proteins outside of this compartment would not interfere this screening process.
In order to achieve that, we constructed a yeast two-hybrid mini library harboring a majority of the previously identi ed lumenal proteins and lumenal domains of various thylakoid membrane proteins. The library was screened against baits including CYP38F, CYP38N and CYP38C, respectively. Interestingly, when we used CYP38F or CYP38N domain to perform the screening, we failed to nd any positive potential interacting proteins.
Validating localization and in vitro pull-down con rmation assay.
AtCYP38 is known to be present in the chloroplast thylakoid lumen, and interacting proteins should be in this subcellular compartment as well. In order to verify the localization of candidate proteins, we selected three predicted lumenal proteins, At1g21500, TLP17.9 (At4g24930) and TLP15 (At5g52970), and fused the green uorescent protein (GFP) tag to their C-terminus. The GFP-fused proteins were transiently expressed in leaf epidermal cells from Nicotiana benthamiana and analyzed by confocal microscopy. As we expected, At1g21500, TLP17.9 and TLP15 are localized in the chloroplast (Fig. 6A).
In order to further con rm the direct interaction between identi ed proteins with AtCYP38, we selected PSBP-1 and FKBP13 to perform the GST pull-down assays. As shown in Fig. 6B, GST-PSBP-1 and GST-FKBP13 were speci cally pulled down by CYP38C-HIS but not GST, con rming the interaction between those two proteins and CYP38C in vitro. At the meantime, we also included two proteins with unknown function, At1g21500 and TLP17.9, to con rm their interaction with AtCYP38 by the same method. GST-21500 or GST-24930 also can be observed an obvious interaction with CYP38C in the pull-down assay (Fig. 6B). Overall, these results indicated that the interactions between AtCYP38 and our candidate proteins are valid, and our screening strategies are reliable and e cient.

Discussion
As a key protein functioning in PSII repair and assembly, AtCYP38 should physically interact with its substrate proteins and cofactor proteins to play its roles in the chloroplast thylakoid lumen. Finding its lumenal interacting proteins is a key initial step to understand how AtCYP38 perform its functions. In this study, we applied multiple approaches to identify lumenal proteins potentially interacting with AtCYP38.
From the web-based programs, we obtained 12 thylakoid lumenal proteins and 3 membrane proteins (Table 1 and 2). Screening against a genome wide cDNA library revealed that 5 lumenal proteins (PSBP-1 detected twice) and 8 thylakoid membrane proteins could potentially interact with AtCYP38 (Table 3, 4 and 5). From the screening against thylakoid lumen mini library, we obtained 9 soluble thylakoid lumenal proteins and 6 membrane proteins (Fig. 5, Table 6). We consider that all of these proteins identi ed by different approaches could be potential interacting proteins of AtCYP38, because they meet functional pro les in assembly and/or maintenance of photosynthetic apparatus.

Possible functional roles of AtCYP38 based on interacting proteins
Identi ed interacting proteins of AtCYP38 could be classi ed into two major groups: thylakoid membrane proteins and thylakoid lumen soluble proteins. Membrane proteins included light harvesting complex proteins (LHCBs), subunit proteins in PSII, PSI, Cytb6f, and ATPase complexes. The identi cation of PSII components strengthens the conception that AtCYP38 is functionally tightly related to PSII's function. The nding of components in other photosynthetic complexes implied that AtCYP38 might have additional functions other than a restricted role in PSII. The other membrane proteins such as HHL1 (At1g67700), LQY1 (At1g75690), and ALB3B (At2g28800) also probably suggested the multiple roles of AtCYP38 in chloroplast.
Remarkably, several thylakoid lumen proteases, including CTPA, CTPB, CTPC, DEG8, and DEG14 are identi ed as interacting proteins of AtCYP38. CTPs are responsible for the pD1 C-terminal extension processing, which is an essential step for PSII assembly and repair [30,31]. DEG8 and DEG14 protein are involved in damaged protein degradation in the chloroplast thylakoid [32,33], indicating that AtCYP38 is very likely to be involved in the photosynthetic apparatus repair process. TAPX4 and PRXQ are essential proteins protecting thylakoid membrane complexes from attack of reactive oxygen species (ROS) [34,35], showing that AtCYP38 might play a role to response against ROS stress. We also found two thylakoid lumen immunophilins, including FKBP18 and FKBP16-3, to be interacting proteins of AtCYP38, indicating that these proteins could be the working partners of AtCYP38. A number of functionally unknown proteins could interact with AtCYP38, including At1g21500 At2g23670 At2g26340 TLP17.9 (At4g24930), TLP15 (At5g52970), and the phosphatase protein, TLP18.3 (At1g54780) [36,37]. Especially, TLP15 and TLP18.3 were identi ed both by the web-based program and the YTH screening. There should be a strong functionally relationship between these two proteins and AtCYP38. The TLP15 protein is also predicted to harbor phosphatase domain, implying a possible phosphatase function in thylakoid lumen. It indicated that AtCYP38 could be involved in protein de-phosphorylation, in agreement with earlier reports that AtCYP38 homolog in spinach is identi ed in a process of chloroplast phosphatase puri cation [19,20,21].
The possible working mechanism of AtCYP38 AtCYP38 protein contains two distinct domains: an N-terminal domain and a C-terminal domain, and these two domains were closely packed to form a strong intramolecular interaction [23]. The N-terminal domain was proposed to be a leucine zipper domain and responsible for the protein-protein interaction [19]. However, in our YTH screening, the N-terminal domain showed a very low a nity to interact with other proteins in YTH screenings. Similarly, the full-length mature AtCYP38 protein also showed a low a nity to interact with other proteins. On the contrary, the C-terminal domain could interact with a substantial number of thylakoid lumen proteins, which could be functionally related to AtCYP38. Therefore, we consider that the C-terminal domain is responsible for binding it functional targets, and the N-terminal domain serves as an inhibitory regulatory domain. The dynamic intramolecular interaction is required for AtCYP38 in vivo function under different energy status in chloroplast. We postulated that thylakoid membrane proteins and proteases in the thylakoid lumen might be substrates of AtCYP38, while the other small lumen soluble proteins would be its cofactors.
In summary, several thylakoid proteins were identi ed to be functionally related to AtCYP38. It is a good initial step to uncover the working mechanism of AtCYP38 in the thylakoid lumen. We understand this research only lay a foundation for future study to characterize how AtCYP38 works in thylakoid lumen. Next step, we will apply molecular biology, genetics and biochemistry tools to study the functional relationship between AtCYP38 and its partners. For those uncharacterized proteins, we plan to use CRISPR-Cas9 technology to create protein de ciency mutants to explore their physiological function and their relationship with AtCYP38. We anticipated that future research on the partner proteins of AtCYP38 will help us to understand its molecular mechanism.

Potential interacting targets prediction
Co-expression network analysis of AtCYP38 was performed by using the analytical tool of ATTED-II database: http://atted.jp [27], from which the associated gene was analyzed. In this study, genes with lower mutual rank value (MR) were considered as the co-expression network modules. AtCYP38 protein interaction network was analyzed through the STRING database on the website: http://string-db.org [28]. Based on biological functions and localizations, we selected interactors that connected with AtCYP38 using the highest con dence interaction score for further network construction.
Yeast two-hybrid library screening against genome wide cDNA library The mature AtCYP38 protein has two domains: an N-terminal α-helical domain and a C-terminal cyclophilin β-sheet barrel domain, which linked by an intermediate acidic loop [23]. DNA fragments encoding mature full-length AtCYP38 (residues 92-437), N-terminal domain (residues 92-232), and Cterminal domain (residues 236-437), were cloned into a pGBKT7 vector to generate pGBKT7-CYP38F, pGBKT7-CYP38N and pGBKT7-CYP38C, respectively. They were transformed into host strain AH109 using the modi ed lithium acetate/PEG method according to the protocol described previously by Fields and Ausubel et al [38,39], and assayed auto-activation tests on a series of nutrient de cient selective plates. The Arabidopsis cDNA library (prey) were transformed into yeast AH109 harboring BD-bait and plated on selective medium for target colonies. Meanwhile, dilution yeast cells spread onto control SD/-Leu/Trp plates to make sure the transfer e ciency, the estimate counting results indicated that we have more than 2×10 7 transformants in our three individual screenings. The colonies showing potential interaction on SD/-Leu/Trp/His plates were further assayed on SD/-Leu/Trp/His/Ade selective medium until new colonies were grown up.
The plasmid DNA of candidate positive yeast colonies was extracted according to manufacturer's instructions of TIANprep Yeast Plasmid DNA kit (TIANGEN Biotech, Beijing), and then retransformed back to Escherichia coli to amplify the cDNA inserts. The colonies were then sequenced with T7 primer: TAATACGACTCACTATAGGGCG.
Yeast two-hybrid analysis against lumen protein library To identify potential interacting partners for AtCYP38 in the chloroplast thylakoid lumen and on the thylakoid membrane, we constructed a yeast two-hybrid mini library harboring chloroplast lumenal proteins/domains. DNA fragments encoding proteins experimentally identi ed or predicted to present in the thylakoid lumen [12,13], and domains in the lumen of PSII subunits [40,41], PSI subunits [42], and Cytb(6)f complex subunits [43] were cloned into the Gal4 AD vector pGADGH. Each AD vector in this library was screened with BD vectors pGBKT7-CYP38F, pGBKT7-CYP38N and pGBKT7-CYP38C individually as described above.

Subcellular localization analysis
For subcellular localization studies, At1g21500, TLP17.9 and TLP15 coding sequences were recombined into modi ed pMDC83 destination vector, respectively. The constructs were transfected into Agrobacteria GV3101, and the strains were in ltrated into tobacco (Nicotinana benthamiana) epidermal cell using a needle-less syringe [44]. The GFP uorescent signal was detected after 48 hours in ltration by using confocal laser scanning microscope. The PCR primers used in this study are listed in supplementary Table 1.

In vitro pull-down assay
The in vitro pull-down assay was performed according to the protocol as detailed by previous described [45]. Recombinant GST-21500F (mature protein, without signal peptide), GST-TLP17.9F (mature protein, without signal peptide), GST-FKBP13, GST-PSBP-1 or CYP38C-HIS was transformed into E. coli strain BL-21 and puri ed using glutathione or Ni sepharose beads, respectively. Reaction components were mixed and incubated in PH 7.4 phosphate buffer solution at 4℃ for 2 hours, the precipitated beads were then washed thoroughly with PBST buffer (PBS buffer with 0.1% Tween 20). Subsequently, washed beads were eluted and analyzed by SDS-PAGE. The PCR primers used in this study are listed in supplementary Table 1

Consent for publication
Not applicable.

Availability of data and materials
All the data on the present study has been included in the tables or supplementary tables in this manuscript. The datasets used and/or analyzed in this study are available from the corresponding author on reasonable request.

Competing interests
Aigen Fu is a member of the BMC plant biology editor board, we declare that we have no other con icts of interest.    Figure 1 The correlation networks between AtCYP38 and its potential interacting proteins. A. The co-expression gene network using the AtCYP38 as query gene on the ATTED-II (http://atted.jp). AtCYP38, shown in yellow module, is co-expressed with multiple photosynthesis related genes. Thylakoid lumen localized proteins are colored in green. B. Protein-protein interaction network is generated by STRING search tool for CYP38 regulatory factors at highest con dent score (>0.970). Yellow modulate indicate the proteins Page 22/27 which are predicted located in thylakoid lumen. Purple line represents the known interactions from experimentally determined. Green, blue, leaf green and black lines represent the predicted interactions from gene neighborhood, co-occurrence, text mining and co-expression, respectively.

Figure 2
The schematic diagram of AtCYP38 protein different domains and interacting proteins identi ed by yeast domain (amino acid residues from 92 to 232) and C-terminal cyclophilin β-sheet domain (amino acid residues from 236 to 437). B. Venn diagram showing the 25 potential AtCYP38-interacting partners are identi ed by YTH screening using 3 different domains.

Figure 3
Con rmation of AtCYP38 candidate partners by yeast two-hybrid system. To test interaction results by yeast two-hybrid screening, both prey and bait vectors were co-transformed into yeast AH109 strain and Page 24/27 inoculated onto SD/-Leu/Trp and SD/-Leu/Trp/His plates. A shows the con rmation of interaction between CYP38F domain and its screening results. B. Con rmation of interaction between CYP38N domain and its screening results. C. Con rmation of interaction between CYP38C domain and its screening results.

Figure 4
Cross examination of interaction of potential targets with AtCYP38 different domains. A. Con rmation of interaction between CYP38C domain and the screening results which identi ed by CYP38F and CYP38N domains. B. Con rmation of interaction between CYP38F or CYP38N domain and screening results by using CYP38C protein as a bait.

Figure 5
Interacting proteins of the AtCYP38 C terminal domain by screening a thylakoid lumen mini-library. A. Identi cation of potential interaction proteins in thylakoid lumen. B. Identi cation of potential interacting proteins on thylakoid membrane with lumenal fragments.
GST, GST-21500F, GST-TLP17.9F, GST-PSBP-1 or GST-FKBP13 was pulled down by CYP38C-HIS immobilized on beads. Protein were detected by immunoblotting using GST antibody, the interaction of CYP38C and GST were used as a negative control.

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