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First published online August 26, 2005; 10.1104/pp.105.065284 Plant Physiology 139:425-436 (2005) © 2005 American Society of Plant Biologists
A Copper Chaperone for Superoxide Dismutase That Confers Three Types of Copper/Zinc Superoxide Dismutase Activity in Arabidopsis1Department of Life Science and Institute of Plant Biology, National Taiwan University, Taipei 10617, Taiwan (C.-C.C., W.-C.L., W.-Y.G., S.-M.P., T.-L.J.); and Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan (L.-J.C., H.-m.L.)
The copper chaperone for superoxide dismutase (CCS) has been identified as a key factor integrating copper into copper/zinc superoxide dismutase (CuZnSOD) in yeast (Saccharomyces cerevisiae) and mammals. In Arabidopsis (Arabidopsis thaliana), only one putative CCS gene (AtCCS, At1g12520) has been identified. The predicted AtCCS polypeptide contains three distinct domains: a central domain, flanked by an ATX1-like domain, and a C-terminal domain. The ATX1-like and C-terminal domains contain putative copper-binding motifs. We have investigated the function of this putative AtCCS gene and shown that a cDNA encoding the open reading frame predicted by The Arabidopsis Information Resource complemented only the cytosolic and peroxisomal CuZnSOD activities in the Atccs knockout mutant, which has lost all CuZnSOD activities. However, a longer AtCCS cDNA, as predicted by the Munich Information Centre for Protein Sequences and encoding an extra 66 amino acids at the N terminus, could restore all three, including the chloroplastic CuZnSOD activities in the Atccs mutant. The extra 66 amino acids were shown to direct the import of AtCCS into chloroplasts. Our results indicated that one AtCCS gene was responsible for the activation of all three types of CuZnSOD activity. In addition, a truncated AtCCS, containing only the central and C-terminal domains without the ATX1-like domain failed to restore any CuZnSOD activity in the Atccs mutant. This result indicates that the ATX1-like domain is essential for the copper chaperone function of AtCCS in planta.
Reactive oxygen species (ROS), including superoxide, hydrogen peroxide, and hydroxyl radicals, are generated as a by-product in cells from physiological reactions such as electron flow in chloroplasts and mitochondria and some redox reactions (Fridovich, 1978  ). Accumulation of ROS might cause damages to living organisms (Imlay and Linn, 1988; Bowler et al., 1992; Mehdy, 1994). Superoxide dismutase (SOD), a group of metalloenzymes, belongs to the enzymatic defense system against ROS and catalyzes the dismutation of superoxide radicals to molecular oxygen and hydrogen peroxide (Beyer et al., 1991). Copper/zinc SOD (CuZnSOD), manganese SOD (MnSOD), and iron SOD (FeSOD) are three types of SODs reported in plants.
MnSOD is localized in mitochondria, whereas FeSOD is localized in chloroplasts (Jackson et al., 1978
Copper chaperones are a novel class of proteins involved in intracellular trafficking and delivery of copper to copper-containing proteins (Harrison et al., 1999
CCS possesses three functionally distinct protein domains. The N-terminal ATX1-like domain, bearing striking homology to ATX1 and containing the MXCXXC copper-binding site, is required for CCS function under strict copper limitation conditions in vivo (Schmidt et al., 1999
In yeast, a truncated yCCS without the ATX1-like domain complemented the lys7 null mutant weakly, compared with the intact yCCS. This result indicated that the ATX1-like domain is important for the yCCS activity (Schmidt et al., 1999 To further study the function of AtCCS, we have obtained an AtCCS knockout mutant. We show here that the mutant has lost all three forms of CuZnSOD activity. Complementation of the mutant with the cDNA encoding the 320-amino acid polypeptide, as annotated by MIPS, restored all three activities. The results suggest that one AtCCS gene encodes both the cytosolic and the chloroplastic forms of AtCCS and activates CuZnSOD activities at different subcellular locations. In addition, we show that the ATX1-like domain of AtCCS is essential for the copper chaperone function in planta.
AtCCS Gene in Arabidopsis In Arabidopsis, the AtCCS gene (At1g12520) is composed of six exons and five introns. Analysis of the genomic sequence relevant to this gene revealed the existence of an additional ATG located upstream of the annotated initiation codon, and this additional ATG is in frame with the open reading frame annotated in TAIR database (Fig. 1A). This additional ATG is the start codon predicted in the MIPS Arabidopsis database (http://mips.gsf.de/proj/thal/db/index.html). In TAIR database, the AtCCS gene is predicted to encode a 254-amino acid polypeptide containing no signal peptide for organelle targeting (designated hereinafter as AtCCScytosolic [AtCCScyt]). Whereas, the AtCCS gene in the MIPS database is predicted to contain an additional 66 amino acids carrying a putative chloroplastic targeting signal (designated hereinafter as AtCCSchloroplastic [AtCCScp]). However, the upstream ATG was not found in four cDNA clones encoding AtCCS (AF179371, AF061517, AY050357, and GSLTSIL10ZC12) identified in the MIPS database. In our results, the upstream ATG is present in a 979-bp reverse transcription (RT)-PCR product amplified from rosette leaves and flowers using a specific primer set, ACCS-15 and ACCS-12 (Fig. 1, A and B). This confirmed the existence of the upstream ATG in the AtCCS transcript. Sequence comparison between the genomic PCR and RT-PCR products amplified by the same primer pair positioned the exons and introns as shown in Figure 1A. Thirteen cDNA clones obtained by 5'-RACE were randomly selected for sequencing. The results indicated that the first ATG existed in the 5'-end of all 13 AtCCS transcripts. Moreover, 10 clones were mapped to one nucleotide upstream, while the other clones were mapped to four, 14, and 26 nucleotides upstream of the first ATG, respectively (Fig. 1C, arrows).
In Figure 1D, bioinformatic searches (on Web sites of http://www.cbs.dtu.dk/services/ChloroP and http://www.cbs.dtu.dk/services/TargetP) revealed that the additional 66 amino acids encoded by the sequence between the first and second ATG comprise a potential chloroplast-targeting transit peptide. The following 67th to 161st amino acids contain a highly conserved region, MXCXXC, which is a homologous domain of the ATX1 copper chaperone. The central domain (162nd to 290th amino acids) shares 19.1% sequence similarity to CSD1, and 22.7% similarity to both CSD2 and CSD3 of Arabidopsis. The last 30 residues of the C-terminal domain are unique to CCS and contain another highly conserved CXC motif.
The pattern for SOD activity in Arabidopsis is shown in Figure 2A. In addition to the two CuZnSOD activities as reported in Kliebenstein et al. (1998)
We then analyzed the expression of the AtCCS gene. In northern-blot analyses, the AtCCS cDNA probe detected only one hybridization signal in flowers but not in rosette leaves (Fig. 2B). However, low levels of expression could be detected in rosette and cauline leaves by RT-PCR (Fig. 2C). In immunoblot analyses for protein expression, for some unknown reasons, our anti-AtCCS serum could detect AtCCS only when used on nondenaturing, but not on denaturing, PAGE (data not shown). As shown in Figure 2D, the anti-AtCCS antiserum detected several signals in stems and flowers when used in nondenaturing PAGE. Very weak signals were sometimes also observed in leaves (Fig. 2, D and E). The AtCCS signals did not result from antiserum cross-hybridization with SOD since they migrated at different positions in the native gels (Fig. 2A, western). Furthermore, overexpression of a cDNA encoding AtCCScyt (AtCCScyt/wild type) increased the AtCCS signals detected (Fig. 2E), supporting that the bands detected on the native gels were indeed AtCCS.
An Atccs mutant (SALK_025986), caused by a T-DNA insertion in the second exon (Fig. 3A), was obtained. Lines homozygous for the T-DNA insertion were identified by PCR and confirmed by Southern-blot analyses (Fig. 3B). Incomplete digestion might be the cause of higher Mr bands seen in the Atccs sample digested with XbaI. In this Atccs mutant, neither the AtCCS transcript nor the AtCCS protein was detected (Fig. 3, C and D).
Almost no CuZnSODs activity was detected in the Atccs mutant (Fig. 4A) even in the presence of CuZnSOD transcripts (Fig. 4B) and proteins (Fig. 4C), which indicated that AtCCS was necessary for the activation of all types of CuZnSOD activity. The protein levels of CSD1 and CSD2 in the Atccs mutant were decreased in all tissues tested compared to the levels in the wild type (Fig. 4C). However, the steady-state level of CSD1 and CSD2 transcripts were not reduced in the Atccs mutant (Fig. 4B).
Different Recovery of CuZnSOD Activities in the Atccs Mutant Complemented with AtCCScyt, AtCCScp, and AtCCScp-mutant Because CSD1, CSD2, and CSD3 activities could be simultaneously detected in flowers, in subsequent studies only results from flower tissue were presented. The Atccs mutant was transformed with AtCCScyt, the TAIR-predicted 254-amino acid open reading frame of the AtCCS gene (the transformants were designated as AtCCScyt/Atccs), or with a 1.64-kb genomic fragment (Fig. 1, A and B) that was sufficient to encode the MIPS-predicted 320-amino acid open reading frame (designated as AtCCScp/Atccs), to test for the recovery of the CuZnSOD activities. Both constructs were driven by the cauliflower mosaic virus 35S promoter. Immunoblot analyses revealed that AtCCS proteins were indeed expressed in AtCCScyt/Atccs and AtCCScp/Atcc transgenic plants (Fig. 5B). The CuZnSOD activity profile in different AtCCScyt/Atccs transgenic lines was the same as that of the wild type except that the chloroplastic CSD2 activity was never recovered (Fig. 5A, AtCCScyt/Atccs 1 to 3). However, T1 transgenic individuals transformed with AtCCScp were able to recover all three types of CuZnSOD activity (Fig. 5A, AtCCScp/Atccs 1 to 3). Recovery of all three types of CuZnSOD activity was also observed when the Atccs mutant was transformed with the AtCCScp cDNA driven by the 35S promoter (data not shown). These results indicated that AtCCScp, but not AtCCScyt, was sufficient to recover all three CuZnSOD activities in the Atccs mutant. To further test the importance of the 66 amino acids at the N terminus of AtCCScp, a mutant construct (AtCCScp-mut, Fig. 6A) was generated by introducing a guanine nucleotide before the second ATG through site-directed mutagenesis. CuZnSOD activity profile in the AtCCScp-mut/Atccs T1 transgenic plants was again missing the CDS2 band (Fig. 6B). This result suggested that the additional 66 amino acids in AtCCScp comprised a plastidic transit peptide necessary to restore the chloroplastic CSD2 activity.
Chloroplastic Localization of AtCCScp To investigate if the first 66 amino acids of AtCCScp function as a chloroplast-targeting transit peptide, 35S-labeled AtCCScyt and AtCCScp were synthesized by in vitro transcription/translation and incubated with isolated pea (Pisum sativum) chloroplasts under import conditions. The AtCCScp cDNA directed the synthesis of two proteins, one 34 kD and one 29 kD (Fig. 7, lane 5). The AtCCScyt cDNA directed the synthesis of only the 29-kD protein (Fig. 7, lane 1), suggesting that the 29-kD product from AtCCScp was a result of internal initiation of translation from the second ATG. The 29-kD protein synthesized from AtCCScyt could not be imported into chloroplasts (Fig. 7, lane 3). In contrast, when the protein products from AtCCScp were incubated with chloroplasts, a 29-kD protein was produced (Fig. 7, lane 7), and this 29-kD protein was fully resistant to thermolysin (Fig. 7, lane 8), indicating its localization within the chloroplasts. Since the 29-kD protein synthesized from AtCCScyt could not be imported into chloroplasts (Fig. 7, lane 3), the 29-kD chloroplast-localized protein produced after import of AtCCScp (Fig. 7, lane 7) most likely resulted from import of the 34-kD protein directed by AtCCScp. These results indicated that the first 66 amino acids of AtCCScp were necessary for the chloroplast import of AtCCScp.
Interaction between AtCCScyt and CSD1 We further investigated the possibility of direct interaction between AtCCScyt and CSD1 using the yeast two-hybrid assay. The AtCCScyt cDNA and CSD1 cDNA were fused with GAL4 DNA binding domain (BD-AtCCScyt) and activation domain (AD-CSD1), respectively. Cells containing both the BD and AD constructs were produced by mating of haploid cells containing individual plasmids. Diploid cells were then streaked on selection media to test for protein interaction. AD-CSD1 and BD-AtCCScyt together resulted in growth of the yeast transformants while cells transformed with individual binding or activation construct or with control plasmids did not grow (Fig. 8). This result suggested that AtCCScyt physically interacted with CSD1.
Importance of the ATX1-Like Domain in Conferring CuZnSOD Activity A truncated AtCCS gene containing only the central and C-terminal domains (hereinafter referred as AtCCSD2D3, encoding the 162th–320th amino acids, Fig. 1D) was transformed into the Atccs mutant (designated as AtCCSD2D3/Atccs lines) to test for the importance of the ATX1-like region. The expression of the AtCCSD2D3 transcript in AtCCSD2D3/Atccs T1 transgenic lines was confirmed by northern-blot analyses (data not shown). Among 70 AtCCSD2D3/Atccs T1 transgenic lines, no CuZnSOD activity was observed in any of the plants even when protein extracts were prepared from detached leaf stalks that had been first incubated with a 1 mM CuSO4 solution at 25°C for 4 h (Fig. 9A). However, a weak CuZnSOD activity did appear if the leaf protein extracts were prepared first and the extracts were incubated directly in a 1 mM CuSO4 solution at 25°C for 4 h (Fig. 9B). This partial restoration of CuZnSOD activity was due to AtCCSD2D3 since no activity was observed in the Atccs mutant without the transgene (Fig. 9B, Atccs). Furthermore, the CSD3 activity, never observed in vegetative tissues, was detected in the leaf extracts of wild type plants under such copper-supplemented conditions (Fig. 9, A and B, wild type, compare control and +CuSO4).
One AtCCS gene was demonstrated to activate three forms of CuZnSODs localized in different subcellular locations in this study. The results obtained from the AtCCScyt/Atccs, AtCCScp/Atccs, and AtCCScp-mut/Atccs transgenic plants and the in vitro protein import analysis indicated that the open reading frame from the first ATG encoded a plastidic AtCCS containing a chloroplastic transit peptide and was responsible for CSD2 activation (Figs. 5–7  ). Import of cytosolically synthesized precursor proteins into chloroplasts is usually very efficient and no precursor proteins are normally detected in the cytosol (Sun et al., 2001). Furthermore, the AtCCS precursor with the chloroplast-targeting transit peptide may not possess the chaperone function before the removal of the transit peptide. Therefore, a cytosolic form of AtCCS without the transit peptide is most likely also produced from AtCCScp from the second ATG for the activation of CSD1 and CSD3. Results from AtCCScyt/Atccs and AtCCScp-mut/Atccs transgenic plants also support that the second ATG has the potential to be used as a translation initiation site. However, detection of the two proteins synthesized from AtCCScp will be difficult since the cytosolic form and the processed mature form in plastids would be almost the same size (Fig. 7).
Several mechanisms have been reported for translation of two proteins from a single gene (Danpure, 1995
Rizhsky et al. (2003)
In the copper-deficient mice and the CCS knockout mice, the level of SOD1 protein was significantly reduced to different levels in different tissues, even though the level of SOD1 transcript was not altered (Prohaska et al., 2003
Protein translocation into chloroplasts and peroxisomes occur posttranslationally (Schnell and Hebert, 2003
The importance of the ATX1-like domain in copper binding has been studied in yeast (Schmidt et al., 1999
Plants, Growth Condition, and CuSO4 Treatment The Atccs mutant (SALK_025986) of Arabidopsis (Arabidopsis thaliana) Columbia ecotype was obtained from the Arabidopsis Biological Resource Center (The Ohio State University). Seeds were sown in BIO-MIXTTING SUBSTRATUM (Agricultural Materials Company), incubated in the dark for 2 to 4 d at 4°C, and transferred to a growth chamber for germination. The seedlings were grown under 16-h light/8-h dark at 23°C/21°C at a light intensity of 60 to 100 µmol m–2 s–1.
For experiments shown in Figure 9, the detached rosette leaf stalks were immersed in a 1 mM CuSO4 solution, or crude protein extracts prepared from rosette leaves were incubated with 1 mM CuSO4. Both treatments were performed at 25°C for 4 h. Protein extracts were then prepared from the detached leaves and samples from both treatments were assayed for CuZnSOD activity as described by Pan et al. (2001)
Sequence information of genes, proteins, and cDNAs were retrieved by searching public databases with the BLAST algorithm (Altschul et al., 1997
Genomic DNA was isolated from rosette leaves according to Dellaporta (1993)
Total RNA was isolated with TRIZOL reagent (Invitrogen) and 10 µg total RNA was separated on a 1.2%-formaldehyde agarose gel and hybridized with AtCCS, CSD1, CSD2, and ACT2 cDNA fragments labeled with Digoxigenin-11-dUTP by PCR. Hybridization signals were detected as described for Southern-blot analyses. The cDNA probes were amplified by PCR with the following gene-specific primers: AtCCS, ACCS-15 and ACCS-12 (as described in Southern-blot analyses); CSD1, 5'-CGCCATGGCGAAAGGAGTTGCAGTTT-3' (the NcoI site is underlined) and 5'-ATCCCGGGGCCCTGGAGACCAATGAT-3' (the SmaI site is underlined); CSD2, 5'-ACGGATCCATCCTCGCATTCTCATCTCCTT-3' (the BamHI site is underlined) and 5'-ACTCTAGAGACGGCACTCATCTTCTGG-3' (the XbaI site is underlined); ACT2, 5'-AGCTCCCGGGCTAAGCTCTCAAGATCAAAGGCTTA-3' (ACT2-376s) and 5'-AGCTCCCGGGTTAACATTGCAAAGAGTTTCAAGGT-3' (ACT2-3'N3). The ACT2 and AtCCS probes detected only one signal each on northern-blot analyses with sizes of approximately 1.7 and 1.1 kb, respectively. Thus, the two probes were mixed together to hybridize with the same membrane.
For RT-PCR, Superscript II (Invitrogen) kit was used according to the manufacturer's instructions. Total RNA (1 µg) was used and primed with the oligo (dT) primer for cDNA synthesis. The RT reaction mixture (1 µL) was used for subsequent PCR with the following gene-specific primers: AtCCS, ACCS-15 and ACCS-12, or ACCS-1, 5'-ATTCTAGAGCCTCTGCGATTCCCATC-3' (the XbaI site is underlined) and ACCS-8, 5'-CTGTGCTGGCTGCTCCGTTTGT-3' (Fig. 1A), and internal control ACT2, ACT2-376 s and ACT2-3'N3, or UBQ10, 5'-GATCTTTGCCGGAAAACAATTGGAGGATGGT-3' (UBQ1) and 5'-CGACTTGTCATTAGAAAGAAAGAGATAACAGG-3' (UBQ2). The 5'-terminal sequence of the AtCCS transcript was obtained using the BD SMART RACE cDNA Amplification kit (CLONTECH) according to the manufacturer's instructions (5'-RACE). Total RNA (1 µg) from the flowers in the wild type was used for cDNA synthesis. The subsequent PCR was amplified with the universal primers mix provided in the kit and the gene-specific reverse primer, ACCS-8. The PCR products of the 5'-RACE were cloned into the yT&A vector (Yeastern Biotech) for DNA sequencing.
Tissues were ground (tissue:medium ratio 1:3, w/v) with 150 mM Tris-HCl (pH 7.2), and the homogenate was centrifuged at 13,000g at 4°C for 10 min. The protein concentration was determined by the method of Bradford (1976)
Total protein (30 µg) was separated on a 10% nondenaturing polyacrylamide gel in Tris-Gly buffer (pH 8.3). A photochemical method modified from Beauchamp and Fridovich (1971)
The coding region of AtCCS gene predicted in TAIR database was amplified by PCR with the gene-specific primers 5'-TCTGGATCCATGGCGACTGCTCTCACT-3' (the BamHI site is underlined) and 5'-TCTCTCGAGGTTATTAAACCTTACTGG-3' (the XhoI site is underlined) that introduced the two restriction sites on the two ends. The PCR fragment was cloned into the pGEX6P-1 (Amersham Biosciences) for recombinant protein expression. The induction and purification of the fusion protein were performed as described by Pan et al. (1999)
For the detection of AtCCS, 60 µg of total protein was separated on a 10% nondenaturing polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Amersham Biosciences), blocked with 5% nonfat-milk in 1x PBST (phosphate-buffered saline containing 0.05% Tween 20) for 1 h, and then incubated with anti-AtCCS antiserum (1:1,000 dilution) for another hour. The washed membrane was incubated with 1:3,000 diluted goat anti-rabbit IgG conjugated with alkaline phosphatase (Perkin-Elmer); the signals in the membrane were detected by colorimetric reaction with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (PerkinElmer). For CuZnSOD immunoblot assay, 30 µg of total protein was separated on a 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane with the anti-CSD1 (1:1,000 dilution) and -CSD2 (1:2,000 dilution) anti-sera as described in Kliebenstein et al. (1998)
The AtCCScyt and AtCCScp cDNA fragments were amplified by RT-PCR with the following specific primers: AtCCScyt, ACCS-1 and ACCS-12; and AtCCScp, ACCS-15 and ACCS-12 (Fig. 1A). The RT-PCR products were cloned into the yT&A vector for DNA sequencing. Then the AtCCScyt and AtCCScp cDNA fragments were subcloned into the KpnI and BamHI sites of pBluescript SK+ (Stratagene). These constructs were used as templates for in vitro transcription/translation using the TNT T7 coupled wheat germ extract system (Promega) in the presence of [35S]Met according to the manufacturer's instructions. Isolation of chloroplasts from pea seedlings (Pisum sativum cv Little Marvel) and import of the labeled proteins into chloroplasts were performed according to Perry et al. (1991)
The AtCCScyt cDNA and the Arabidopsis CSD1 cDNA were amplified by PCR and subcloned into the pGBKT7 and pACT2 yeast (Saccharomyces cerevisiae) expression vectors (Matchmaker 3, CLONTECH), in-framed with the GAL4 DNA BD and the GAL4 AD, respectively. The resulting BD-AtCCScyt and AD-CSD1 vectors were then transformed into the yeast strain AH109 (MAT a) and Y187 (MAT
The pPZP200GB with
The AtCCScp and AtCCScp-mut genomic fragments were amplified by PCR with the following specific primers: AtCCScp, ACCS-15 and ACCS-12; AtCCScp-mut, ACCS-17, 5'-ATAATGGCATCAAGTTCTCAGGTCAGT-3' (the first ATG is boxed and the introduced extra G nucleotide is underlined), and ACCS-12. The PCR products were cloned into the yT&A vector for DNA sequencing. Then the KpnI/SacI fragments corresponding to AtCCScp and AtCCScp-mut were cloned into pBIB-HYG (Becker et al., 1992
Plasmids for plant transformation were transformed into Agrobacterium tumefaciens C58 by electroporation. Agrobacterium cells containing each plasmid were transformed into the Atccs mutant by the floral-dipping method (Clough and Bent, 1998 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers DQ003054 to DQ003058.
We thank Dr. Hans Bohnert (University of Illinois, Urbana-Champaign) and Dr. John Walker (University of Missouri, Columbia) for providing the AtCCS cDNA clone (BE038022) and the pSK-35S-BAR plasmid, respectively. We also thank Dr. Robert Last (Cornell University, Ithaca, NY) for kindly providing the Arabidopsis SOD anti-sera, and the Arabidopsis Biological Resource Center for the Atccs mutant (SALK_025986). We are grateful to Dr. Chu-Yung Lin and Dr. Wen-Ju Yang (National Taiwan University, Taipei) and Dr. John Walker and Dr. Kevin Lease (University of Missouri, Columbia) for critically reading and for revising the manuscript. Received May 7, 2005; returned for revision June 13, 2005; accepted June 18, 2005.
1 This paper is dedicated to memory of the late Dr. Shu-Mei Pan.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.065284. * Corresponding author; e-mail jinnt{at}ntu.edu.tw; fax 886–2–23638598.
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ABSTRACT
RESULTS
) using a modified lithium acetate method. For positive and negative controls, yeast cells were transformed with the plasmids pACT2-T antigen (AD-T antigen) and pGBKT7-53 (BD-p53), or with the plasmids pACT2-T antigen (AD-T antigen) and pGBKT7-Lam (DB-Lamin C), respectively. Yeast cells were inoculated into yeast peptone dextrose medium and cultured overnight. Cells were than streaked on synthetic dextrose medium lacking Leu, Trp, His, and Ade. 
-glucuronidase and BAR (BASTA resistance gene) cassettes was derived from pBI221 (CLONTECH) and pSK-35S-BAR (John Walker, University of Missouri Columbia). This binary vector was used to clone AtCCScyt and AtCCSD2D3. The cDNA fragment corresponding to AtCCScyt and AtCCSD2D3 was amplified by PCR with the following gene-specific primers: AtCCScyt, 5'-ATTCTAGAATGGCGACTGCTCTCACT-3' (the XbaI site is underlined) and 5'-GCGAGCTCTTAAACCTTACTGGCCACG-3' (the SacI site is underlined); and AtCCSD2D3, 5'-TCTGGATCCATGTCAGCTGCAGTAGCAGAATTC-3' (the BamHI site is underlined) and 5'-GCGAGCTCTTAAACCTTACTGGCCACG-3' (the SacI site is underlined). The PCR products were cloned into the XbaI/SacI sites of pPZP200GB by replacing the