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RESEARCH ARTICLE Ecm11protein of yeast Saccharomyces cerevisiae is regulated by sumoylation during meiosis Apolonija Bedina Zavec1, Aleksandra Comino1, Metka Lenassi2 & Radovan Komel1 1 Laboratory for Biosynthesis and Biotransformation, National Institute of Chemistry, Hajdrihova, Ljubljana, Slovenia; and 2Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov Trg, Ljubljana, Slovenia Correspondence: Apolonija Bedina Zavec, Laboratory for Biosynthesis and Biotransformation, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia. Tel.: 1386 1 476 0200; fax: 1386 1 476 0300; e-mail: [email protected] Received 15 March 2007; revised 19 July 2007; accepted 26 July 2007. First published online 20 September 2007. DOI:10.1111/j.1567-1364.2007.00307.x Editor: Ian Dawes Keywords SUMO; meiosis; Ecm11. Abstract Damaged regulation of the small ubiquitin–like modifier (SUMO) system contributes to some human diseases; therefore, it is very important to identify the SUMO targets and to determine the function of their sumoylation. In this study, it is shown that Ecm11 protein in Saccharomyces cerevisiae is modified by SUMO during meiosis. It is known that Ecm11 is required in the early stages of yeast meiosis where its function is related to DNA replication and crossing over. Here it is shown that the level of Ecm11 protein is low in mitosis, but high in meiosis. The highest level of Ecm11 is in the early-middle phase of sporulation. A specific site of sumoylation was identified in Ecm11 at Lys5 and evidence is provided that sumoylation at this site directly regulates Ecm11 function in meiosis. On the other hand, no relationship was observed between sumoylation of Ecm11 and its role during vegetative growth. It was shown that Ecm11 interacts with Siz2 SUMO ligase in a two-hybrid system; although Siz2 is not essential for the Ecm11 sumoylation. Introduction Sumoylation is one of the covalent posttranslational modifications, such as acetylation, methylation and ubiquitylation, which plays an important role in controlling protein function. SUMO modification affects many biological processes and is required for cell viability in yeast Saccharomyces cerevisiae, nematodes and higher eukaryotes (Fraser et al., 2000). Mammalian SUMO-1 is involved in a wide range of important cellular processes: p53 and c-jun transcriptional activation, signal transduction, inflammatory and immune responses (Melchior, 2000), maintenance of genome integrity (Muller et al., 2001; Hickson, 2003), and recombination (Shen et al., 1996). The SUMO protein is structurally similar to ubiquitin and is a member of the ubiquitin-like proteins. Sumoylation, like conjugation of all ubiquitin-like proteins, occurs as the result of the sequential action of specific enzymes: activating enzyme (E1), conjugating enzyme (E2) and ligase (E3). In the processing of SUMO to react with the target protein, SUMO is transferred from E1 to E2. E3 accelerates the rate of SUMO modification and confers specificity and regulation of the sumoylation process. SUMO is attached to a lysine (K) in the substrate, mostly within the SUMO consensus sequence h-K-X-E/D, where h 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c represents a large hydrophobic residue (usually L, I or V) and X is any residue (Johnson, 2004). In S. cerevisiae the SUMO homologue is known as Smt3 and is encoded by a single essential gene, whereas in vertebrates, four distinct paralogues (SUMO1-4) have been identified. Because yeast cells with deleted SMT3 gene are complemented by the human SUMO1 gene, it is assumed that the SMT3 gene is a yeast orthologue of the human SUMO1 (Johnson, 2004). In yeast SUMO conjugation process, Siz1 and Siz2 SUMO ligases are required for the majority of substrates (Johnson & Gupta, 2001), although new E3 were found in yeast recently (Zhao & Blobel, 2005; Cheng et al., 2006). The first SUMO-linked substrates characterized in budding yeasts were septins (Cdc11, Cdc3, and Shs1), but eliminating septin sumoylation has no detectable phenotypic effect (Hochstrasser, 2002). Topoisomerase II, proliferating cell nuclear antigen (Pol30), Pds5 and Ysc4 (two proteins involved in chromosome cohesion), and kinetochore proteins Ndc10, Bir1, Ndc80 and Cep3 were also shown to be sumoylated (Bachant et al., 2002; Stead et al., 2003; D’Amours et al., 2004; Montpetit et al., 2006). Recently, with a proteome-wide analysis of sumoylated proteins, many new potential sumoylated proteins were found in budding yeast; however, in most of these FEMS Yeast Res 8 (2008) 64–70 65 Yeast protein Ecm11 is sumoylated during meiosis SUMO substrates, the biological role of their sumoylation is not known. Ecm11 is a protein of S. cerevisiae with a strong meiotic phenotype (Zavec et al., 2004). Homozygous deletion of the ECM11 gene causes delay in the process of meiosis, lower efficiency of asci formation and lower spore viability. It was concluded that Ecm11 affects meiotic DNA synthesis and recombination. Cells with deleted ECM11 are also hypersensitive to calcofluor white, zymolase, papulacandin B and hygromycin B, indicating defects in glucan biosynthesis (Lussier et al., 1997). In a wide search of protein–protein interactions, it was found that Ecm11 interacts with SUMO (Smt3) in the two-hybrid system (Ito et al., 2000). Ecm11 protein has two lysine residues, K5 and K101, with corresponding surrounding sequence IKTE that could accept SUMO. In this paper it is shown that yeast Ecm11 belongs to the group of sumoylated proteins. The SUMO-attachment site in the Ecm11 protein and the biological role of Ecm11 sumoylation were identified. Materials and methods Gene disruption and site-directed mutagenesis Genes ECM11, SIZ1 or SIZ2 were deleted and replaced by the kanMX4 gene as described by Wach et al. (1996). Deleted strains were generated by transforming the yeast strain with linear PCR constructs containing the kanMX4 gene flanked by terminal sequences homologous to the ECM11, SIZ1 or SIZ2 gene. Replacement of the genes was verified by PCR analysis with specific oligonucleotides. For insertion of HA-tag (three HA) in the ECM11 gene and for mutagenizing Lys5 and Lys101 of Ecm11 to Asn, the ‘Delitto perfetto’ system was used (Storici et al., 2001). Long oligonucleotides of CORE cassette and oligonucleotides that eliminate the CORE cassette were designed. Strains were made by transformation, selection and counter selection (URA3, kanMX4). Mutations were verified by sequencing. Sequences of oligonucleotides are available upon request. Calcofluor white sensitivity The mutant strains in the logarithmic phase were plated on various concentrations of calcofluor white in YPD and observed for hypersensitivity relative to the isogenic wildtype strain, as described by Lussier et al. (1997). Strains The S. cerevisiae strains used for sporulation tests were as follows: yC66 (MATa, can1-100, his3-11,15, leu1-12, lys2-1, trp1-1, tyr1-2, ura3-1), yC67 (MATa, cyh2r, his3-11,15, leu1c,met13-c, trp1-1, tyr1-2, ura3-1) (G. Tevzadze); W303-1A (MATa, ade2 can1-100r his3-11,15 leu2-3112 trp1-1 ura3-1), W303-1B (MATa, ade2 can1-100r his3-11,15 leu2-3112 trp11 ura3-1) (YGSC, Berkely). In haploid strains, the ECM11 locus was mutated or disrupted; the mutated or disrupted strains were then mated to give the homozygous diploid strains. For the two-hybrid assay, yeast strain EGY48 (ura3, his3, trp1, LexAop-leu2) was used. Cloning experiments were performed with the Escherichia coli strain DH5a, using standard media (Sambrook et al., 1989). Media and growth conditions Strains were grown at 30 1C in standard yeast peptone dextrose (YPD), YPA or minimal medium lacking appropriate amino acids. X-Gal plates were supplemented with 80 mg X-Gal mL 1. To induce sporulation, cells were grown to 3 107 cells mL 1 YPA at 160 r.p.m., washed and transferred into half that volume of sporulation medium (SPM) (0.3% potassium acetate, 0.02% raffinose) and shaken at 180 r.p.m. (Kassir & Simchen, 1991). Alternatively, sporulation was induced by replica plating patches of diploid cells, grown overnight on YPD plates, onto 1% potassium acetate agar plates. Sporulation efficiency was determined microscopically by counting at least 200 cells sample 1. FEMS Yeast Res 8 (2008) 64–70 Western immunoblotting Cells were grown to the mid-log phase; pellets were washed in 1 mL of 20% tricarboxylic acid (TCA) and frozen. They were then resuspended in 1 mL of 20% TCA and lysed with beads and three vortexings of 1 min each at 2 min intervals on ice. The lysate was separated from beads, beads were washed twice and the combined lysate centrifuged for 10 min at 16 000 g. The pellet was resuspended in Laemmli buffer [50 mM Tris pH 6.8, 2% sodium dodecyl sulphate (SDS), 10% glycerol, 2% b-mercaptoethanol] and heated at 100 1C for 3 min. Twenty microlitres of final sample was run on 10% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes (Amersham Pharmacia Biotech). Primary antibodies used for Western blots were anti-HA (1 : 1000 dilution; Sigma). Blots were visualized with HRPconjugated secondary antibodies (Bio-Rad). SeeBlue Plus2 (Invitrogen) molecular weight markers were used. Immunoprecipitation analysis Cell lysates were prepared as described (Yaakov et al., 2003). Protein concentration was measured by the Bradford method with Nanoquant reagent (Roth). After preclearing of supernatants with G-sepharose (Sigma), 2 mg of rabbit antiHA or anti-Smt3 antibodies were added and the mixture incubated at 4 1C overnight. The antibody complexes were pulled down using G-sepharose, and washed five times with the lysis buffer. The G-sepharose–protein complexes were resuspended in 30 mL of 5 protein loading buffer 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 66 (Fermentas) and boiled for 5 min before loading. For the cell lysate samples, an amount of lysate equal to the 25 mg of protein was boiled for 5 min in the same loading buffer before loading. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) in 10% polyacrylamide gel and transferred to polyvinylidine fluoride membrane (Roth). Immunodetection with monoclonal mouse anti-HA (sc-7392) and polyclonal anti-Smt3 (sc-11845) antibodies (Santa Cruz Biotechnology) and polyclonal anti-mouse or anti-goat secondary antibodies conjugated with HRP (Santa Cruz Biotechnology) was performed using the enhanced chemoluminescence detection system (Pierce). Two-hybrid assay The yeast strain EGY48, plasmids for the two-hybrid system pEG202, pJG4-5, pSH18-34, pJK101 and pSH17-4, and yeast genomic library ligated in pJG4-5 were provided by Roger Brent’s laboratory (R.L. Finley and R. Brent, 1994). LexA-ECM11 fusion derivative was generated from genomic DNA by PCR, using modified primers, and cloned into the XhoI and EcoRI sites of pEG202. The YNL137 gene was amplified by PCR from genomic DNA and cloned into the XhoI and EcoRI sites of pEG202. This construct served as a negative control for two-hybrid interaction with positive clones. A.B. Zavec et al. The yeast strain containing HA-tagged Ecm11 was grown to the logarithmic phase and to different phases of meiosis (Fig. 1a). Bands recognized by HA-antibodies were highly specific, but weak, because HA-tagged ECM11 was expressed under an endogenous promoter. During meiosis, the amount of HA-tagged Ecm11 protein increased significantly after 4 and 8 h in sporulation medium. The bands at 38 kDa probably represent the HA-Ecm11 protein (34 kDa13 kDa), whereas the bands at 46 kDa probably represent the HA–Ecm11–SUMO complex (34 kDa13 kDa111 kDa). Another band appears in samples taken after 4 h in sporulation medium, probably corresponding to a complex of HA-Ecm11 with several covalently bound SUMO proteins. After 12 h and especially after 24 h in sporulation medium the amount of Ecm11 decreased. In logarithmic phases of vegetative cell cycle the amount of Ecm11 protein was below the level of detection. In a previously published experiment Ecm11 had been found to interact with Smt3 in two-hybrid system (Ito et al., 2000). To prove the sumoylation of the Ecm11 protein with an alternative approach, immunoprecipitation studies were performed (Fig. 1b). With the HA reactivity, it was shown Results Ecm11 protein is sumoylated and its level is significantly elevated during meiosis The level of Ecm11 protein during the process of meiosis was determined using Western blot analysis. Ecm11 was epitope-tagged with three HA and located with anti-HA for Western blot analysis (anti-Ecm11 antibodies are not available). The ECM11 gene in the haploid strains YCa and YCa was tagged by site-directed mutagenesis. In these strains ECM11-HA was expressed from its normal chromosomal location. Strains were constructed with HA-tagged Ecm11 at the C-terminus, at the N-terminus or with the epitope tag inserted at the hydrophilic region in the middle of the gene. The biological function of tagged Ecm11 in the vegetative cell cycle was tested by the strain sensitivity on calcofluor white, as described by Lussier et al. (1997). To test the biological function of tagged Ecm11 in meiosis, the haploid mutated strains were mated and used for tests of sporulation efficiency, since it is known from previous studies that strains with deleted ECM11 have a 20% lower sporulation level (Zavec et al., 2004). The modified versions of Ecm11 on both terminuses were nonfunctional. Ecm11 with an HA-tag inserted in the middle of the protein was found to be functional in vegetative cell cycle, but not in meiosis. The last mutant was used to trace the level of Ecm11 in the cells. 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c Fig. 1. (a) Kinetics of Ecm11 formation and decay during sporulation. HA-tagged Ecm11 protein was visualized by Western blot analysis, using anti-HA antibodies. Cells with tagged Ecm11 in different phases of meiosis (0, 4, 8, 12, 24 hours in sporulation medium), in logarithmic phases of mitosis (log), and YC wild-type cells as a negative control (Ctl). (b) Demonstration of HA-Ecm11 sumoylation during meiosis with immunoblot analysis of HA-immunoprecipitates. Whole cell lysates from wild-type YC and isogenic mutant cells containing HA-tagged Ecm11 were immunoprecipitated using rabbit anti-HA and blotted for reactivity with mouse anti-HA and goat anti-Smt3 antibodies. Samples taken after 4 and 8 h in sporulation medium served as controls for HA antibody reactivity. (1) YC wild-type cells as negative control, (2) 4 h sporulation sample, (3) 8 h sporulation sample, (4) anti-HA immunoprecipitate of 4 h samples, (5) anti-HA immunoprecipitate of 8 h samples. FEMS Yeast Res 8 (2008) 64–70 67 Yeast protein Ecm11 is sumoylated during meiosis that HA-Ecm11 protein was efficiently immunoprecipitated and with the Smt3 reactivity, the sumoylation was proven. Beside the band at 46 kDa, a higher band 57 kDa, probably representing the double sumoylation of HAEcm11 (34 kDa13 kDa111 kDa111 kDa), was detected also by immunoprecipitation. (a) 60 % of asci 50 40 30 20 Sumoylation of the N-terminus of Ecm11 is essential for Ecm11 functioning in meiosis The importance of Ecm11 sumoylation for progression through meiosis was investigated by studying the effect of mutation of predicted SUMO consensus sites in Ecm11 on sporulation efficiency. Lysines K5 and K101 in the predicted SUMO consensus sites were mutated to the uncharged amino acid asparagine. Strains were constructed with mutations at K5, at K101 or at both lysines. The haploid strains with mutated sumoylation sites were diploidized and sporulized. The kinetics and maximal level of sporulation of mutant K101 did not differ significantly from those of the wild-type strain. The strains mutated at K5 and at both lysines, however, exhibited the same course of sporulation as the strain with deleted ECM11 (Fig. 2a). Lys5 is clearly essential for Ecm11 function in meiosis and is thus identified as the major in vivo SUMO attachment site on Ecm11 during meiosis. This was additionally confirmed by the immunoprecipitation studies on cell extracts of wild-type strain and K5, K101 and K5K101 mutant strains (Fig. 2b). First proteins were concentrated attached to Smt3 by immunoprecipitation with rabbit anti-Smt3 antibodies, then the proteins were separated and Western blot analysis performed using goat anti-Smt3 antibodies. In the case of K5 and K5K101 mutant strains no signals were observed at 45 and 56 kDa bands corresponding to sumoylated Ecm11 protein. 10 0 K101N WT K5N K101N & K5N deleted ECM11 strain (b) 1 3 2 4 kDa 50 anti-Smt3 Fig. 2. (a) The effect on the efficiency of asci formation of mutations of SUMO recognition sequences in Ecm11. The wild-type strain YC (WT), the isogenic strains bearing one or both substitution mutations at K5 and K101 in the ECM11 gene and the strain with deleted ECM11 were sporulated for 3 days on solid media and the percentage of asci was determined microscopically. The averages were obtained from three measurements. (b) Demonstration of importance of K5 residue for Ecm11 sumoylation with immunoblot analysis of Smt3-immunoprecipitates. Whole cell lysates from wild-type YC cells (1), K101 (2), K5 (3), and K5K101 (4) mutant cells were immunoprecipitated using rabbit antiSmt3 and blotted for reactivity with goat anti-Smt3 antibodies. The effect of Ecm11 sumoylation during vegetative growth Deletion of the ECM11 gene causes cells to become calcofluor white sensitive (Lussier et al., 1997); otherwise mutant cells have the same generation time as wild-type strains during vegetative growth (Zavec et al., 2004). To examine possible effects of mutation of sumoylation sites in Ecm11 on vegetative cells, the sensitivity of the mutant strains to calcofluor white was tested. Mutation in the predicted SUMO sequences at K5 and K101, as well as in both of them, did not affect the calcofluor white sensitivity of the mutant strains, while a significant defect in growth on calcofluor white plates was detected on the strain with deleted ECM11 (Fig. 3). The same results were obtained with the W303 and YC mutant strains. FEMS Yeast Res 8 (2008) 64–70 Fig. 3. The effect of substitution mutations of SUMO recognition sequences in Ecm11 on the sensitivity to calcofluor white: (1) K5N, (2) K101N, (3) K5N and K101N, (4) deleted ECM11, (5) wild-type W303. Protein Ecm11 interacts with Siz2 SUMO ligase in the two-hybrid system In order to identify proteins interacting specifically with Ecm11, a two-hybrid screen was performed using Ecm11 as 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 68 A.B. Zavec et al. a bait, and for interactions were searched with the yeast genomic library. Only one positive clone exhibited galactose-dependent activation of both reporter genes. A DNA fragment from the positive clone was sequenced and the nucleotide query sequence was compared with a yeast nucleotide sequence database by BLAST. The clone contained 80% of the SIZ2 ORF. The deletion of neither SIZ2 nor SIZ1 affects sporulation To determine whether Siz2 is the only SUMO ligase for Ecm11 protein during meiosis, a strain with deleted SIZ2 gene was constructed. Sporulation did not differ from that of the isogenic wild type. A minor effect on sporulation level was observed with deletion of the second yeast SUMO ligase gene, SIZ1. Only deletions of both SUMO ligase genes simultaneously strongly diminished asci formation (Fig. 4). Discussion Sumoylation and the process of meiosis Herein it was demonstrated that the Ecm11 protein in S. cerevisiae is sumoylated during meiosis. There are many known proteins that are sumoylated; however, only a few of them have a role in meiosis. Only recently, it was shown that SUMO1 has a role in mammalian spermatogenesis (Vigodner & Morris, 2005; Vigodner et al., 2006), SUMO in budding yeast regulates synaptonemal complex formation during meiosis (Cheng et al., 2006; Hooker & Roeder, 2006) 50 40 % of asci Sumoylation is requisite modification for Ecm11 functioning in meiosis Protein Ecm11 has a different function in meiosis and in vegetative cells, and its modification with SUMO is important only in meiosis, but not during vegetative growth. Mutation of the predicted sumoylation site K5 affects the biological function of Ecm11 in meiosis. Mutation of K5 led to the reduction of sporulation to the same level as in the mutant with deleted ECM11 gene, while mutation of K101 does not affect sporulation level (Fig. 2a). Additionally, no sumoylation was observed in the case of K5 or K5K101 mutated Ecm11 protein by immunoprecipitation (Fig 2b). These results suggest, firstly, that the N-terminus of Ecm11 is modified by SUMO during meiosis and, secondly, that sumoylation is essential for the biological role of Ecm11 in meiosis. The only known effect of deletion of ECM11 in vegetative cells is that they become calcofluor white sensitive (Lussier et al., 1997; Zavec et al., 2004). To examine the possible effects of mutation of Ecm11 sumoylation sites on cells during vegetative growth, the sensitivity of the mutant strains was tested on calcofluor white. But no relationship between sumoylation of Ecm11 and its role in cell wall biogenesis was observed. The level of Ecm11 protein and its sumoylation status during meiosis 60 30 20 10 0 WT Deleted SIZ2 Deleted SIZ1 Deleted SIZ1 & SIZ2 strain Fig. 4. The effect of deletion of SUMO ligases SIZ1 and SIZ2 on the efficiency of asci formation of the YC isogenic strains. The strains were allowed to sporulate for 3 days on solid media and the percentage of asci was determined microscopically. The averages were obtained from three measurements. 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c and has a role in meiotic recombination (Koshiyama et al., 2006). These results represent additional confirmation that sumoylation is important for the process of meiosis. Western blot analysis revealed a marked increase in the level of Ecm11 after 4 and 8 h in sporulation medium, followed by a decrease in the later phases of meiosis (Fig. 1a). These results are consistent with the mRNA measurement by microarray hybridization, available on the Stanford Genome Database. Based on the time in meiosis at which genes are expressed in the process of meiosis (Vershon & Pierce, 2000), Ecm11 was classified into the early-middle group of sporulation genes. By immunoprecipitation, it was confirmed that Ecm11 is sumoylated during meiosis and that Ecm11 interacts with SUMO covalently. The HA antibodies recognized the HAtagged Ecm11, as well as additional 10 and 20 kDa larger species, which correspond to the molecular mass of one or two copies of mature SUMO (Fig. 1). Mono and double sumoylation of Ecm11 was shown also with the anti-Smt3 reactivity on anti-HA immunoprecipitated samples. Multiple sumoylation was already observed for other sumoylated proteins, identified in a study of posttranslational modifications in yeast proteome (Wykoff & O’Shea, 2005). It was FEMS Yeast Res 8 (2008) 64–70 69 Yeast protein Ecm11 is sumoylated during meiosis found that the majority of Ecm11 protein in the cell is sumoylated during meiosis. Siz2 is SUMO ligase for Ecm11 These results showed that Ecm11 interacts with the SUMO ligase Siz2 in a two-hybrid system. In all organisms examined so far, single activating (E1) and conjugating (E2) enzymes for sumoylation have been detected, but multiple ligases (E3), indicating that the latter determine substrate specificity (Johnson, 2004). There are more E3 ligases in budding yeast, but Siz1 and Siz2 are required for most SUMO conjugation, because double siz1/siz2 deletion results in the elimination of c. 99% of the SUMO conjugates (Johnson & Gupta, 2001). It was found that the SIZ2 deletion had no effect on sporulation regarding the isogenic wild type, so that Siz2 is clearly not essential for Ecm11 sumoylation. Deleting the SIZ1 gene also had minor effect on sporulation regarding the isogenic wild type. Yeast mutants lacking both SUMO ligases, Siz1 and Siz2, have an unusual phenotype, with sectors of enlarged cells that are arrested in the G2/M phase (Chen et al., 2005). In this context, this result with sporulation test was as expected: simultaneous deletion of both SUMO ligase genes, SIZ1 and SIZ2, had much greater effect on sporulation than deletion of ECM11, and strongly diminished asci formation (Figs 2 and 4). These results confirm those described by Johnson & Gupta (2001). They found that mutants with deleted SIZ1 or SIZ2 mated efficiently, and that single mutants sporulated with the same efficiency as the isogenic wild type to produce viable spores; however, in their experiment the double mutant had not been tested for sporulation. It can be surmised that Siz2 is the major ligase for Ecm11 protein, but could be replaced by some other SUMO ligase, most probably Siz1, when Siz2 is missing. Conclusions Many proteins in budding yeast have been reported to be modified by SUMO, but the experimental evidence for the biological role of sumoylation in most of these cases has proven inconclusive. 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