Title: Identification of a structural motif that confers specific interaction with the WD40 repeat domain of Arabidopsis COP1
Abstract: Article15 January 2001free access Identification of a structural motif that confers specific interaction with the WD40 repeat domain of Arabidopsis COP1 Magnus Holm Magnus Holm Department of Molecular, Cellular and Developmental Biology, Yale University, OML 354, Yale University, PO Box 20-8104, 165 Prospect Street, New Haven, CT, 06520-8104 USA Search for more papers by this author Christian S. Hardtke Christian S. Hardtke Department of Molecular, Cellular and Developmental Biology, Yale University, OML 354, Yale University, PO Box 20-8104, 165 Prospect Street, New Haven, CT, 06520-8104 USA Search for more papers by this author Rachelle Gaudet Rachelle Gaudet Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, 02138 USA Search for more papers by this author Xing-Wang Deng Corresponding Author Xing-Wang Deng Department of Molecular, Cellular and Developmental Biology, Yale University, OML 354, Yale University, PO Box 20-8104, 165 Prospect Street, New Haven, CT, 06520-8104 USA Search for more papers by this author Magnus Holm Magnus Holm Department of Molecular, Cellular and Developmental Biology, Yale University, OML 354, Yale University, PO Box 20-8104, 165 Prospect Street, New Haven, CT, 06520-8104 USA Search for more papers by this author Christian S. Hardtke Christian S. Hardtke Department of Molecular, Cellular and Developmental Biology, Yale University, OML 354, Yale University, PO Box 20-8104, 165 Prospect Street, New Haven, CT, 06520-8104 USA Search for more papers by this author Rachelle Gaudet Rachelle Gaudet Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, 02138 USA Search for more papers by this author Xing-Wang Deng Corresponding Author Xing-Wang Deng Department of Molecular, Cellular and Developmental Biology, Yale University, OML 354, Yale University, PO Box 20-8104, 165 Prospect Street, New Haven, CT, 06520-8104 USA Search for more papers by this author Author Information Magnus Holm1, Christian S. Hardtke1, Rachelle Gaudet2 and Xing-Wang Deng 1 1Department of Molecular, Cellular and Developmental Biology, Yale University, OML 354, Yale University, PO Box 20-8104, 165 Prospect Street, New Haven, CT, 06520-8104 USA 2Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, 02138 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:118-127https://doi.org/10.1093/emboj/20.1.118 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Arabidopsis COP1 is a photomorphogenesis repressor capable of directly interacting with the photomorphogenesis-promoting factor HY5. This interaction between HY5 and COP1 results in targeted deg radation of HY5 by the 26S proteasome. Here we characterized the WD40 repeat domain-mediated interactions of COP1 with HY5 and two new proteins. Mutational analysis of those interactive partners revealed a conserved motif responsible for the interaction with the WD40 domain. This novel motif, with the core sequence V-P-E/D-φ-G (φ = hydrophobic residue) in conjunction with an upstream stretch of 4–5 negatively charged residues, interacts with a defined surface area of the β-propeller assembly of the COP1 WD40 repeat domain through both hydrophobic and ionic interactions. Several residues in the COP1 WD40 domain that are critical for the interaction with this motif have been revealed. The fact that point mutations either in the COP1 WD40 domain or in the HY5 motif that abolish the interaction between COP1 and HY5 in yeast result in a dramatic reduction of HY5 degradation in transgenic plants validates the biological significance of this defined interaction. Introduction Arabidopsis seedlings follow two dramatically different developmental patterns depending on the presence or absence of light (Kendrick and Kronenberg, 1994). Light-grown seedlings undergo photomorphogenesis and have short hypocotyls with photosynthetically active and highly differentiated cotyledons. In contrast, seedlings grown in darkness are etiolated and develop elongated hypocotyls with unexpanded cotyledons. Genetic screens have identified both positive and negative regulators mediating this light control of seedling development in Arabidopsis (Deng and Quail, 1999; Nagy and Schafer, 2000; Neff et al., 2000). Two of these regulators, HY5 and COP1, act as critical components of this developmental switch (Osterlund et al., 1999). The bZIP transcription factor HY5 promotes photomorphogenesis in the light, whereas COP1 represses photomorphogenic development in darkness. The COP1 protein contains three distinct domains, a Zn2+-binding RING finger domain, a coiled-coil domain and seven WD40 repeats in the C-terminal half of the protein (Deng et al., 1992; McNellis et al., 1994a). All characterized lethal cop1 alleles contain mutations within the WD40 domain, whereas the weak alleles either lack the WD40 domain or have mutations N-terminal to the domain (McNellis et al., 1994a). COP1 is nuclear localized in the dark, whereas its nuclear abundance decreases upon exposure to light (von Arnim and Deng, 1994, 1996). COP1 interacts directly with HY5, both genetically and physically, through its WD40 domain, negatively regulating HY5 activity (Ang and Deng, 1994; Ang et al., 1998; Torii et al., 1998). Recent evidence suggests that the interaction results in a dark-dependent degradation of HY5 (Osterlund et al., 2000). HY5 accumulation is very low in dark-grown seedlings and is ∼20-fold higher in the light. The level of HY5 protein correlates directly with photomorphogenic development and inversely with the nuclear abundance of COP1. The regulated HY5 degradation is likely to be mediated by the 26S proteasome and depends on its interaction with COP1 (Osterlund et al., 2000). Taken together, the results suggest that HY5 interacts with the WD40 domain of COP1 and that the interaction negatively regulates the abundance of HY5 in the dark. Many regulatory proteins have WD40 domains that mediate protein–protein interactions. WD40 domains usually contain 4–8 WD40 repeats and were originally identified in the β-subunit of heterotrimeric G-proteins (Gβ) (Fong et al., 1986). The repeats have since been found in a large number of proteins, including transcriptional repressors, such as TUP1 and Groucho (Paroush et al., 1994; Komachi and Johnson, 1997), as well as several E3 ubiquitin ligases (Tyers and Jorgensen, 2000). Crystal structures of representative WD40 domains alone and in complex with interacting proteins have been determined (Wall et al., 1995; Gaudet et al., 1996; Lambright et al., 1996; Sondek et al., 1996; Sprague et al., 2000). The WD40 repeats form four antiparallel β-strands producing β-sheets, which in turn fold into a propeller-like structure, denoted the β-propeller (Sondek et al., 1996). The β-propeller gains rigidity from hydrophobic interactions between the β-sheets, and solvent-exposed residues in the loops between the β-sheets are free to interact with other proteins. Residues in the loops of Gβ interact with Gα and phosducin, as well as with several other effectors (Gaudet et al., 1996; Lambright et al., 1996; Ford et al., 1998). Furthermore, loop residues in TUP1 are important for the interaction between TUP1 and the DNA-binding protein α2 (Komachi and Johnson, 1997). In this study, we characterize the interaction between the WD40 domain of COP1 and HY5 as well as with two new interaction partners, STO and STH. We define a novel WD40 domain-interacting motif conserved in all three COP1-interacting proteins. Furthermore, we identify residues important for the interaction both in the motif and in the WD40 domain of COP1, and reveal key features of the underlying interaction. Results Mutations in the COP1 WD40 domain abolish COP1–HY5 interaction in yeast Deletion analyses have shown that the WD40 domain of COP1 is required for its interaction with the bZIP protein HY5 (Ang et al., 1998; Torii et al., 1998). In Arabidopsis, truncated COP1 proteins lacking the WD40 domain (cop1-4), containing a small deletion in the WD40 domain (cop1-8) or possessing a single amino acid substitution (cop1-9) in the WD40 domain cause a loss-of-function phenotypic defect (Figure 1A; McNellis et al., 1994a). Thus, we initially examined the ability of HY5 to interact with the COP1 proteins found in the three cop1 alleles above, using a yeast two-hybrid assay system. To this end, we fused amino acids 25–60 of HY5, sufficient for COP1 interaction in yeast (Hardtke et al., 2000), to the LexA DNA-binding domain (LexA-BD) and assayed the interaction with activation domain-fused wild-type COP1, COP1-4, COP1-8 and COP1-9 proteins. All these COP1 proteins were expressed to similar levels in yeast (data not shown). As shown in Figure 1, the COP1-interacting domain of HY5 interacts well with wild-type COP1 but is unable to interact with the COP1-4, COP1-8 and COP1-9 proteins, suggesting that these mutations in the WD40 domain interfere with the ability of COP1 to interact with HY5. Figure 1.The WD40 domain of COP1 is required for interaction with STO, STH and HY5. (A) Diagrams of wild-type COP1 and three mutated versions. The total number of amino acids in each protein are indicated on the right. (B) Summary of the detected interactions between the four versions of COP1 and the three interactive partners. The STO and STH experiments were performed with Gal4-BD-fused COP1 proteins and the HY5 experiments were performed with AD-fused COP1 proteins. 'Yes' represents >50-fold activation over background for HY5 and 5- to 15-fold activation over background for STO and STH; 'no' represents background activation. See text for additional detail. Download figure Download PowerPoint Identification of two new proteins interacting with the WD40 domain of COP1 To identify novel COP1-interacting proteins, we used full-length COP1 protein fused to the Gal4 DNA-binding domain (Gal4-BD) to screen a library made from 3-day-old dark-grown Arabidopsis seedlings (Kim et al., 1997). Fourteen of ∼170 000 independent transformants allowed auxotrophic growth and showed β-galactosidase activity in plate assays. The plasmids recovered from 10 of the transformants allowed COP1-dependent auxotrophic growth and β-galactosidase activity when re-introduced in yeast. Sequencing and restriction mapping revealed that the 10 plasmids were encoding five different cDNAs. One of the plasmids contained a truncated salt tolerance (STO) cDNA (Lippuner et al., 1996), whereas two independent plasmids contained a cDNA encoding a homolog of STO (STH) (Figure 2). The STO homolog has been sequenced by the Arabidopsis genome initiative and is located on chromosome II. The predicted protein (DDBJ/EMBL/GenBank accession No. AAD26481) is one amino acid shorter than the protein encoded by the STH cDNA due to an error in exon–intron boundary prediction. To confirm the interactions, we fused the coding regions of STO and STH to the Gal4 activation domain (Gal4-AD) in pGAD (a different two-hybrid vector, see Materials and methods). The STO and STH proteins expressed from the pGAD vector again interact well with wild-type COP1. However, like HY5, STO and STH were unable to interact with the COP1-4, COP1-8 and COP1-9 proteins (Figure 1), suggesting that the WD40 domain of COP1 is also required for interaction with the STO and STH proteins. Figure 2.Alignment of the STO and STH proteins. Identical amino acids are boxed and the tandem repeated cysteine-rich motifs are underlined. Arrows indicate the first amino acid of the protein encoded by pGAD-STO and the truncated STH cDNA in pACT-STH2, respectively. The respective DDBJ/EMBL/GenBank accession Nos are AF323666 (STH) and X95572 (STO). Download figure Download PowerPoint The STO and STH proteins share 70% overall amino acid identity and contain two tandem cysteine-rich motifs in their N-termini (Figure 2). The cysteine motifs, predicted to form Zn2+ fingers, are homologous to motifs found in CONSTANS (CO) and resemble the DNA-binding Zn2+ finger domain in the GATA1 transcription factor (Putterill et al., 1995). The cysteine motifs are directly adjacent in CO but are spaced by nine amino acids in the STO and STH proteins. However, the STO protein and the truncated STH protein recovered in the screen lacked the cysteine motifs (Figure 2), indicating that this domain is dispensable for the interaction with COP1. Taken together, HY5, STO and STH interact with the WD40 domain of COP1 in yeast. The N-terminal half of HY5 and the C-terminal half of STO and STH are sufficient to mediate this interaction. The mutations in strong and lethal cop1 alleles are likely to distort the structure of the WD40 domain The mutations in the WD40 domain of COP1-8 and COP1-9 abolish interaction with the HY5, STO and STH proteins. So far, all characterized strong and lethal cop1 alleles that still accumulate COP1 protein contain mutations within the WD40 domain (Figure 3A; McNellis et al., 1994a). To gain insight into the molecular nature of the WD40 domain mutations in COP1, we modeled the COP1 WD40 repeats after the β-propeller of Gβ transducin (Sondek et al., 1996; Figure 3B). Secondary structure analysis of COP1 predicts 28 β-strands between amino acids 361 and 673 of COP1 (Figure 3C), suggesting that COP1, like Gβ, has seven β-sheets. The deletions in cop1-1, cop1-8, cop1-10 and cop1-11 remove multiple β-strands, whereas the substitution in cop1-9 (Figure 3A and C) replaces Gly524 with a glutamic acid. In Gβ, this conserved glycine is imbedded in the hydrophobic interface between two β-sheets. A large, charged residue in this position would interfere with the hydrophobic interactions and could potentially disrupt the β-propeller. Thus, it is likely that all mutations found in the strong and lethal cop1 alleles interfere with the formation of a β-propeller. Figure 3.Alignment of the WD40 repeats in COP1. (A) Schematic representation of cop1 alleles with WD40 domain mutations adapted from McNellis et al. (1994a). (B) Schematic representation of the β-propeller in Gβ adapted from Sondek et al. (1996). The positions of β-strands A–D are indicated. (C) Alignment of the WD40 repeats in COP1. The β-strands predicted by jpred (Cuff, 1998) are shaded and substituted amino acids are boxed. The coordinates of the amino acid positions are indicated on the left. Download figure Download PowerPoint COP1 proteins with substitutions of putative protein-interacting residues in the WD40 domain are partially functional in plants A change in the overall structure of the WD40 domain, as predicted for the strong and lethal cop1 alleles, is likely to interfere with the ability of the domain to interact with multiple interacting proteins. To examine the interaction between the COP1 WD40 domain and the HY5, STO and STH proteins, we generated point mutations in the WD40 domain that could interfere specifically with the interactions of the domain without affecting its β-propeller structure. To this end, we generated five independent amino acid-substituted COP1 proteins (Figure 3C). The substitutions are located in four different D–A loops, thus potentially involved in protein–protein interactions and unlikely to interfere with the folding of the domain. One of the substitutions, W467A, replaces a large hydrophobic side chain with a single methyl group, whereas the others, K422E, R465E, K550E and E592R, are charge reversals. To examine the effect of the WD40 domain substitutions in vivo, we transformed constructs overexpressing each of the substituted COP1 proteins into heterozygous cop1-5 plants. Transgenes segregating as a single locus were selected and selfed to generate lines homozygous for both cop1-5 and the transgenes. At least three independent transgenic lines for each construct were examined, and western blot analysis indicated that the levels of mutated COP1 proteins were similar in all the lines and to that of the wild-type COP1 overexpressor control. All five amino acid-substituted COP1 proteins were able to rescue the lethal phenotype of the cop1-5 null mutation when overexpressed in plants (Figure 4). However, when the seedlings were grown in constant white light, the hypocotyl length differed between the lines. At higher light intensity, 200 μmol/m2/s, the E592R substitution results in slightly longer hypocotyls than wild type, whereas no significant difference was observed between wild-type seedlings and the seedlings overexpressing either COP1 proteins with the K422E, R465E, W467A and K550E substitutions or wild-type COP1, respectively (Figure 4D). At lower intensity light, 40 μmol/m2/s, the E592R substitution still results in a longer hypocotyl than wild type, comparable to seedlings overexpressing wild-type COP1, whereas the K422E, R465E, W467A and K550E substitutions display a significantly shorter hypocotyl than wild-type seedlings (Figure 4A and C). Dark-grown seedlings expressing the substituted COP1 proteins all showed elongated hypocotyls (Figure 4B and data not shown), but ∼70% of the seedlings expressing the W467A-substituted COP1 protein had open cotyledons (Figure 4B). The ability of the substituted COP1 proteins to partially rescue the cop1-5 null mutation indicates that the overexpressed COP1 proteins are functional in plants and that the WD40 domains are properly folded. Furthermore, the phenotypes observed for the substituted COP1 proteins are much weaker as compared with those of the genetically identified mutations, which are likely to affect the overall structure of the WD40 domain. This suggests that the substitutions might affect specific protein–protein interaction surfaces on the COP1 WD40 domain; thus, each substitution potentially may interfere with the interaction of only a subset of COP1 target proteins. Figure 4.The amino acid-substituted COP1 proteins can largely rescue the cop1-5 null mutation. (A) Six-day-old cop1-5 null mutant, wild-type (WT), cop1-5 seedlings homozygous for transgenes expressing COP1 proteins with the indicated substitution and seedlings overexpressing wild-type COP1 in a wild-type background (OE) grown in 40 μmol/m2/s constant white light. The scale bar represents 2 mm. Western analysis indicated that all COP1-overexpressing lines accumulated similar levels of COP1 protein. (B) A 7-day-olddark-grown wild-type (WT) seedling and a cop1-5 mutant with 35S::COP1W467A transgene. The scale bar represents 2 mm. (C) Graphical representation of the hypocotyl length of seedlings grown in 40 μmol/m2/s and (D) 200 μmol/m2/s constant white light. Error bars represent standard deviations, n = 30. Data from two representative lines from each construct are presented in (C) and (D). Similar results were observed in all three lines examined for each construct. Download figure Download PowerPoint Substitutions of loop residues affect the interactions between COP1 and the three interaction partners We examined the effect of the COP1 substitutions on the interactions with HY5 and the STO/STH proteins in yeast two-hybrid assays. Due to technical reasons (see Materials and methods), we assayed the COP1–HY5 interactions in the LexA system and the COP1–STO/STH interactions in the Gal4 system. The amino acid-substituted COP1 proteins expressed as well as wild-type COP1 in both two-hybrid systems (data not shown). The use of different two-hybrid systems prevents a direct comparison between the HY5 and the STO/STH proteins; however, the relative effect that the WD40 domain substitutions have on the interaction with each partner can still be compared (Figure 5). Two of the substituted COP1 proteins, K422E and R465E, interact differently with HY5 and the STO/STH proteins. The K422E substitution results in a >5-fold higher interaction with HY5 compared with wild-type COP1, but allows only a weak interaction with the STO and STH proteins. The R465E-substituted protein interacts at wild-type level with HY5 but fails to interact with the STO/STH proteins. The finding that the K422E substitution enhances the interaction with HY5 and decreases the interaction with the STO/STH proteins resembles results seen with Gβ. A study by Ford et al. (1998) has shown that the substitution of loop residues in Gβ could increase, decrease or abolish interactions with different effectors, indicating that a change in the WD40 interaction surface can have different effects on different target proteins. Figure 5.Substitutions of loop residues in COP1 affect its interaction with HY5 and STO/STH proteins. The HY5 experiments were performed with AD-fused COP1 proteins in the LexA system, and the STO and STH experiments were performed with Gal4-BD-fused COP1 proteins in the Gal4 system. To allow comparison between the different two-hybrid systems, we set the relative β-galactosidase activities between the wild-type COP1 and its partners to 100%. Error bars represent standard deviations, n = 6. Download figure Download PowerPoint Interestingly, three of the substitutions have similar effects on the interactions with HY5 and the STO/STH proteins. The COP1E592R protein interacts slightly better than wild-type COP1 with HY5, STH and STO, whereas COP1W467A and COP1K550E are unable to interact with either HY5 protein or STO/STH. The inability of W467A and K550E to interact with HY5 or STO/STH suggests that these residues may be critical for the interaction of COP1 with all three proteins. The identical pattern of interactions between STO and STH with the substituted COP1 proteins suggests that these two proteins interact with a common surface on COP1. Furthermore, the finding that the interaction pattern of the STO and STH proteins overlaps with that of the HY5 protein suggests that STO/STH and HY5 interact with partially overlapping surfaces on the COP1 WD40 domain. A novel motif mediates interaction with the COP1 WD40 domain If the HY5 and STO/STH proteins interact with partially overlapping surfaces on the COP1 WD40 domain, it is possible that they contain common sequence features able to mediate the interaction with COP1. Upon examining the amino acid sequence of the HY5 and STO/STH proteins, we found a short sequence within the COP1-interacting domain in HY5 that resembles the sequence in the very C-terminus of the STO and STH proteins (Figure 6A and B). The conserved sequence motif is a stretch of negative amino acids and a spacer of three amino acids followed by the motif V-P-E/D-Φ-G, where Φ designates a hydrophobic residue (Figure 6B). The motif is conserved in tomato HY5, STF proteins from soybean, in a bZIP protein from fava bean and in STO/STH homologs from rice (Figure 6A and B; Cheong et al., 1998; Song et al., 1998). Figure 6.HY5, STO and STH interact with COP1 through a novel motif. (A) Schematic representation of Arabidopsis HY5 and HY5 homologs from tomato (THY5; DDBJ/EMBL/GenBank accession No. AJ011914), soybean (STF1 and STF2; DDBJ/EMBL/GenBank accession Nos L28003 and L28004) and fava bean (Vf-bZIP; DDBJ/EMBL/GenBank accession No. X97904), and the Arabidopsis STO and STH proteins with their rice homologs (Os-zf; DDBJ/EMBL/GenBank accession Nos AB001883 and AB001885). The bar represents the position of the motif. Protein sizes (in amino acids) are indicated on the right. (B) Alignment of the motifs in the proteins in (A); the V-P-E/D-Φ-G motif and the cluster of negative amino acids are shaded. (C) Schematic representation of interactions between wild-type and amino acid-substituted COP1, STH and HY5 proteins. +++ represents >350-fold activation over the background level; ++ represents ∼70-fold activation for HY5 and 5- to 15-fold activation for STH over the background level; + represents 3- to 5-fold activation over background; and − represents background levels of activation. The STH experiments were performed with Gal4-BD-fused COP1 proteins and the HY5 experiments were performed with AD-fused COP1 proteins. Download figure Download PowerPoint To examine whether this conserved motif is important for the interaction with COP1, we substituted the conserved valine–proline (VP) pair in the HY5 and STH protein with alanines. As shown in Figure 5C, alanine substitution of the VP pair completely abolishes the ability of both STH and HY5 to interact with wild type or any of the amino acid-substituted COP1 proteins, indicating that this motif is indeed important for COP1 interaction. The location of the motif, in the N-terminal half of HY5 and in the C-terminus of STO and STH, suggests that its function is position independent. A salt bridge is involved in the interaction between COP1 and the conserved motif We have shown that the COP1R465E and COP1K550E proteins are unable to interact with the STO/STH proteins (Figure 5). Since both substitutions are positive to negative charge reversals, it is possible that an acidic amino acid in or close to the motif in the STO/STH proteins could form a salt bridge with either R465 or K550 in wild-type COP1. To test this possibility, we generated four STH proteins with single amino acid substitutions reversing the charge of amino acids 228, 229, 230 and 236. The substitutions are unlikely to be structurally disruptive since the motifs in both the STH and HY5 proteins are predicted to form a loop. Three of the amino acid-substituted STH proteins (E228K, E229K and E230K) were able to interact with the same set of COP1 proteins as wild-type STH (Figure 7A). Although the mutations have small quantitative effects on the interaction with wild-type COP1 and COP1K422E, no qualitative differences were observed (Figure 7A). This result suggested that each individual residue in this upstream negatively charged amino acid stretch is dispensable. However, as a whole, this stretch of negatively charged amino acids is essential for COP1–HY5 interaction (Ang et al., 1998). In contrast, the STH-D236K protein is unable to interact with wild-type COP1 but allows a weak interaction with COP1K550E (Figure 7A and B). Thus, the K550E-substituted COP1 protein is unable to interact with wild-type STH and the D236K-substituted STH protein is unable to interact with wild-type COP1. However, when the charge is reversed in both proteins, their interaction is restored, suggesting that amino acid K550 in COP1 interacts directly with amino acid D236 in STH. To test whether the motif in HY5 behaves similarly, we substituted the reciprocal amino acid (E45) with a lysine or an arginine. As shown in Figure 7C and D, the E45K and E45R substitutions in HY5 render the protein unable to interact with wild-type COP1 but allow a strong interaction with COP1K550E. Taken together, this new COP1-interacting motif found in the N-terminal half of HY5 and in the C-terminus of STO and STH requires the amino acids V-P-E/D-Φ-G. Furthermore, we identify direct amino acid contacts between K550 in COP1 and E45 in HY5 and D236 in STH. Figure 7.Amino acid K550 in COP1 may form a salt bridge withthe amino acids D236 in STH and E45 in HY5. (A) Schematic representation of interactions between wild-type and amino acid-substituted COP1 and STH. ++ represents 5- to 15-fold activation for STH; + indicates 3- to 5-fold activation over background; and − indicates background levels of activation. (B) Interactions between wild-type and amino acid-substituted COP1 proteins and the STH-D236K, (C) HY5-E45K and (D) HY5-E45R proteins, respectively. The STH experiments were performed with Gal4-BD-fused COP1 proteins and the HY5 experiments were performed with AD-fused COP1 proteins. Error bars represent standard deviation, n = 6. Download figure Download PowerPoint Mutations affecting the COP1 and HY5 interaction also interfere with HY5 accumulation A recent study showed that the HY5 protein is degraded in the absence of light, suggesting that the COP1–HY5 interaction mediates a COP1-dependent, negative regulation of the abundance of HY5 protein in the dark (Osterlund et al., 2000). If the residues W467 and K550 in the COP1 WD40 domain are important for the interaction between COP1 and the novel motif in vivo, it is expected that substitutions of W467, K550 or in the motif would interfere with the COP1-mediated degradation of HY5 in Arabidopsis. To test this assumption, we used two independent approaches. First, we assayed the stability of endogenous HY5 protein in the lines expressing the substituted COP1 proteins. Secondly, we examined the ability of endogenous COP1 protein to affect the stability of overexpressed HY5 protein with or without substitutions in the defined motif. As shown in Figure 8A, HY5 is readily detectable in 4-day-old light-grown seedlings overexpressing wild-type COP1 or either of the five amino acid-substituted COP1 proteins in the cop1-5 null mutant background. Transfer of the seedlings to darkness for 20 h results in a reduction in the amount of endogenous HY5 protein as previously observed (Osterlund et al., 2000). Interestingly, the W467A and K550E substitutions result in a delayed dark-dependent HY5 reduction, suggesting that the COP1 proteins with these two substitutions are less efficient in targeting HY5 for degradation. The observed reduced degradation of HY5 in seedlings expressing the W467A- and K550E-substituted COP1 proteins corresponds well with the loss of interaction in yeast two-hybrid assays (Figure 5) and with the short hypocotyl seen when these seedlings were grown in low-intensity white light (Figure 4A and D). On the other hand, the E592R mutation resulted in a reduced HY5 level in the light, also consistent with its elongated hypocotyl