The Cell Biology of the COP/DET/FUS Proteins. Regulating Proteolysis in Photomorphogenesis and Beyond?

Schematic presentation of the light control of seedling development and the role of COP/DET/FUS loci. Wild-type seedlings grown in the light display photomorphogenic development, i.e. a short hypocotyl and green expanded cotyledons. In complete darkness, wild-type seedlings develop skotomorphogenically, characterized by a highly elongated hypocotyl and small, closed, undifferentiated cotyledons. In contrast, constitutive photomorphogenic seedlings (cop/det/fus mutants) are hyperphotomorphogenic in the light and display a light-grown morphology in darkness. Flow diagram of light signal transduction of Arabidopsis. Only a portion of the known components is listed. Light is perceived by the different photoreceptors that sense light of particular wavelengths. The light signal is then transduced by early response signaling intermediates to the pleiotropicCOP/DET/FUS loci. The COP/DET/FUS genes integrate the various light signals and modulate the activity of downstream effectors such as HY5 and additional unknown factors (X and Y). These factors govern the changes in metabolism and gene expression that will eventually result in the photomorphogenic responses. Early response mechanisms are additionally activated directly by the phytochromes. These include modulation of downstream transcriptional effectors such as PIF3. PIF3 directly promotes light-induced gene expression, bypassing any intermediate signaling input from the pleiotropicCOP/DET/FUS genes. Also, substrates for phytochrome-associated kinase activity such as NDPK2 and PKS1 may directly influence downstream effectors or modulate the activity of early signaling components and pleiotropic COP/DET/FUSgenes. Several loci acting as negative regulators of photomorphogenesis have been isolated from genetic screens that sought to identify mutants that display characteristics of light-grown seedlings in complete darkness. They were named constitutive photomorphogenic (COP; Deng et al., 1991) or de-etiolated (DET;Chory et al., 1989). A number of these loci turned out to be identical to previously isolated fusca (FUS) mutants (Misera et al., 1994), which were named after their purple seed color resulting from anthocyanin accumulation. Thus this group of genes is collectively referred to as the pleiotropic COP/DET/FUS loci of Arabidopsis (Wei and Deng, 1999). Mutants in the 11 pleiotropic COP/DET/FUS loci display a photomorphogenic phenotype in complete darkness (Fig. 1). This phenotype is not only characterized by morphological changes such as open, expanded cotyledons, suppression of hypocotyl growth, and anthocyanin accumulation, but also corresponding changes in subcellular features such as chloroplast differentiation and de-repressed expression of normally light-induced genes. The mutants in all 11COP/DET/FUS loci are recessive and can be generally grouped into two classes: mutations in eight loci, which lack the COP9 signalosome (Chamovitz et al., 1996; Karniol et al., 1999; Serino et al., 1999; Deng et al., 2000) and mutations in three loci that do not (Fig. 2). It is interesting that all cop/det/fus mutants lacking the signalosome are lethal at the seedling stage. Although this is also true for severe alleles of the non-signalosome mutantscop1, cop10, and det1, their weak alleles are viable (Chory et al., 1989; Kwok et al., 1996; Vogel et al., 1998). Thus the COP9 signalosome seems to have a more general role in Arabidopsis development. This update will focus on the most recent advances, whereas earlier progress can be found in several previous reviews (Wei and Deng, 1996, 1999; Osterlund et al., 1999;Schwechheimer and Deng, 2000). Recent progress in understanding the molecular basis ofCOP/DET/FUS gene action has come from a better understanding of the nature of the interaction between the COP1 and HY5 (long hypocotyl 5) (Koornneef et al., 1980) proteins. So far,HY5 is the only genetically characterized positive regulator of photomorphogenesis that acts downstream of the bottleneck comprised by the COP/DET/FUS loci. HY5 encodes a bZIP transcription factor that binds to the G-box in the promoters of light-inducible genes such as RBCS1A or CHS1, and thereby plays a vital role in their activation during seedling morphogenesis (Oyama et al., 1997; Ang et al., 1998; Chattopadhyay et al., 1998). The hy5 mutant has been re-isolated as an extragenic suppressor of cop1, and molecular analysis has confirmed that both gene products also interact physically (Ang and Deng, 1994;Ang et al., 1998). HY5 is a constitutively nuclear localized protein, whereas COP1 is excluded from the nucleus in the light, but nuclear localized in darkness (von Arnim and Deng, 1994). Thus an early working hypothesis suggested that the physical interaction between COP1 and HY5 could only take place in darkness and should negatively regulate HY5 activity (Ang et al., 1998). The molecular basis for the negative regulation remained elusive, however, and had to await a more detailed characterization of the HY5 protein. Analysis of HY5 in Arabidopsis seedling development indicated highly elevated HY5 protein levels in light-grown seedlings as opposed to dark-grown seedlings (Osterlund et al., 2000a, 2000b). HY5 accumulates to about 20-fold within 15 h after shifting seedlings from darkness into light, and this accumulation is reversible by the opposite light shift. This finding did not correspond, however, to HY5 mRNA levels, which show only a 2- to 3-fold difference between dark and light. In transgenic lines ectopically overexpressing HY5, HY5 protein levels are independent from endogenous HY5 mRNA levels. It is striking that the same light-dark dependence of HY5 abundance was observed again. By uncoupling any effects on mRNA translation from protein stability by the application of a protein synthesis inhibitor, enhanced degradation of HY5 in darkness could be established as the primary determinant for HY5 abundance. Analysis of HY5 levels in cop1 mutant backgrounds showed that HY5 abundance in dark-grown cop1 mutant seedlings is similar to the levels in light-grown seedlings. This finding by itself could be considered a consequence of the light-grown morphology ofcop1 mutants in the dark rather than one of its causes. However, the accumulation dynamics of HY5 argues against the former. A steep rise in HY5 abundance upon illumination of skotomorphogenic seedlings precedes any striking changes in seedling morphology other than apical hook opening. This suggests that photomorphogenesis is at least in part promoted by elevated HY5 activity, in line with thehy5 mutant phenotype. Thus cop1 mutants seem to be unable to degrade HY5 in the dark. Moreover, a truncatedHY5 transgene that lacks the COP1 interaction domain is not subject to differential degradation in the dark any more. Since this observation coincides with the nuclear localization of COP1 only in the dark, the results suggest that interaction between COP1 and HY5 results in targeted degradation of the HY5 protein. A working model for COP1 action in Arabidopsis photomorphogenesis. It is assumed that COP1 acts as part of an E3 ubiquitin ligase complex. Ubiquitin is activated in the cell by E1 ubiquitin-activating enzymes, which pass the ubiquitin to E2 ubiquitin conjugating enzymes. The E2 enzyme in turn associates with an E3 ubiquitin ligase that directs the transfer of the ubiquitin to target proteins. In its active form, COP1 is associated with other proteins in an E3 ligase complex. Within the E3 complex COP1 provides specificity of the ubiquitination machinery toward selected substrates involved in photomorphogenesis such as the HY5 transcription factor. The contact between COP1 and its substrates is mediated via its WD40 repeat domain. The other functional domains of COP1 process distinct signaling inputs from a number of CIP. The CIP8 protein is incorporated into the E3 complex by interaction with the RING-finger domain of COP1 and drives E2-dependent ubiquitin chain assembly on the target proteins recruited by COP1. In darkness this results in targeting of HY5 for degradation by the 26S proteasome. Upon illumination COP1 might be inactivated by an early response mechanism mediated by CIP7 and/or CIP4 (CIP7/4), nuclear factors whose transcription is highly inducible by light. CIP7/4 act by interacting with the coiled coil domain of COP1, which is necessary for COP1 homodimerization. Because dimerization is necessary for COP1 activity, its disruption results in a stabilization of HY5. Long-term inactivation of COP1 is triggered by light through nuclear exclusion of COP1 protein. Anchoring of COP1 in the cytoplasm might be mediated by CIP1, a cytoskeleton-associated protein, which also interacts with the COP1 coiled coil domain. Moreover, the COP1 substrates may be under control of additional inputs themselves. In the case of HY5, phosphorylation within its COP1-binding domain by a CKII activity results in a physiologically less-active protein. This phosphorylated HY5 is at the same time less susceptible to degradation, since its interaction with COP1 is impaired. This mechanism ensures the maintenance of a small pool of HY5 in darkness. In the light this pool gets rapidly activated due to the inhibition of the CKII activity by light. In combination with the light-induced reduction of COP1 activity this results in an accumulation of more active unphosphorylated HY5 in the light, which promotes the onset of photomorphogenesis. In the case of HY5, phosphorylation of the COP1-binding domain on the one hand seems to influence HY5 stability, since unphosphorylated HY5 interacts better with COP1 and is a preferred substrate for degradation. On the other hand, unphosphorylated HY5 is also physiologically more active. This is evidenced by transgenic rescue of a hy5 null mutant with a transgene whose gene product cannot be phosphorylated in the COP1 interaction domain. In these transgenic lines a slightly hyperphotomorphogenic phenotype, as well as a delay in lateral root outgrowth, is observed, which is essentially an overcompensation of hy5 mutant traits. The overcompensated root phenotype by a HY5, which is a better substrate for COP1-mediated degradation represents a paradox, since COP1 is constitutively nuclear localized in root cells even in the light (von Arnim and Deng, 1994). However, it must be considered that the hyperphotomorphogenic phenotype might result in an altered carbon-to-nitrogen ratio due to elevated photosynthesis and thus a reduction in lateral root growth as a secondary effect. In any case, in the context of photomorphogenesis the physiologically more active unphosphorylated HY5 seems to be the prime target for COP1. Thus in darkness unphosphorylated HY5 gets degraded, whereas elevated CKII activity ensures the maintenance of a small pool of less active phosphorylated HY5, which can be rapidly activated upon illumination. The combination of regulated HY5 abundance and HY5 phosphorylation thus further extends the HY5 activity differential between dark- and light-grown seedlings. A hint toward the specific role of COP1 in its interaction with HY5 comes from the finding that HY5 degradation can be blocked by proteasome inhibitors in vitro (Osterlund et al., 2000a). Thus the proteasome pathway seems likely to be responsible for HY5 degradation. Proteins targeted for degradation by the proteasome pathway are in general marked by the covalent attachment of ubiquitin. Ubiquitin is a highly conserved 8-kD protein found in all eukaryotes examined to date and gets attached to a Lys in target proteins via its C terminus. Additional ubiquitin molecules are subsequently linked to an internal Lys in the ubiquitin itself, eventually generating a chain of interconnected ubiquitin molecules on one or more lysines in the target protein. A protein marked this way is recognized as a substrate by the 26S proteasome, a multiprotein complex that eventually chops the target protein into small peptides. Attachment of ubiquitin moieties to target proteins is mediated by a system of three enzymes (Fig. 3). In a first step, so-called E1 enzymes activate ubiquitin molecules, which then are transferred to E2 ubiquitin conjugating enzymes. The ubiquitin is eventually transferred from the E2 enzyme to the target protein and to achieve this, the E2 enzyme in numerous cases teams up with a so-called E3 ubiquitin ligase. Most E3 ubiquitin ligases are multisubunit protein complexes and critical for the ubiquitin tagging of substrates since they provide substrate specificity to the reaction by selecting the appropriate target proteins in vivo. Within this scenario COP1 would occupy a place as an integral part of an E3 ligase and mediate interaction between the ubiquitin ligase machinery and specific target proteins. The presence of a RING-finger domain in its N terminus and a number of WD40 repeats in its C terminus is consistent with such a role. Although the N terminus might be required for assembly into the E3 complex, the WD40 repeat domain could serve as an interaction surface for target proteins (see below). Apart from HY5, a number of proteins interacting with COP1 have been isolated by the application of two-hybrid interaction screening and far-western analysis. These proteins can generally be classified according to where the interaction with COP1 takes place. Although some proteins seem to interact with domains (RING-finger or coiled coil) in the N terminus of COP1, others seem to interact with the WD40 repeat domain of COP1 (Fig. 3). A protein interacting with the coiled coil domain in the N terminus of COP1 is the CIP1 (Matsui et al., 1995). CIP1 does not have primary sequence features pointing out any functionally defined domains; however, it seems to have the capacity to form a coiled coil structure itself. More revealing, CIP1 seems to be a cytosolic protein associated with the cytoskeleton and thus might be involved in light-induced nuclear depletion of COP1 by anchoring it in the cytoplasm. Another protein interacting with the coiled coil domain of COP1 is CIP7 (Yamamoto et al., 1998). Unlike CIP1, CIP7 is a nuclear protein and contains coiled coil domains that mediate interaction with COP1. Although CIP7 possesses some transcription activation potential, it is not clear whether CIP7 indeed functions as a transcriptional regulator.CIP7 mRNA is hardly detected in the dark, but highly induced by light. Since the kinetics of COP1 nuclear exclusion upon exposure to light are clearly lagging behind the corresponding photomorphogenic changes in the seedling, an early inactivation mechanism for COP1 distinct from nuclear exclusion has been suggested (von Arnim et al., 1997). It is interesting that proper function of COP1 requires its homodimerization, which is also a prerequisite for COP1-HY5 interaction (Torii et al., 1998). Further, COP1 homodimerization is mediated through its coiled coil domain. Thus disruption of COP1 dimerization through occupation of this domain by CIP7 could be a means of rapid COP1 inactivation in response to a light stimulus (Fig. 3). A similar role could be envisioned for the CIP4 protein, which also interacts with the coiled coil domain in COP1 (Yamamoto et al., 2000) and could mediate distinct signaling inputs not covered by CIP7. However, the transactivation potential of CIP4 is much higher than that of CIP7, and an alternative or additional function of CIP4 in transcription control should not be ruled out. An additional N-terminal interactor of COP1 is CIP8, a RING-finger protein that specifically interacts with the RING finger of COP1 (Torii et al., 1999). Clues as to the function of this interaction are by recent on the role of RING-finger proteins in the ubiquitination The RING-finger protein has been established as a of the E3 ubiquitin ligase complex et al., 1999; et al., et al., 1999). Moreover, several RING-finger proteins have been in E2 conjugating enzyme ubiquitination et al., 1999). This might also be the case for CIP8 and Deng, Thus CIP8 might function as part of an E3 ubiquitin ligase complex that COP1 (Fig. 3). The interacting proteins contact COP1 via its WD40 repeat domain. Apart from HY5, a number of other factors that specifically interact with the WD40 repeat domain of COP1 have been in two-hybrid screens and Deng, 1999; and Deng, These additional for COP1. Thus the WD40 repeat domain could serve as an interaction surface for various substrates specifically selected by COP1 for degradation. WD40 repeat domain components are likely responsible for substrate in a number of E3 ubiquitin ligases such as the or complexes 1999). does the COP9 signalosome into this In eukaryotes from to the COP9 signalosome has been in a number of for involved in the of the cell et al., 1999; et al., transcription et al., and et al., 1995). It is interesting that mutants for signalosome have been found in to date and the only mutant in is a lethal in et al., 1999). Thus the COP9 signalosome might not only be for the development of but also of other the of the signalosome in it seems likely that it also takes part in numerous in Arabidopsis. In the signalosome and of Arabidopsis et al., which are of the transcriptional and et al., are also as in other signalosome These for be involved in other than photomorphogenesis. However, in the of viable mutants these are to It is that transgenic with the COP9 signalosome by of one of its could a photomorphogenic response by as observed for et al., 1998). The might be that a constitutive photomorphogenic phenotype is the of a lack of signalosome Thus the of genetic or with signalosome activity by the application of inducible will be necessary to the function of the signalosome at of Arabidopsis development. cop1, all other mutants are unable to degrade HY5 in darkness (Osterlund et al., 2000a). This result might an consequence of the of COP1 from the nucleus since COP1 is localized in the in all the COP9 signalosome mutants even in the dark. It has been suggested that the signalosome could be involved directly or in the regulation of the nuclear machinery responsible for of COP1 (Chamovitz et al., On the other hand, cytosolic COP1 in the dark could also be considered a consequence of the photomorphogenic development in these triggered by an to degrade positive regulators of photomorphogenesis such as HY5. However, an system to COP1 is hypothesis can be for the time role of the COP9 signalosome in protein degradation. of the Arabidopsis COP9 signalosome and the proteasome of and their corresponding in the are opposite one and their primary sequence is of loci or genes corresponding to signalosome are indicated The for the signalosome is indicated on the for the role of the signalosome as an alternative in the of the 26S proteasome. The proteasome substrates that are selected by the The specifically substrates by a of E3 ubiquitin ligases such as an unknown which the factor for degradation. In an alternative the is by the The signalosome a different of proteasome substrates targeted for degradation by a different of E3 ligases such as a factor by a ligase In this of E3 a ligase COP1 is which HY5 for degradation (see 3). that the specificity of the and the signalosome could also be by with the specific E3 ligase rather than substrate These suggest the working hypothesis that the COP9 signalosome is involved in degradation of a selected of substrate by as an alternative of the (Fig. this were indeed the case one could that it might process a specific of proteasome substrates by a number of distinct E3 ligase An E3 ligase complex COP1 could be one of and it might be an E3 ligase in early Arabidopsis development. This would the constitutive photomorphogenic phenotype of the signalosome mutants as the of a lack of signalosome In these cop/det/fus at the same or in development, inputs from more E3 ligase would not be and the combination of these could result in the disruption of This could only lethal alleles of signalosome mutants have been found in Arabidopsis. The would suggest that the substrates directly interact with the COP9 between HY5 and the signalosome have been to however, this might be due to two-hybrid might not these since the integral structure of the signalosome could be required rather than an Moreover, in could be to due to the likely nature of the contact between E3 ligase complexes and the of the proteasome have been found in in and 2000) and it will be interesting to similar can be established for the COP9 as in cop1 and the COP9 signalosome COP1 is also localized in the in the dark in seedlings. Thus both of these loci could also be involved in the of COP1, or are not components of the COP9 signalosome (Chamovitz et al., other can be also been isolated as an extragenic suppressor of and 1997). to the interaction of cop1, this genetic interaction seems to be However, whereas the hy5 has influence on the de-repressed gene expression in at least transcription is to wild-type levels in a encodes a nuclear protein of unknown function to date et al., 1994), but it is that it might be a critical involved in light-induced gene expression, acting on genes under control of HY5. It is clear that a more detailed analysis of the nature of the interaction between and HY5 is to this Also, the role within the context of HY5 degradation Although the mutant any other pleiotropic cop/det/fus mutant with to HY5 accumulation, for cop10, for det1, it is not clear whether it is involved in HY5 degradation. of the gene will this It is interesting that the of has been to a et al., 1999). Although the mutants are to light, they display hardly any morphological phenotype in darkness as with Arabidopsis Since seems to be only gene in has been ruled out as a for this it has been suggested that and act as of whose might be different in the two However, it is also that the mutants are weak Although a about the molecular basis of the function of the COP/DET/FUS genes is from recent a number of from the the role of in the Also, some earlier to be into the is a cop1 mutant that an N-terminal but lacks all of the WD40 repeat domain less severe and whereas a in the WD40 repeat domain can result in et al., et al., A better understanding of the of COP1 within an E3 ligase context might this the must be Although is a to the proteasome on number and primary recent results that the structure of the signalosome complex seems to be rather distinct from the proteasome et al., 2000). Thus whereas a function for the signalosome as an alternative proteasome is a working alternative functions should be More toward a better of the might be from the of the pleiotropic COP/DET/FUSgenes. However, loci one might whether additional genes involved in the system to a different phenotype, for due to or have not been found for other genetic The nature of the of all for the pleiotropic COP/DET/FUS proteins is from In the for the of their in vitro might not their more complex of action in vivo. recent on E3 ligase function that the RING-finger protein of the multisubunit E3 ligase complex is to mediate E2-dependent ubiquitination in vitro et al., 2000). Thus CIP8 or even the RING finger in COP1 would these It is clear that more cell and are to understanding of the mechanisms involved in the photomorphogenic the recent progress in understanding the COP1-HY5 interaction can be as a the COP1-HY5 as a and on the results from of the COP9 signalosome will and into further and Serino for on the