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Annals of Botany 83 : 593–599, 1999 Article No. anbo.1999.0878, available online at http :\\www.idealibrary.com on BOTANICAL BRIEFING ATP-dependent Clp Proteases in Photosynthetic Organisms— A Cut Above the Rest ! A D R I A N K. C L A R KE* Department of Plant Physiology, Uniersity of Umeab , Umeab S-901 87, Sweden Received : 25 January 1999 Returned for revision : 16 February 1999 Accepted : 2 March 1999 Proteases are critical regulatory factors for many metabolic cellular processes as well as being vital for degrading proteins damaged during environmental stresses. Many of those responsible for targeted protein degradation require the hydrolysis of ATP, and one class that has attracted much attention recently are the Clp proteases. They are among the best characterized proteases to date, and were the first shown to rely on an ATPase regulatory subunit possessing molecular chaperone activity, which functions both within the proteolytic complex and independently. A range of Clp proteins has been identified from many different bacteria and eukaryotes, with by far the greatest number and diversity of forms in oxygenic photobionts such as cyanobacteria and higher plants. Functionally, Clp proteins have also evolved into one of the more critical proteolytic enzymes within photobionts, and it is now somewhat of a paradox that we currently know least about Clp protease functions in the photosynthetic organisms, where they have their most important roles. This discrepancy is now being addressed, with studies on Clp protein in cyanobacteria and, in an increasing number, in higher plants. # 1999 Annals of Botany Company Key words : Chloroplasts, Clp proteins, cyanobacteria, molecular chaperones, proteolysis. INTRODUCTION It is only over the last few years that most plant biologists have become aware of the existence of Clp proteins in cyanobacteria and plants, and even today many remain unaware of their importance for protein catabolism in photobionts. This probably stems from the early characterizations of the Clp protease, done almost exclusively in E. coli, where genetic evidence suggested it is a minor enzyme whose loss is readily compensated for by other ATPdependent proteases. The ubiquity of Clp proteins was also unrecognized for many years and has only recently become evident as a result of both directed cloning efforts by individual groups and broad scale genomic sequencing. From this background, it has emerged that Clp proteins are indeed present throughout nature, in all eubacteria examined so far, including cyanobacteria, and in higher eukaryotes such as plants and mammals. Moreover, such studies have demonstrated that the importance of Clp proteases is significantly greater in many organisms than in certain bacteria such as E. coli. It is now well recognized that proteases perform vital functions throughout the cell. As housekeeping enzymes they are critical for cell homeostasis, removing polypeptides that denature either spontaneously or as a result of fluctuating growth environments. Selective protein turnover is also an important regulatory mechanism, influencing the activity of short-lived metabolic enzymes and regulatory proteins, as well as facilitating systemic responses that involve the removal of pre-existing proteins concomitant with the synthesis of new ones (Gotesman, 1996). Because * Fax 46 90 7866676, e-mail Adrian.Clarke!plantphys.umu.se 0305-7364\99\060593j07 $30.00\0 of the potential risk proteases pose to normal protein components, they need to be tightly controlled within the cell. As a result, many of the proteases involved in such targeted proteolysis rely on metabolic energy in the form of ATP. Three proteolytic systems are now emerging as important means for protein turnover in bacteria and eukaryotes. Structurally, all three have similar architecture, and involve not only proteolytic subunits but also accessory proteins that facilitate recognition, binding and presentation of substrates ready for degradation. The ubiquitin\ proteasome system in archeabacteria and within the cytosol and nucleus of eukaryotes is probably the best known of these (Hershko and Ciechanover, 1998), with the HslVU (or ClpQY) protease as its eubacterial counterpart (Rohrwild et al., 1996), and the ClpP protease widespread in eubacteria and higher eukaryotes. THE CLP PROTEASE FROM E. COLI – THE BASIC MODEL Most of what we know about Clp protease comes from studies of the enzyme from E. coli, which has become the basic model for understanding variants in other organisms. The protease from E. coli was first identified as catalyzing in itro degradation of the substrate casein (KatayamaFujimura, Gottesman and Maurizi, 1987), and hence gained its designation as Caseinolytic protease (Clp). It consists of two functionally distinct subunits : a serine-type proteolytic subunit, ClpP (21 kD), and one of two regulatory ATPase subunits, ClpA (83 kD) or ClpX (46 kD). Alone, ClpP is incapable of degrading polypeptides longer than six amino acids, and requires either ClpA or ClpX to become # 1999 Annals of Botany Company 594 Clarke—Clp Proteases in Cyanobacteria and Plants proteolytically active (Woo et al., 1989 ; Wojkowiak, Georgopoulos and Zylicz, 1993). The proteolytic complex consists of two central heptameric annuli of ClpP flanked by one or two hexameric rings of ClpA or ClpX. ClpA has twice the affinity of ClpX for binding to ClpP, although mixed complexes of single ClpA and ClpX hexamers attached to either end of the ClpP core are possible in itro (Grimaud et al., 1998). The ClpAP protease is an endopeptidase that degrades polypeptide substrates without apparent sequence specificity to short peptides of seven to ten amino acids (Thompson and Maurizi, 1994 ; Thompson, Singh and Maurizi, 1994), with the process requiring both Mg#+ and ATP hydrolysis (Katayama et al., 1988). ATP binds at two distinct domains of the ClpA protein, one bound ATP is required for ClpA oligomerization, while hydrolysis of the other ATP is required for proteolysis (Singh and Maurizi, 1994). ClpA also recognizes and binds the protein substrate in some fashion, and then consumes ATP in steps facilitating the accessibility and binding of the protein substrate to the ClpP proteolytic active sites (Wang, Hartling and Flanagan, 1997). Both ClpA and ClpX are now known to be members of a new family of molecular chaperones known as Clp\Hsp100. This family consists of two broad groups, which are separated further into different types based on specific sequence signatures (Schrimer et al., 1996). The first group contains proteins between 85 to 105 kD with two distinct ATP-binding domains. These are further divided into five subtypes, designated ClpA to -E. ClpA is found exclusively in certain Gram-negative bacteria like E. coli, ClpB is present in all bacteria and eukaryotes, ClpC in most Grampositive bacteria, cyanobacteria and plants, ClpD exclusively in plants, and ClpE in certain Gram-positive bacteria. The second group has only two representatives to date : ClpX and ClpY. Both these proteins differ from the first group in having only a single ATP-binding domain, one that is more structurally similar to the ATP-2 domain of the Group 1 Clp proteins (Gottesman et al., 1993). ClpX has been found in all eubacteria and eukaryotes so far, whereas ClpY (or HslV) appears to be restricted to certain eubacteria, functioning as part of the HslVU protease. Excluding ClpY, varying degrees of evidence for ClpP association and involvement in Clp proteolysis exist for ClpA, ClpC, ClpE and ClpX. ClpB is the main exception, however, and is unlikely to participate directly within a Clp protease complex (Clarke, 1996). It is currently unclear whether ClpD possesses the dual protease regulatory\chaperone activity of most other Hsp100 proteins or if it functions independently of ClpP as does ClpB. Determining the structure of the Clp protease has been crucial for our understanding of its action. The architecture of the holo-ClpAP protease from E. coli resembles that of the eukaryotic 26S proteasome, while the ClpP peptidase component resembles that of the 20S proteasome (Kessel et al., 1995). The two heptomeric ClpP rings are stacked backto-back to form a hollow cylinder (Flanagan et al., 1995 ; Shin et al., 1996), with the conserved serine-type protease catalytic sites all facing the inside of the cylinder (Wang et al., 1997). This central catalytic chamber, 50 A/ in diameter, is bounded by solid-walls with only two axial openings (Fig. 1). The width of the axial apertures, however, is only 11 A/ , which prevents most native polypeptides from entering the cavity. Activation of ClpP proteolysis is therefore thought to require unfolding chaperone activity of Hsp100 proteins such as ClpA for efficient translocation of the protein substrate into the ClpP active site domain (Beuron et al., 1998 ; Hoskins et al., 1998). Once bound inside, the unfolded polypeptide is rapidly degraded due to the number and distribution of the active sites within the ClpP cylinder (Wang et al., 1997). Although compelling, several aspects of this proteolytic model still require experimental verification, in particular how the Clp\Hsp100 partners interact with ClpP at its apical surface, and how they recognize, bind and translocate the protein substrate into the proteolytic chamber. ISOMERIC FORMS OF CLP PROTEINS IN CYANOBACTERIA As more clp genes are identified in different bacteria, it becomes evident that the number and functional importance of Clp proteins is far more diverse than previously thought. Although most is known about E. coli Clp proteins, their genetic disruption causes few phenotypic changes, either during normal growth or under stresses such as heat shock (Kroh and Simon, 1990 ; Maurizi et al., 1990 a). In many other bacteria such as Bacillus subtilis, however, Clp proteases are intricately involved in a variety of processes, ranging from developmental changes to stress tolerance (reviewed in Porankiewicz, Wang and Clarke, 1999). As in these bacteria, Clp proteins in cyanobacteria are also vital for growth and stress acclimation. As in most bacteria, cyanobacteria possess two different Clp\Hsp100 proteins likely to participate in Clp proteolytic complexes. In addition to ClpX, cyanobacteria have a homologue to the E. coli ClpA, known as ClpC. Little is yet known about the exact functions of either protein, or their involvement in different Clp proteolytic complexes in cyanobacteria. The cyanobacterial ClpC, however, is approx. 90 % similar (i.e. 80 % identical and 10 % functionally conserved) in amino acid sequence to ClpC in plant chloroplasts, and both proteins are constitutively expressed and their levels remain essentially unaffected by different stress conditions (Shanklin, DeWitt and Flanagan, 1995 ; Clarke and Eriksson, 1996). Genetic interference with ClpC synthesis is also lethal for both kinds of photobionts (Shanklin et al., 1995 ; Clarke and Eriksson, 1996), demonstrating that its chaperone activity, whether independent or as part of a Clp protease, is essential. An added complexity within cyanobacteria is the existence of isomeric forms of ClpP. In the unicellular strains, Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942, three distinct ClpP isomers have been found (Kaneko et al., 1996 ; Clarke, Schelin and Porankiewicz, 1998 ; Clarke and Schelin, unpubl. res.). Originally a fourth clpP gene was assigned within the Synechocystis genome (Kaneko et al., 1996), although the sequence similarity to other clpP genes was relatively low. Analysis of the predicted polypeptide, however, shows it lacks all three conserved residues that constitute the serine-type catalytic active site, and is thus Clarke—Clp Proteases in Cyanobacteria and Plants 595 F 1. Structure of E. coli ClpP complex. Shown are space-filling representations of the ClpP tetradecamer as seen from end-on, depicting the narrow opening to the proteolytic chamber (A), the side, showing the arrangement of the two heptameric ClpP rings (B), and a cutaway view, exposing the large, cylindrical proteolytic chamber (C). Although each subunit is identical, for clarity each is shown in a different colour. Pictures come from Wang, Hartling and Flanagan (1999) and are reproduced with permission. unlikely to function within a Clp protease complex. This protein may still function as a protease with characteristics different from ClpP and is, as such, distinguished as ClpR (Porankiewicz et al., 1999). Interestingly, genes coding for two distinct polypeptides homologous to ClpR have been identified from Arabidopsis thaliana (Clarke, unpubl. res.), suggesting these proteins may constitute a new protease family unique to photosynthetic organisms. Of the three cyanobacterial genes, two are monocistronic (clpP1 and clpP3) while the other (clpP2) is apparently arranged in an operon with clpX, similar to the E. coli homologue (Kaneko et al., 1996 ; Clarke et al., 1998). All studies on cyanobacteria Clp proteins have so far come from Synechococcus sp. PCC 7942, of which to date most is known about ClpP1. ClpP1 is synthesized constitutively in Synechococcus at a relatively low level but, surprisingly, it is not heat-shock inducible, as are the known ClpP proteins in all other eubacteria, nor is it involved in acquired thermotolerance (Clarke et al., 1998). Instead, ClpP1 appears to play an important role during steady-state growth and for long-term acclimation to other types of environmental changes, rather than being involved in resistance to severe, transient stresses. In cyanobacteria and plants, light is a critical environmental variable. ClpP1 content increases dramatically during both short exposures to extreme high light resulting in photoinhibition, and during acclimation to moderate but non-inhibitory light. Inactivation of clpP1, however, has no effect on the susceptibility of Synechococcus to photoinhibition but does produce pleiotrophic changes during steady-state growth. These include slower growth rates, particularly at higher light intensities, as well as changes in pigment composition and the formation of many filamentous cells similar to those formed during stationary phase in wild type cells (Clarke et al., 1998). Reduced growth rates and filamentous morphology were also observed in the corrsponding ∆clpP strain from B. subtilis (Msadek et al., 1998 ; Gerth et al., 1998) suggesting protein turnover mediated by this class of ClpP protein is involved in cell division in some bacteria. In addition to high light, ClpP1 is also strongly induced during acclimation of Synechococcus to temperature drops (37 to 25 mC) or to moderate UV-B supplementation of the growth light (Porankiewicz, Schelin and Clarke, 1998). In these instances, inactivation of ClpP1 synthesis produced a much more severe phenotype. While wild type Synechococcus readily acclimates to both treatments, the ∆clpP1 strain could not, being incapable of either resuming growth or recovering photosynthetically after the initial stress period (Porankiewicz et al., 1998). This again demonstrates the importance of ClpP proteolysis in stress acclimation in cyanobacteria, to a greater extent than in any other eubacteria studied. It is an apparent paradox that isomeric forms of ClpP exist in certain organisms, given that ClpP lacks substratebinding domains and alone is proteolytically inactive. One explanation is differential associations of the ClpP isomers with the various Clp\Hsp100 proteins. This proposal arises from theoretical studies whereby the amino acid differences between each cyanobacterial ClpP protein isomer and ClpP from E. coli are highlighted on the structural model for the E. coli protein (Porankiewicz et al., 1999). This comparison clearly shows that for each cyanobacterial ClpP, most amino acid variations are on the outer surfaces of the heptameric ring structure, away from the central proteolytic cavity, suggesting each cyanobacterial homologue possesses similar proteolytic activity. When the variations distinguishing the three cyanobacterial ClpP proteins are compared, significant differences appear on the apical surface, the region thought to interact with the Hsp100 partner in the holocomplex. This suggests each ClpP isomer may have different affinities for the different Clp\Hsp100 proteins. Although this proposal is appealing, there are only two likely Clp\Hsp100 partners in cyanobacteria (i.e. ClpC and ClpX) to interact with the three ClpP isomers. Despite the two ClpB isomers in cyanobacteria, there is no evidence to date from any organism to suggest ClpB can associate with ClpP to form an active protease. One solution to this stoichiometric conundrum may be the apparent promiscuous affinity of ClpP for other chaperones that have protein 596 Clarke—Clp Proteases in Cyanobacteria and Plants F. 2. Diagrammatic representation of the types, sizes, genetic origin and localization of Clp proteins in the higher plant, Arabidopsis thaliana. Intracellular distribution of Clp proteins is based on experimental observations except for those indicated by question marks where currently only predicted sequence evidence supports localization assignments. Clarke—Clp Proteases in Cyanobacteria and Plants unfolding activity, as was shown in itro by the activation of E. coli ClpP proteolysis by GroEL (Kandor et al., 1994). Another possible explanation for the multiple ClpP isomers is that they may differ in their specificities for short peptides, since one unresolved question about ClpP is whether its peptidase activity has any functional significance in io, perhaps in the degradation of peptide fragments. Despite the attraction of such suggestions, it cannot yet be excluded that the sequence variations between the ClpP isomers have simply accumulated during evolution and thus have no functional significance, especially since most are situated outside of the proteolytic cavity. The main difference between these proteins may be in their genetic regulation, with each being differentially expressed during growth and in response to various stresses. DIVERSITY AND MULTITUDE OF CLP PROTEINS IN HIGHER PLANTS ATP-dependent proteolysis within chloroplasts was demonstrated over 15 years ago (Hammond and Preiss, 1983), but specific proteases have only been identified within the last 5 years. Three main compartmentalized proteolytic systems are known from chloroplasts : soluble Clp proteases in the stroma (Shanklin et al., 1995), FtsH protease within thylakoid membranes (Lindahl et al., 1996 ; Ostersetzer and Adam, 1997), and DegP protease inside the thylakoid lumen (Itzhaki et al., 1998). Other chloroplastic proteolytic activities have been reported (Sokolenko et al., 1997), but specific proteins or corresponding genes for such proteases have not yet been identified. To date four distinct Clp proteins have been found inside plant chloroplasts, two Hsp100 types (ClpC and ClpD) which are both nuclear-encoded, and two ClpP proteins (Fig. 2). The best characterized of these so far is ClpC together with one of the two ClpP proteins. As in cyanobacteria, ClpC appears to be the main Clp\Hsp100 protein in plant chloroplasts, and the homologues share similar characteristics (Shanklin et al., 1995 ; Clarke and Eriksson, 1996). Although two clpC genes were identified in tomato (Gottesman et al., 1990), both code for near identical proteins and thus there is probably only one functional form of ClpC protein. In contrast to the nuclear-encoded ClpC, the first ClpP protein identified in plants was one encoded by the plastid genome (pClpP) (Gray, Hird and Dyer, 1990 ; Maurizi et al., 1990 b). In most plant species, the pclpP gene is cotranscribed along with two ribosomal protein genes (5hrps12 and rpl20), and is later post-transcriptionally processed to produce the mature monocistronic transcript (Koller et al., 1987 ; Clarke, Gustafsson and Lidholm, 1994) ; the main exception to this pattern is pclpP from Arabidopsis thaliana, which is monocistronic and split into two exons. Like ClpC, the pClpP protein is localized in the stroma and is synthesized constitutively in all plant tissues (Shanklin et al., 1995 ; Ostersetzer and Adam, 1996 ; Ostersetzer et al., 1996). Its function is also apparently essential for chloroplasts since its disruption prevents phototrophic growth in green algae (Huang et al., 1994). There is now evidence for a structural association between 597 pClpP and ClpC (Halperin and Adam, 1996 ; Desimone et al., 1997 ; Sokolenko et al., 1998), suggesting they form an active Clp protease in the chloroplast stroma. The potential for such a protease has already been demonstrated in itro, with recombinant plant ClpC being able to activate E. coli ClpP proteolytic activity, and conversely E. coli ClpA activating plant pClpP proteolysis (Shanklin et al., 1995). Less is known about the remaining chloroplast Clp proteins, although their existence implies there is more complexity in the types of Clp proteases than previously thought. The ClpD protein is synthesized constitutively but to a much lesser degree than ClpC (Zheng and Clarke, unpubl. res.). It was first identified in Arabidopsis as a desiccation-inducible protein, Erd1 (Kiyosue, YamaguchiShinozaki and Shinozaki, 1993), and has since been shown to also be induced by high salt concentrations, dark-induced etiolation, and at the onset of senescence (Nakashima et al., 1997). Apart from the plastid-encoded protein, a second ClpP protein occurs in plant chloroplasts. This ClpP protein (nClpP1) is nuclear-encoded but post-translationally imported into the organelle, where it is again localized in the stroma (Sokolenko et al., 1998 ; Zheng and Clarke, unpubl. res.). This ClpP isomer was first identifed from a gene clone from a tomato wound-inducible cDNA library (Schaller and Ryan, 1995), although recent data suggests nClpP1 is primarily a constitutively synthesized protein like pClpP (Zheng and Clarke, unpubl. res.). The nClpP1 protein also appears to form oligomeric structures with ClpC, as well as mixed complexes with pClpP (Sokolenko et al., 1998), suggesting it may participate in Clp proteolytic activity within plant chloroplasts. The importance of individual Clp proteins for chloroplast function has been demonstrated through genetic inactivations (Huang et al., 1994 ; Shanklin et al., 1995), and structural associations between different chloroplastic Clp subunits have been shown. Nevertheless, functional evidence for an active stromal Clp protease remains scant. The specific role of such a Clp protease remains speculative, whether it functions mainly in housekeeping to remove and recycle damaged or otherwise aberrant polypeptides, or whether it instead targets specific stromal enzymes and regulatory proteins. If the latter, then one potential stromal target is Rubisco, whose subunit levels are regulated proteolytically to maintain correct stoichiometry (Schmidt and Mishkind, 1983). Incorrectly processed precursors imported from the cytosol are also degraded in the stroma by enzymes with the functional characteristics of Clp proteases (Halperin and Adam, 1996). Recognition of such mistargeted polypeptides may be further facilitated by the apparent association of a small proportion of ClpC with the chloroplast inner-membrane import machinery (Nielsen et al., 1997), possibly enabling it to efficiently screen precursor proteins following membrane translocation. Supracomplexes within thylakoid membranes may also be subject to Clp proteolysis under certain conditions, particularly the cytochrome b \f complexes during nitrogen starvation in ' algal chloroplasts (Vallon, pers. comm.). Apart from the chloroplast proteins, there are at least four other ClpP isomers in higher plants, all of which are 598 Clarke—Clp Proteases in Cyanobacteria and Plants nuclear-encoded and are known to date from Arabidopsis (Fig. 2). Little is yet known about any of these additional ClpP isomers, although circumstantial evidence suggests one or more may form part of a Clp protease within mitochondria. A similar possibility has already been proposed in humans, where the single known ClpP protein is localized within mitochondria (Corydon et al., 1998). Sequence alignments of all known ClpP proteins show that one of the isomers from Arabidopsis (nClpP2) is closely related to the human mitochondrial protein, and also includes a potential transit peptide (Porankiewicz et al., 1999). Furthermore, in humans, the only known Hsp100\ Clp partner for the lone ClpP protein is ClpX, for which a cDNA clone was recently identified. A similar clpX cDNA was recently cloned from Arabidopsis, and although the protein product was originally thought to be chloroplastic, studies now suggest it may instead be localized inside mitochondria (Adam, pers. comm.). A ClpXP protease, therefore, is likely to be present in human and plant mitochondria, meaning that Clp proteases are not exclusive to plastids in plants. FUTURE PERSPECTIVES Genetic evidence to date suggests ATP-dependent Clp proteases are vital for protein catabolism in both cyanobacteria and higher plants, under both optimal and stress conditions. Despite recent progress in this area, many questions still remain unanswered, especially the specific biochemical functions and compositions of different Clp proteases. Future work must characterize the regulatory Clp\Hsp100 subunits, which confer protein substrate recognition, binding and translocation into the ClpP proteolytic cavity. Similarly, the identity and nature of those protein substrates remain unclear in both cyanobacteria and plant organelles. Another puzzling feature is the number of Clp proteins in photobionts ; what are the functions of these isomers ? In the case of plant Clp\Hsp100 proteins, an obvious role would be to confer differences in substrate specificities and to accommodate sub-organellar compartmentalization of different Clp proteases. For ClpP isomers, the possible functions are less evident, although one possibility is differential specificities for Clp\Hsp100 proteins. Regulatory questions also need investigation, to determine whether the different Clp isomers are differentially expressed or assembled within cyanobacteria and plants, and if so, exactly which signals are responsible ; whether developmental or environmental. Details of the specific cellular locations and targets for these multiple Clp proteases in photosynthetic organisms will greatly advance our knowledge about this new and undoubtedly important family of proteases. A C K N O W L E D G E M E N TS The author would like to thank Dr Jimin Wang for the structural models of the E. coli ClpP complex shown in Fig. 1, Drs Zach Adam and Olivier Vallon for access to unpublished data, and Dr Douglas Campbell for critical reading of the manuscript. This work is supported by grants from the Swedish Natural Science Research Council, the Swedish Agricultural and Forestry Resource Council and the Centre for Forest Biotechnology and Chemistry. LITERATURE CITED Beuron F, Maurizi MR, Belnap DM, Kocsis E, Booy FP, Kessel M, Steven AC. 1998. At sixes and sevens : Characterization of the symmetry mismatch of the ClpAP chaperon-assisted protease. Journal of Structural Biology 123 : 248–259. Clarke AK. 1996. Variations on a theme : Combined molecular chaperone and proteolysis functions in Clp\HSP100 proteins. Journal of Biosciences 21 : 161–177. Clarke AK, Eriksson M-J. 1996. The cyanobacterium Synechococcus sp. 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