26S proteasome pathway, the complex last chapter in the life of many plant proteins

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The ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins Richard D. Vierstra Cellular and Molecular Biology Program and the Department of Horticulture, 1575 Linden Drive, University of Wisconsin-Madison, Madison, WI 53706, USA

Plants use a repertoire of methods to control the level and activity of their constituent proteins. One method, whose prominence is only now being appreciated, is selective protein breakdown by the ubiquitin/26S proteasome pathway. Remarkably, recent analyses of the near-complete Arabidopsis thaliana genome identified > 1300 genes, or , 5% of the proteome, involved in the ubiquitin/26S proteasome pathway, making it one of the most elaborate regulatory mechanisms in plants. Molecular genetic analyses have also connected individual components to almost all aspects of plant biology, including the cell-cycle, embryogenesis, photomorphogenesis, circadian rhythms, hormone signaling, homeosis, disease resistance and senescence. Consequently, it appears that the ubiquitin/26S proteasome pathway rivals transcription complexes and protein kinase cascades as the main player in plant cell regulation. ‘Birth, taxes and death’ is a phrase often used to describe the human condition. Recent scouring of the Arabidopsis thaliana genome suggests that plants live by this same creed. We have long known that the birth of proteins – the intricate processes of transcription and translation – is crucial for most if not all aspects of plant biology. Plants appear to contain at least 1500 factors that affect transcription [1]. Subsequently, it has become apparent that taxation – post-translational processes that regulate a protein’s location, association and activity – is also influential. For example, the Arabidopsis genome is full of protein kinases and phosphatases that could reversibly modify an unlimited number of substrates [2]. However, only in the past few years have we begun to understand protein death fully. In some situations, protein death can be as influential as birth and taxation in regulating plant physiology, growth and development [3,4]. This emerging appreciation of protein death can be attributed to our recent understanding of what is arguably the main proteolytic pathway in eukaryotes, the ubiquitin (Ub)/26S proteasome system [3– 6]. In this pathway, the highly conserved 76-amino-acid protein Ub serves as a reusable tag for selective protein breakdown. It becomes covalently attached to specific protein targets via an ATP-dependent reaction cascade (Fig. 1). The resulting Corresponding author: Richard D. Vierstra ([email protected]).

Ub – protein conjugates are then recognized and degraded by the multisubunit 26S proteasome with the concomitant release of the Ub moieties for reuse. Through this cycle, the Ub/26S proteasome pathway helps remove abnormal proteins and thus performs an essential housekeeping function. By also targeting appropriate normal proteins for breakdown, the pathway provides an important control point by eliminating rate-limiting enzymes and key regulatory factors, and by dismantling crucial signaling networks. For plants, the Ub/26S pathway appears to be particularly important. This view is strengthened by recent discoveries that various pathway components control key aspects of plant growth, development and defense [3,4], and the realization that a sizable fraction of the plant genome is devoted to encoding these proteins [7 – 9]. Counting just the genes that encode core components of the Ub/26S pathway, there are . 1300 participating loci in the Arabidopsis genome, which account for ,5% of the proteome (Table 1) [7 –11]. The purpose of this review is to help to disabuse the notion that proteolysis is a pedestrian aspect of a protein’s life by highlighting recent advances in the field. Table 1. Genomic organization of the Arabidopsis Ub/26S proteasome pathway Protein

Number of genesa

Ub E1 E2 and E2-like E3 HECT SCF F-box RBX –Cullin–ASK Ring finger U-box APC DUBs 26S proteasome 20S CP 19S RP

16 (4) 2 (1) , 45 (13) 7 (5) 694 (14) 33 (3) , 387 (47) 37 (1) . 20? (12) 32 (17) 23 (14) 31 (17) Total . 1327 (148)

Abbreviations: APC, anaphase-promoting complex; CP, core protease; DUBs, deubiquitination enzymes; E1, Ub-activating enzyme; E2, Ub-conjugating enzyme; E3, Ub-protein ligase; HECT, homology to E6-AP C-terminus; RP, regulatory particle; SCF, SKP1, CDC53 and F-box protein complex; Ub, ubiquitin. a Numbers in parentheses indicate the predicted number of Saccharomyces cerevisiae genes.

http://plants.trends.com 1360-1385/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1360-1385(03)00014-1

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Fig. 1. The ubiquitin (Ub)/26S proteasome pathway. The pathway begins with the ATP-dependent activation of Ub by E1, followed by transfer to an E2, and finally attachment of the Ub to the target protein with or without the help of an E3. Once a conjugate is assembled bearing a chain of multiple Ubs, it is either recognized by the 26S proteasome and degraded in an ATP-dependent process or the conjugate is disassembled by deubiquitinating enzymes (DUBs), releasing the Ub and target intact. Abbreviation, K, lysine.

Ubiquitin conjugation cascade Cell fractionation studies and database searches using orthologs from yeast and animals as queries have resulted in the detection and isolation of most of the core components of the plant Ub/26S proteasome system over the past decade [3,5,7]. The Ub conjugation cascade involves a ‘bucket brigade’ of three enzyme families, E1, E2 and E3, that ultimately ligates multiple Ubs to its substrates (Fig. 1). In the initial reaction, E1 (or Ub-activating enzyme) activates the Ub by coupling ATP hydrolysis to the formation of an E1 – Ub intermediate in which the C-terminal glycine of Ub is linked via a thiolester bond to the E1. Next, activated Ub is transferred to an E2 (or Ub-conjugating enzyme) by transesterification. This intermediate then delivers the Ub moiety to the substrate, usually using an E3 (or Ub-protein ligase) as the catalyst. The E3 recruits the substrate, positions it for optimal transfer of the Ub moiety, and then initiates conjugation. In the final product, the C-terminal glycine carboxyl group of Ub is attached via an isopeptide bond to a free lysl e-amino group in the target. Through reiterative rounds of conjugation, a chain of multiple Ubs is often assembled using various Ub lysines (29,48,63) as the site for concatenating additional Ubs [12]. The importance of each of the three enzymatic steps in the hierarchy of target selection is evident by the genomic complexity of their corresponding genes: the Arabidopsis genome encodes only two E1s, at least 45 E2 or E2-like proteins and almost 1200 E3 components (Table 1). http://plants.trends.com

To date, five E3 types have been described in yeast and animals, based on subunit composition and mechanism of action: HECT, SCF, VBC-Cul2, Ring/U-box and APC (Fig. 2). The recent structural solution of representative HECT, Ring, SCF and VBC-Cul2 E3s have provided great insights into how these enzymes work [13 – 16]. These structures show that the E3 forms an arched scaffold that simultaneously docks the Ub– E2 intermediate and target in juxtaposition, presumably to promote Ub transfer to accessible target lysine(s). My research group and others have detected representatives of all but the VBC-Cul2 E3s in the Arabidopsis genome by searching for signature sequence motifs (Fig. 2). The HECT E3s are single polypeptides that were originally identified by the presence of a conserved 350-amino acid C-terminal HECT domain (for Homology to E6-AP C-Terminus) [6]. This domain contains the binding site for the Ub–E2 complex and a positionally conserved cysteine that accepts the Ub moiety to form an E3–Ub thiolester intermediate before final ubiquitination of the target (Fig. 2). HECT E3s typically are .100 kDa (two from Arabidopsis are 405 kDa [17]), with the region N-terminal to the HECT domain containing various protein-interacting motifs (e.g. C2, WW, SH3, Ring-finger, RCC1, UBA and coiled-coil) that might be important for target recognition, Ub-binding and/or localization. Whereas yeast and Arabidopsis contain only five and seven HECT E3s, respectively, .50 have been detected in humans, implying a preferential expansion of this E3 type in mammals (B.P. Downes, unpublished).

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Fig. 2. Organization and structure of (a) the HECT, (b) Ring/U-box, (c) SCF and (d) anaphase-promoting complex (APC) E3s in association with a ubiquitin (Ub)-E2 intermediate. (b) The proposed structure of the Ring-finger motif is shown. Positions 4 and 5 contain histidines in the Ring H2 class, whereas in the Ring HC class, position 4 contains a histidine and position 5 contains a cysteine (C). The pathway of Ub transfer from the E2 to accessible lysines (K) in the target is indicated by the arrows.

The Ring/U-box E3s are a loosely defined collection of polypeptides bearing a signature Ring-finger motif [18]. For the Ring E3s, this motif is formed by an octet of cysteines and histidines that binds zinc in either a RING– H2 or RING –HC arrangement (Fig. 2). Almost 400 potential Ring E3s have been detected in Arabidopsis [9], with the photomorphogenic regulator COP1 serving as the paradigm for structure and activity [19]. Often N-terminal to the Ring finger are other motifs that presumably participate in target recognition (e.g. WW, WD-40 domains). A smaller group of possible U-box E3s has also been identified (, 37 in Arabidopsis) that are proposed to have a similar Ring-finger-like structure called the U-box [20]. Instead of using zinc chelation by cysteine or histidines, U-boxes presumably exploit other intramolecular interactions to stabilize a finger-like motif. The Ring/ U-box E3s do not directly participate in Ub ligation. Instead they serve as a docking platform with the Ring finger interacting with Ub– E2 in a way that allosterically activates the transfer of Ub to the substrate lysine(s) [18]. The SCF E3s (and their related VBC– Cul2 E3s) are complexes of four polypeptides that together have Ubligase activity (Fig. 2). The first complex of this type was shown to participate in the prompt removal of several yeast cell cycle-checkpoint proteins, and was called SCF based on the name of three of its subunits, S KP1, C DC53 (or Cullin), and F-box protein [21]. The fourth subunit, RBX1 (or ROC1 and HRT1), contains a Ring H2-type Ring finger that binds E2 –Ub. Like Ring/U-box E3s, the SCF E3s function as scaffolds that bring the activated Ub – E2 intermediate together with the targets to promote transfer without forming an E3 – Ub intermediate. Specificity of the SCF complex is conferred by the F-box subunit, which contains one of several protein-interaction motifs at its C-terminus and a signature F-box motif at its N-terminus [8]. The F-box motif binds SKP, thus anchoring the recognition motif to the core Ub-ligase activity conferred by the other two subunits, RBX1 and Cullin [16]. The unique combinatorial organization of SCF E3s provides an http://plants.trends.com

effective mechanism for recognizing many substrates simply by exchanging F-box subunits. Genome analyses show that eukaryotes contain numerous SCF complexes, with the number increasing with organism complexity. For example, yeast contains 14, Drosophila contains 24, Caenorhabditis contains 337 and Arabidopsis contains at least 694 different F-box proteins [8]. The diverse array of C-terminal domains within the Arabidopsis F-box protein family (such as leucine-rich repeat, kelch, lectin binding, Armadillo, tetratricopeptide repeats, Jumonji-C, DEAD box and several novel domains) imply that they are capable of recognizing a wide variety of targets [8]. Coupled with the presence of distinct Cullin and SKP (CUL and ASK in Arabidopsis) subunits [11,21– 23], Arabidopsis has the potential to generate an almost infinite array of distinct SCF subtypes, each with unique specificities and/or regulation. The expectation that they have a wide-ranging impact on plant biology has been supported by rapidly accumulating genetic data demonstrating that specific F-box proteins are involved in a variety of activities, from hormone responses and floral homeosis to circadian rhythms and pathogen defense (Table 2) [3,4]. Most known targets of SCF complexes in animals and yeast need to be phosphorylated before F-box recognition [6,18]. Consequently, it is conceivable that the role of many plant protein kinases is to modulate target recognition by F-Box proteins, thus intimately connecting these two protein superfamilies in proteolytic regulation. The most elaborate of the E3s is the anaphase promoting complex (or APC), first identified in yeast and metazoans by its role in the timed breakdown of cyclins and several mitotic checkpoint proteins [24]. It contains 12 or more subunits, including a Cullin and a Ring-finger protein that together presumably form the core ligase activity. Many of the APC genes can be easily identified in the Arabidopsis genome. Only one gene encodes an APC-related Cullin subunit, suggesting that a single or a small set of APC isoforms exists in plants [23]. Its expected role in the plant cell cycle was supported recently by the analysis of the Arabidopsis hobbit mutant. This mutation

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Table 2. Function of selected Ub/26S proteasome pathway component genes from Arabidopsis Gene

Protein type

function

Refs

UFO TIR1 COI1 FKF1/ZTL/LKP2 EID1 ORE9/MAX2 SON1 SKP2;1 ASK1 CUL1 RBX1 SGT1 COP10 COP1 CER3 PRT1 SINAT5 UPL3 HOBBIT RPN12a RPN10 UBP1 and 2 UBP14

E3 (F-box) E3 (F-box) E3 (F-box) E3 (F-box) E3 (F-box) E3 (F-box) E3 (F-box) E3 (F-box) E3 (SKP) E3 (Cullin) E3 (RBX) E3 (SGT1 –SCF) E2-like E3 (Ring HC) E3 (Ring HC) E3 (Ring HC) E3 (Ring HC) E3 (HECT) E3 (APC) 26S proteasome lid 26S proteasome base DUB DUB

Flower development Auxin responses Jasmonate responses Circadian rhythms Photomorphogenesis Senescence Pathogen resistance E2F degradation Auxin responses Embryogenesis Auxin responses Pathogen resistance Photomorphogenesis Photomorphogenesis Wax biosynthesis N-end rule substrates Auxin responses Trichome development Cell division Cytokinin responses ABA responses Abnormal protein degradation Embryogenesis

[61] [43] [62] [63,64] [65] [66,67] [52] [68] [69] [23] [45,70] [48 –50] [55] [19] [71] [72] [46] (B.P. Downes, unpublished) [25] [29] [28] [11] [37,38]

Abbreviations: ABA, abscisic acid; APC, anaphase-promoting complex; DUB, deubiquitination enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; HECT, homology to E6-AP C-terminus; SCF, SKP1, CDC53 and F-box protein complex; Ub, ubiquitin.

affects the CDC27 subunit of the APC and causes a severe dwarf phenotype characteristic of reduced cell division rates [25]. 26S proteasome Once a Ub-protein conjugate is assembled, its most likely fate is recognition and degradation by the 26S proteasome, an ATP-dependent self-compartmentalized protease [26,27]. Although most work on this 2-MDa complex is derived from animals and yeast, evidence is accumulating that the plant version is similarly organized [10,28,29]. The 26S proteasome can be further divided into two particles, the 20S core protease (CP) and the 19S regulatory particle (RP) (Fig. 3). The CP is a broad-spectrum ATP- and Ub-independent peptidase created by the assembly of four, stacked heptameric rings of related a and b subunits in a a1 – 7b1 – 7b1 – 7a1 – 7 configuration. The protease-active sites within the b1, b2 and b5 polypeptides are sequestered in a central chamber. Access to this chamber is restricted by a narrow gated channel created by the a-subunit rings that allows only unfolded proteins to enter [26]. Each end of the CP is capped by a RP; the RP confers both ATP-dependence and substrate specificity to the holoenzyme, especially with respect to those bearing the poly-Ub tag (Fig. 3) [26,27]. Whereas the structure and activity of the CP is known at the atomic level, our understanding of the RP is still rudimentary. The RP presumably helps identify appropriate substrates for breakdown, releases the attached Ubs, opens the a-subunit ring gate, and directs entry of unfolded proteins into the CP lumen for degradation. Its 18 principal subunits can be further divided into two subparticles, the Lid and the Base (Fig. 3). The Base contains three non-ATPase subunits (RPN1, RPN2 and RPN10) and a ring of six AAA-ATPase subunits (RPT1 – RPT6) that contacts the a-subunit ring http://plants.trends.com

and probably assists in target unfolding and transport [30]. One AAA-ATPase, RPT5, might also have a role in recognizing poly-ubiquitinated proteins [31]. The Lid binds to each end of the Base and contains nine additional RPN subunits (RPN3, RPN5– RPN9 and RPN11 – RPN13) (Fig. 3) [26,27,30]. The functions of most of the RPN subunits are unknown. RPN11 and RPN13 (or UCH37) have proteolytic activities that appear to help release poly-Ub chains from substrates before degradation [32,33]. RPN1 can recognize Ub-like proteins that might help shuttle substrates to the 26S proteasome [34]. RPN10 helps tether the Lid to the Base and possibly helps identify ubiquitinated proteins via a Ub-interacting motif [28,30]. The Lid subunits appear to be evolutionarily related in organization and subunit sequences to the COP9/signalosome (CSN) and EIF3 complexes, which are involved in various signaling processes and translation, respectively [30,35]. An intriguing possibility is that CSN and maybe EIF3 can substitute for the Lid in creating new forms of 26S proteasome with distinct specificities. Deubiquitination Plants, animals and yeast also contain a family of deubiquitination enzymes (DUBs) capable of specifically removing covalently bound Ubs. These DUBs help regulate the Ub/26S proteasome pathway by generating free Ub moieties from their initial translation products, recycling Ubs during breakdown of the Ub– protein conjugates, and/or removing Ubs from specific targets and thus preventing their turnover by the 26S proteasome (Fig. 1) [5,36]. In Arabidopsis, for example, 32 DUB genes are present that can be organized into 16 distinct subfamilies [7,11]. In animals, DUBs have been implicated in a variety of processes, including eye development, neuronal function, endocytosis, DNA replication, gene silencing and various pathologies, suggesting that specific

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Fig. 3. Organization and structure of the 26S proteasome. (a) Organization of the core protease (CP) and (b) the regulatory particle (RP) with its Lid and Base subparticles. The dimensions of the CP were obtained from the crystal structure of the yeast complex [73]. The N-terminal threonine residues that form the protease active sites in the b1, b2, and b5 subunits are indicated. Abbreviations: N, RP non-ATPase subunits; T, RP AAA-ATPase subunits. (c) Proposed structure and sequence of events that lead to the degradation of a ubiquitinated protein by the 26S holoprotease.

Ub conjugates are targets [36]. Three DUBs within the Arabidopsis UBP subfamily have roles in removing damaged proteins (UBP1 and UBP2 [11]) and in recycling Ub chains (UBP14 [37]). Surprisingly, loss of UBP14 generates an embryo-lethal phenotype, with the globularstage embryos containing enlarged endosperm cells with polyploid nuclei [37,38]. The ubp14 phenotype could reflect either a stabilization of one or more repressors of embryogenesis, or a general block on 26S proteasome activity caused by the accumulation of free Ub chains that act as competitive inhibitors. Lessons from genomic analysis of the Ub/26S proteasome system Even without genetic evidence, the shear number of the Ub/26S proteasome pathway components in Arabidopsis argues that it must play an important role in plant biology. Indeed, the family of F-box proteins represents the largest protein class identified to date in plants [8]. Moreover, this genomic fraction (5% of the proteome) is more than twice the size of that in yeast or Drosophila for example (Table 1 and R.D. Vierstra, unpublished), implying that plants have placed a particular emphasis on this proteolytic system for cellular control. Like other Arabidopsis gene families [39], those encoding components of the Ub/26S proteasome pathway appear to have evolved from local and large-scale duplications followed by sequence divergence. For the F-box family, related genes are often clustered, indicating that recent tandem chromosomal duplications played an important role [8]. Although many of the components have obvious orthologs in other organisms, several Arabidopsis components are of sufficient sequence http://plants.trends.com

diversity to imply new roles even though they can group into the same protein class [7,8,11]. Not surprisingly, much of the complexity of the Arabidopsis Ub/26S proteasome system is within the enzyme families (E3s) that help choose which proteins should be ubiquitinated [, 1200 E3 component genes (Table 1)]. Because sequence comparisons and limited genetic information suggest that many of these E3s are not redundant (Table 2) [8], it is also possible that Arabidopsis has an equally large number of targets. Based on estimates that 10% of total eukaryotic proteins are regulated by the Ub/26S proteasome system [40], as many as 2500 substrates are possible. Extrapolated further, with . 1200 different E3 components assisting in the ubiquitination of 2500 targets, it is conceivable that most Arabidopsis targets have their own ubiquitination cascades. These cascades could in turn recognize unique degradation signals as a way to confer precise specificity. Such a possible one-to-one correspondence between target and recognition machinery was unimaginable during the early days of proteolysis research when just a few proteases were thought to turnover most substrates by a limited number of general target attributes (e.g. size, pI and heat stability) [41]. Functions of the Ub/26S proteasome pathway Accumulating genetic analyses also support the notion that the Ub/26S proteasome pathway plays a pervasive role in plant biology. Mutations affecting specific factors have been shown to block processes as diverse as embryogenesis, hormonal responses, entrainment of circadian rhythms, floral homeosis, photomorphogenesis,

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trichome differentiation, senescence and pathogen defense (Table 2). Characterizing some of these mutations has offered clues as to how the Ub/26S proteasome system is controlled to degrade the right protein at the appropriate time and place. These studies have also led to the identification of additional components, indicating that the entire Arabidopsis Ub/26S proteasome pathway is even more elaborate than the current estimate in Table 1. The best-understood case is the participation of the Ub/26S proteasome pathway in responses induced by the hormone auxin. Auxin acts in part by directing the degradation of the family of short-lived AUX/IAA proteins; these proteins serve as repressors of auxin action by binding to and blocking the ARF family of transcription factors that upregulate auxin-inducible genes [4]. In the presence of auxin, the SCF complex containing the TIR1 F-box protein (SCFTIR1) triggers AUX/IAA degradation by recognizing a conserved domain (Domain II) within the AUX/IAA targets [42,43]. Presumably this works by SCFTIR1 ubiquitinating the AUX/IAA proteins at a conserved lysine in Domain II, but such intermediates have not yet been detected in vivo. How auxin promotes recognition of AUX/IAA proteins by SCFTIR1 is unknown. One possibility is that auxin induces a modification of the AUX/IAA proteins (e.g. phosphorylation) or that the hormone directly activates the SCFTIR1 complex. With regard to activation of the SCFTIR1 complex, Mark Estelle and co-workers have shown that the Cullin subunit (CUL1) of the SCFTIR1 complex is post-translationally modified by a second peptide tag called RUB (for Related to Ub) [44]. RUB attachment involves a parallel but distinct set of conjugation enzymes that covalently link RUB to a specific lysine within CUL1. Arabidopsis mutants that block RUB attachment attenuate auxin sensitivity, indicating that this modification is required for optimal SCFTIR activity and subsequent auxin responsiveness [44,45]. Downstream of TIR1, the auxin signal for lateral root production is transduced by the transcriptional activator NAC1. Levels of this protein are negatively regulated by the nuclear-localized Ring E3 SINAT5 [46]. Together, a picture is emerging that auxin induces a cascade of modifications that ultimately affects the turnover of auxin-signaling repressors and activators for appropriate up and downregulation. The involvement of the Ub/26S proteasome pathway in pathogen resistance was first inferred by the discovery that the half-life of the Arabidopsis resistance (R) protein (RPM1) is dramatically decreased during infection by compatible pathogens [47]. Given the role of RPM1 in eliciting the hypersensitive response (a localized cell death triggered by pathogen invasion), it appears likely that induced turnover of RPM1 is necessary for mounting appropriate plant defenses. In searches for proteins that interact with RAR1 (a barley protein required for resistance signaling triggered by multiple R genes [48,49]) and for Arabidopsis mutations that abrogate R-gene-mediated defenses [50], several laboratories independently identified plant orthologs of yeast SGT1 as also being important in R protein-mediated resistance. Yeast SGT1 was first identified as a kinetochore protein that also associates with the SKP and Cullin subunits of the SCF complexes, http://plants.trends.com

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presumably to enhance their activity. Plant SGT1s are necessary for early defense against a variety of pathogens, suggesting that they participate in a convergent reaction directed by various R proteins [48,50,51]. Like yeast SGT1, they also associate with SCF complexes [49]. It is an intriguing possibility that R proteins are targets of SGT1 – SCF complexes whose association and breakdown are triggered by a compatible pathogen infection. It is likely that other steps in the defense response also require the Ub/26S proteasome pathway. For example, a SCF complex involving the SON1 F-box protein was recently identified as an important negative regulator of more downstream defense responses independent of systemic signals [52]. During photomorphogenesis, the light-regulated removal of multiple regulators by the Ub/26S proteasome pathway appears crucial. It has long been known that the level of the phytochrome A (phyA) photoreceptor is dramatically reduced in planta upon illumination. This loss is triggered by the Pfr-specific ubiquitination of the photoreceptor and involves a degradation signal near the chromophore-binding pocket [53]. Conversely, light also triggers the stabilization of members of the HY5 family of bZIP transcription factors; they are responsible for the light-induced upregulation of numerous genes required for photosynthetic potential [19,54]. In the dark, these factors are rapidly degraded, thus preventing photomorphogenesis. Breakdown requires a suite of COP (for constitutive photomorphogenic) proteins, first identified in genetic screens by mutations that allow photomorphogenesis to proceed in the dark [35]. Degradation of HY5 is directed by a complex containing the Ring E3 COP1 and the E2-like protein COP10 [55]. Light inhibits HY5 degradation by controlling the intracellular location of COP1 and possibly the phosphorylation state of HY5 [56,57]. Whereas COP1 is free to enter the nucleus and reach targets such as HY5 in the dark, this nuclear import is blocked in the light, thus stabilizing these targets. Another set of COP proteins assembles together to form the eight-subunit CSN complex [35]. The CSN is present in animals, plants and fission yeast (with a variant possible in Saccharomyces cerevisiae) and performs a variety of essential physiological functions that are intimately connected to the Ub/26S proteasome pathway. CSN interacts with SCF complexes such as SCFTIR1 [58] and contains an activity that can remove RUB bound to the Cullin subunit [59]. It has been proposed that the CSN complex is responsible for deactivating associated SCF E3s such as SCFTIR1 in agreement with the observations that weak CSN mutants are auxin insensitive [58]. Given its architectural similarity to the Lid [30,35], the CSN might also substitute for the Lid in the 26S proteasome, thus creating a supercomplex containing both Ub-ligating and targetdegrading functions. It had been assumed that much of the selectivity of the Ub/26S proteasome pathway is achieved by the ubiquitination reactions. However, recent genetic analyses have also implicated the RP of the 26S proteasome as an important contributor. Mutants affecting individual Arabidopsis RP subunits display a wide range of phenotypes, consistent with each participating in the destruction of

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distinct sets of targets [28,29] (J. Smalle unpublished). For example, a mutant affecting RPN12a confers a decreased sensitivity to cytokinins whereas a mutant affecting RPN10 confers a hypersensitivity to abscisic acid (ABA). Furthermore, the rpn10-1 mutant is able to degrade HY5 and phyA normally but dramatically stabilizes the ABA-response regulator ABI5, showing that RPN10 only affects a subset of Ub/26S proteasome substrates [28]. How the individual RP subunits are able to discriminate between substrates is not yet clear, but for subunits such as RPN10 and RPN1, it could involve specific interactions with shuttle proteins (e.g. RAD23, DSK1 and DSK2 [28,34]) that might help deliver substrates to the 26S proteasome. Conclusion Even though our understanding of the Ub/26S proteasome pathway in plants has progressed rapidly over the past few years, much remains to be solved. It is especially important to identity the targets and define how these targets are recognized. All short-lived intracellular proteins are potential suspects, but to date only a handful (i.e. phyA, HY5/HYH, AUX/IAA, NAC1, E2F and ABI5) have been confirmed to be substrates and only phyA has been demonstrated to be ubiquitinated in planta. The more-complete list of targets will ultimately require proteomic methods such as mass spectrometry to characterize the transient Ub-conjugate pool, an extraordinary challenge given its heterogeneity, short half-life and low abundance. As extensive and complex as the Ub/26S proteasome pathway is in plants, it should be emphasized that it is not the sole proteolytic system. It is obvious that the Arabidopsis genome also encodes families of other proteases and alternative pathways for protein recycling [5]. For example, under starvation conditions and during senescence, plants use an autophagic process to indiscriminately engulf and deliver cytoplasmic and organellar proteins to the vacuole where they are degraded by resident proteases [60]. As we continue to study protein death in plants, a dizzying array of mechanisms should spring to life and thus further elevate its status in the pantheon of plant regulatory processes. Acknowledgements I apologize for not including all pertinent references because of space constraints. Research in my laboratory is supported by grants from USDA-NRICGP (00 – 35301 – 9040), DOE (DE-FG02 – 88ER-13968) and the NSF Arabidopsis 2010 program (MCB-00115870).

References 1 Riechmann, J.L. et al. (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290, 2105 – 2110 2 Shiu, S.H. and Bleecker, A.B. (2001) Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. Proc. Natl. Acad. Sci. U. S. A. 98, 10763 – 10768 3 Callis, J. and Vierstra, R.D. (2000) Protein degradation in signaling. Curr. Opin. Plant Biol. 3, 381 – 386 4 Hellmann, H. and Estelle, M. (2002) Plant development: regulation by protein degradation. Science 297, 793– 797 5 Vierstra, R.D. (1996) Proteolysis in plants: mechanisms and functions. Plant Mol. Biol. 32, 275 – 302 http://plants.trends.com

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