Pretty Complex Interactions in Diverse Signaling Pathways

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44 Grant, M. et al. (2000) The RPM1 plant disease as a signal in plant disease resistance. Nature 63 Scheel, D. et al. (2000) Receptor-mediated signal resistance gene facilitates a rapid and sustained 394, 585–588 transduction in plant defense. In Biology of increase in cytosolic calcium that is necessary for 54 Durner, J. et al. (1998) Defense gene induction in Plant–Microbe Interactions (Vol. 2) (De Wit, the oxidative burst and hypersensitive cell death. tobacco by nitric oxide, cyclic GMP and cyclic ADP P.D.G.M. et al., eds), pp. 131–135, International Plant J. 23, 441–450 ribose. Proc. Natl. Acad. Sci. U. S. A. 95, Society of Plant-Microbe Interactions 45 Mithöfer, A. et al. (1999) Transgenic aequorin 10328–10333 64 Ligterink, W. et al. (1997) Receptor-mediated monitors cytosolic calcium transients in soybean 55 Clarke, A. et al. (2000) NO way back: nitric oxide activation of a MAP kinase in pathogen defense cells challenged with β-glucan or chitin elicitors. and programmed cell death in Arabidopsis of plants. Science 276, 2054–2057 Planta 207, 566–574 thaliana suspension cultures. Plant J. 24, 667–677 65 Yang, K-Y. et al. (2001) Activation of a mitogen- 46 Xu, H. and Heath, M.C. (1998) Role of calcium in 56 Foissner, I. et al. (2000) In vivo imaging of an activated protein kinase pathway is involved in signal transduction during the hypersensitive elicitor-induced nitric oxide burst in tobacco. disease resistance in tobacco. Proc. Natl. Acad. response caused by basidiospore-derived infection Plant J. 23, 817–824 Sci. U. S. A. 98, 741–746 of the cowpea rust fungus. Plant Cell 10, 585–597 57 Clough, S.J. et al. (2000) The Arabidopsis dnd1 66 Kovtun, Y. et al. (2000) Functional analysis of 47 Romeis, T. et al. (2000) Resistance gene- ‘defense, no death’gene encodes a mutated cyclic oxidative stress-activated mitogen-activated dependent activation of a calcium-dependent nucleotide-gated ion channel. Proc. Natl. Acad. protein kinase cascade in plants. Proc. Natl. Acad. protein kinase in the plant defense response. Sci. U. S. A. 97, 9323–9328 Sci. U. S. A. 97, 2940–2945 Plant Cell 12, 803–815 58 Köhler, C. et al. (1999) Characterization of a novel 67 Petersen, M. et al. (2000) Arabidopsis MAP kinase 48 Heo, W.D. et al. (1999) Involvement of specific gene family of putative cyclic nucleotide- and 4 negatively regulates systemic acquired calmodulin isoforms in salicylic acid-independent calmodulin-regulated ion channels in Arabidopsis resistance. Cell 103, 1111–1120 activation of plant disease resistance responses. thaliana. Plant J. 18, 97–104 68 Frye, C.A. et al. (2001) Negative regulation of Proc. Natl. Acad. Sci. U. S. A. 96, 766–771 59 Cardinale, F. et al. (2000) Differential activation of defense responses in plants by a conserved 49 Scheel, D. (2001) Oxidative burst and the role of four specific MAPK pathways by distinct elicitors. MAPKK kinase. Proc. Natl. Acad. Sci. U. S. A. reactive oxygen species in plant–pathogen J. Biol. Chem. 275, 36734–36740 98, 373–378 interactions. In Oxidative Stress in Plants 60 Nühse, T.S. et al. (2000) Microbial elicitors induce 69 Johnson, P.R. and Ecker, J.R. (1998) The ethylene (Inzé, D. and van Montagu, M., eds), pp. 137–153, activation and dual phosphorylation of the gas signal transduction pathway: a molecular Harwood Academic Publishers Arabidopsis thaliana MAPK 6. J. Biol. Chem. perspective. Annu. Rev. Genet. 32, 227–254 50 The Arabidopsis Genome Initiative (2000) 275, 7521–7526 70 Ichimura, K. et al. (2000) Various abiotic stresses Analysis of the genome sequence of the flowering 61 Romeis, T. et al. (1999) Rapid Avr9- and Cf-9- rapidly activate Arabidopsis MAP kinases plant Arabidopsis thaliana. Nature 408, 796–815 dependent activation of MAP kinases in tobacco ATMPK4 and ATMPK6. Plant J. 24, 655–665 51 Kawasaki, T. et al. (1999) The small GTP-binding cell cultures and leaves: convergence of resistance 71 Osusky, M. et al. (2000) Transgenic plants protein Rac is a regulator of cell death in plants. gene, elicitor, wound, and salicylate responses. expressing cationic peptide chimeras exhibit Proc. Natl. Acad. Sci. U. S. A. 96, 10922–10926 Plant Cell 11, 273–287 broad-spectrum resistance to phytopathogens. 52 Ono, E. et al. (2001) Essential role of the small 62 Zhang, S. and Klessig, D.F. (1998) The tobacco Nat. Biotechnol. 18, 1162–1166 GTPase Rac in disease resistance of rice. Proc. wounding-activated mitogen-activated protein 72 Gao, A-G. et al. (2000) Fungal pathogen protection Natl. Acad. Sci. U. S. A. 98, 759–764 kinase is encoded by SIPK. Proc. Natl. Acad. Sci. in potato by expression of a plant defensin 53 Delledonne, M. et al. (1998) Nitric oxide functions U. S. A. 95, 7225–7230 peptide. Nat. Biotechnol. 18, 1307–1310

PCI complexes: pretty complex interactions in diverse signaling pathways

Tae-Houn Kim,Kay Hofmann, Albrecht G. von Arnim and Daniel A. Chamovitz

Three protein complexes (the proteasome regulatory lid, the COP9 (‘C’) and the translation initiation factor eIF3 (‘I’). signalosome and eukaryotic translation initiation factor 3) contain protein All three are structurally related and are highly subunits with a well defined protein domain, the PCI domain. At least two (the conserved among higher eukaryotes. The situation COP9 signalosome and the lid) appear to share a common evolutionary origin. in Saccharomyces cerevisiae is less clear, because Recent advances in our understanding of the structure and function of the there is an obvious proteasome regulatory lid and a three complexes point to intriguing and unanticipated connections between smaller but obvious eIF3, but no recognizable CSN. the cellular functions performed by these three protein assemblies, especially All three complexes are ~500 kDa. Subunits of all between translation initiation and proteolytic protein degradation. three complexes contain one of two domains, the PCI domain or the Mpr1–Pad1 N-terminal (MPN) The PCI complexes are a recently discovered family domain1–4. The primary sequence of the PCI domain of multisubunit protein complexes that regulate is not well conserved, which hinders classic development and signal transduction. There are alignment and phylogenetic analysis. Instead, the three known PCI complexes: the regulatory lid of the PCI domain is characterized by a conserved 26S proteasome (‘P’), the COP9 signalosome (CSN) secondary structure that is a largely α-helical fold.

Table 1. MPN and PCI-containing proteins encoded in the Arabidopsis genomea Csn5a 98 Domain Identity Accession no. Rpn11 MPN RPN8 At3g11270

MPN RPN11 At5g23540 eIF3h MPN CSN5 At1g22920, At1g71230 100 MPN CSN6 At4g26430, At5g56280 Csn6 MPN eIF3f At2g39990 40 MPN eIF3h At1g10840 Rpn8 MPN unknown At5g55940 MPN Similar to AMSHb At1g10600, At1g48790 eIF3f MPN unknown At1g80210, At3g06820 TRENDS in Plant Science MPN Similar to Prp8 At4g38780, At1g80070 PCI RPN3 At1g20200, At1g75990 Fig. 1. Phylogenetic analysis of the MPN- domain subunits from the PCI RPN5 At5g09900, At5g64760 COP9 signalosome (Csn5, Csn6), the proteasome regulatory lid PCI RPN6 At1g29150 (Rpn11, Rpn8) and eukaryotic initiation factor 3 (eIF3h, eIF3f) from Arabidopsis. The multisequence alignment was constructed using PCI RPN7 At4g24820 ClustalX. The unrooted tree branch-point confidence values are based PCI RPN9 At5g45620 on 1000 bootstrap replications. PCI CSN1 At3g61140, Q9LTV1 PCI CSN2 At2g26990 complex (such as CSN4 and Rpn5, which are CSN and lid subunits, respectively), whereas others might PCI CSN3 At5g14250 be closer to subunits within the same complex. PCI CSN4 At5g42970 PCI CSN7 At1g02090 Proteasome lid complex PCI CSN8 At4g14110 The eukaryotic proteasome is a ~2.5 MDa complex PCI eIF3a At4g11420 that specifically directs ubiquitin-mediated protein PCI eIF3c At3g56150, At3g22860 degradation8. The proteasome plays a crucial role in PCI eIF3e At3g57290 diverse processes including cell-cycle regulation, PCI Unknown At3g02200, At5g15610 DNA repair, circadian rhythms and responses to PCI Unknown At2g19560 extracellular signals. In plants, the proteasome has 9–11 aThe predicted proteins from the Arabidopsis genome been implicated in light and hormone signaling . (ftp://ftpmips.gsf.de/cress/arabiprot/) were searched for MPN and The proteasome holoenzyme (also known as the 26S PCI-containing motifs. Only obvious matches are listed. Several of proteasome) can be divided into two major the proteins result from duplicated genes. subcomplexes: the 20S core particle (CP), which bAMSH, associates molecule with the SH3 domain of STAM. contains the protease subunits, and the 19S regulatory particle (RP), which regulates the The MPN domain is more easily identified and forms function of the CP. The RPcan be further dissected an α/β fold. The significance of these domains is not into two multisubunit structures: a regulatory lid understood, although the PCI domain might and an ATPase-containing base. In S. cerevisiae, the stabilize protein–protein contacts within the regulatory lid is composed of eight subunits that can complex5–7 and the MPN domain might have a more be separated from the base as a distinct 500 kDa general role in transient protein–protein complex3. Putative orthologs for each of these interactions. proteins are encoded in the Arabidopsis genome The similarities between the CSN, eIF3 and the (Table 1). To date, no distinct function or enzymatic

Tae-Houn Kim proteasome regulatory lid suggest that the three activity has been shown for any of the lid subunits. Albrecht G. von Arnim complexes, or their subunits, share a common Given that attachment of the lid is necessary for Dept Botany, The evolutionary ancestor. Although both the proteasome proteolysis of a ubiquitinated protein, the lid University of Tennessee, lid and the CSN contain eight subunits, six of which probably interacts with the polyubiquitin chain12. Knoxville, TN 37996-1100, 4 USA. contain the PCI domain, and two MPN proteins , Alternatively, the lid might affect the cooperativity of eIF3 is more distantly related (Table 1). Only five of the ATPase activity of the base13. The lid is part of the Kay Hofmann Bioinformatics Group, the eIF3 subunits have the PCI or MPN signature RP that is most exposed to the cytoplasm, and MEMOREC Stoffel, domains. It is clear that, at least for the MPN- therefore is an ideal candidate to interact with other D50829 Koeln, Germany. containing subunits, the proteasome and CSN cellular factors that could fine tune its function or Daniel A. Chamovitz* subunits are more closely related to each other than affect its subcellular localization. Dept Plant Sciences, they are to the eIF3 proteins (Fig. 1). The S. cerevisiae has been the most widely used system Tel Aviv University, phylogenetic relationship among the PCI proteins is for studying the regulatory lid, but plants are serving Tel Aviv 69978, Israel. *e-mail: less clear. For example, for some PCI proteins, the as adequate higher eukaryotic systems. In plants, the [email protected] closest relative appears to be a subunit of another base, but not the lid, of RP has been studied in the

http://plants.trends.com Review TRENDS in Plant Science Vol.6 No.8 August 2001 381 moss Physcomitrella patens14. As previously shown in involved in numerous pathways19. This pleiotropic yeast, the P. patens base subunit MCB1 (Rpn10) has nature implies that the CSN acts at the link between affinity for polyubiquitin chains in vitro, suggesting multiple signal inputs and a variety of downstream that it might contribute to the recognition of regulatory cascades controlling specific aspects of ubiquitinated substrates. The yeast ortholog of MCB1 cellular differentiation. Thus, the CSN could be a also helps to tether the lid to the base of the RP general developmental regulator whose activity is (Ref. 3). Interestingly, in the moss, and unlike S. modulated by other signals in addition to light, and cerevisiae, disruption of the gene encoding MCB1 by whose targets extend beyond light regulation. homologous recombination caused developmental The hypothesis that the CSN is a general arrest, which could be partially rescued by exogenous developmental regulator is further supported by the application of auxin and cytokinin. This is an identification of the CSN in animal systems. Analysis important result because it confirms for a of the CSN from mammals20,21 and Drosophila7, as multicellular plant that an RP subunit has a well as from cauliflower (Brassica oleracea)22 and conditional role within the proteasome. Arabidopsis23, indicate that this complex comprises In Arabidopsis, a 500 kDa complex (PR500) with eight core subunits. Although a readily recognizable many attributes expected of the proteasome lid has CSN is apparently absent from S. cerevisiae4,24, a been purified15. PR500 contains at least three complex containing several homologs of the CSN was components: Rpn3, Rpn5 and Rpn6, in common with identified in the fission yeast Schizosaccharomyces a larger, 800 kDa complex, which might be the RP. pombe25. Genetic analyses have also begun to The lid-like PR500 complex can be isolated in a free elucidate the role of the CSN in animal systems. The form, independent of the proteasome base, in wild- CSN is essential for the developmental transition of type plants. However, in Arabidopsis mutants Drosophila melanogaster from larva to adult7. This defective for the CSN, PR500 was absent, suggesting phenotype is curiously reminiscent of the Arabidopsis that its subunits had become incorporated into the CSN mutants because the lethality is revealed only 19S RP or the 26S holoenzyme. These data after successful embryogenesis and several days of foreshadow interactions between the two PCI development. In fission yeast, although a CSN complexes that will be discussed in more detail below. mutant was viable, it had cell-cycle defects and overexpression of the CSN5 subunit caused drug CSN resistance26, a phenotype also seen upon The CSN is a highly conserved complex that has a overexpression of the c-Jun-related transcription major role in regulating the development of factor Pap1 (AP-1)27. eukaryotes. The CSN was first identified in The mammalian CSN is thought to associate with Arabidopsis, and most research on CSN has been in a kinase that phosphorylates the transcription factor Arabidopsis4,16. Mutations in the Arabidopsis CSN c-Jun, implying a role for the CSN in regulating the result in dark-grown seedlings that mimic light- activity of c-Jun, possibly by stabilizing c-Jun against grown wild-type seedlings in almost all proteasome-mediated degradation20. Moreover, developmental parameters that have been overexpression of the CSN2 subunit increased the monitored. Genetic studies indicated that the CSN level of c-Jun-dependent gene expression28. These acts to repress photomorphogenesis in the dark, and results mirror the findings in fission yeast of a genetic that light reverses this repression. Among the CSN interaction between the human CSN5 subunit Jab1 subunits, mutations in CSN1, CSN4, CSN7 and and the c-Jun-related Pap1 protein26. Additional CSN8 (originally described as FUS6, COP8, FUS5 evidence supports a role for the CSN in regulating and COP9, respectively) all lead to almost identical signaling pathways mediated by mitogen-activated- phenotypes, underscoring the close cooperation of protein kinase (MAP kinase) in mammalian cells, these gene products. CSN5 is encoded by two genes because several of the CSN subunits have been that are presumably functionally overlapping17. A independently identified as putative regulators of specific direct role for the CSN in light signaling was MAP kinase signaling, cell-cycle control and hormone highlighted by showing that overexpression of CSN1 responses29. The direct connection between these or CSN8 causes mild gain-of-function phenotypes in pathways and the CSN is unknown. the repression of light-regulated development18. Although some of the CSN subunits are found Although it was originally described as a master exclusively in the complex (e.g. CSN1 and CSN8), repressor of photomorphogenic development in studies in Drosophila and Arabidopsis have shown plants, the CSN has a more general role. Mutations in that at least three subunits (CSN4, CSN5 and CSN7) the CSN lead to lethality following the transition from are also found in other forms7,17,23,30. This raises the embryo to seedling development, even under normal possibility that some of the subunits might have roles light conditions, indicating that the complex has an independent of the complex. In particular, in essential role in normal light-grown development. Arabidopsis, the CSN5 subunit exists in an Furthermore, several non-light-regulated genes are uncomplexed, and possibly cytoplasmic, form6,17, also misregulated in one of the csn mutants, further whereas the assembled plant CSN was found in the indicating that the CSN is a pleiotropic regulator nucleus22. Mammalian CSN5 interacts with many http://plants.trends.com 382 Review TRENDS in Plant Science Vol.6 No.8 August 2001

c-Jun p27Kip1 eIF2 Bcl-3 eIF4B SRC-1 LFA1 eIF2B rLHR eIF1 MIF1 eIF5 eIF3a eIF3k CSN4

CSN8 eIF3g eIF3c eIF3j CSN5 CSN1 eIF3i

eIF3b CSN7 eIF3e eIF3d CSN3

CSN2 CSN6 eIF3h eIF3f Scd1(Ral1) Tax Vpr Rfp Cul1 TR/RXR P56 ICSBP Rpn11 Rpn5 Cul1 Rpn3

Rpn7 Rpn12 Rpt5 MPN Core 19S Lid Rpn6

Rpn9 Rpn8 PCI Non-core 19S Rpn10

TRENDS in Plant Science

Fig. 2. Protein interaction map for the three PCI complexes. Diverse Taken together, the biochemical and genetic data patterns of protein–protein interactions suggest a role for the PCI from diverse eukaryotic model organisms are complexes as integrators of different signal transduction pathways. converging on a consensus for the role of the CSN as a Solid lines denote interactions between the subunits, which were obtained from yeast two-hybrid and direct protein–protein interaction key regulator involved in numerous signaling assays in yeast, human or Arabidopsis. Known interactions between pathways and developmental transitions. Some of the PCI complexes with other cellular signaling factors are indicated by these interactions appear to have been conserved broken lines. The eIF3 structure is based on the human complex; in Arabidopsis , eIF3j is not present, but an eIF3l subunit is present. Only through evolution along with the protein sequences the five core components are conserved in Saccharomyces cerevisiae. of the CSN subunits. More investigations are needed to complete the full set of interactions and the figure does not represent the actual three-dimensional eIF3 positions of each subunit within the protein complexes. Abbreviations: c-Jun, a subunit of the AP-1 transcription factor29; Bcl-3, an Only one other PCI complex is currently known: oncoprotein29; Cul1, SCF component Cullin1 (Ref. 54); ICSBP, interferon- eIF3. In eukaryotes, translation initiation is a consensus-sequence-binding protein51; LFA1, integrin29; MIF1, multistep process32. Briefly, when the 40S ribosomal macrophage migration inhibitory factor29; p27Kip1, cyclin-dependent- kinase inhibitor29; P56, interferon-induced protein62; Rfp, Ret finger subunit is associated with tRNA–methionine, it is protein57; rLHR, lutropin/choriogonadotropin receptor29; Scd1(Ral1), brought into contact with the 5′ end of the mRNA, Ras1 effector66; SRC-1, steroid receptor co-activator29; Tax, followed by 5′-to-3′ scanning to select the AUG start transactivator of the human T cell leukemia virus type I (Ref. 65); codon. The 60S ribosomal subunit then joins it, TR/RXR, thyroid hormone receptors/retinoid X receptor67; Vpr, HIV-1 accessory gene product68. which permits binding of the second tRNA and progression into the translation elongation phase. cellular proteins in both the cytoplasm and the Among the numerous eukaryotic translation nucleus29 (Fig. 2). CSN5 might well function as an initiation factors (eIFs) involved, four play key roles: adapter or a recruitment device for the CSN. How eIF4F, eIF2, eIF5 and eIF3. CSN5 might exert its regulatory role is brought into Structurally, eIF3 is the most complex of the eIFs focus most clearly in connection with p27Kip1, a because of its three fundamental roles33: repressor of the G1–S phase cell-cycle transition. The • It prevents association of the 40S and 60S ribosomal interaction between CSN5 and p27Kip1 results in the subunits in the absence of mRNA. relocalization of nuclear p27Kip1 to the cytoplasm, • It facilitates loading of the 40S subunit with the suggesting a direct or indirect role for CSN5 in ternary eIF2–tRNA–Met–GTP complex. regulating protein relocalization31. • It interacts with eIF4B and the eIF4F mRNA

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cap-binding complex, thus arranging the contact phenotype is not understood, it is intriguing that between the mRNA and the 40S subunit. overexpression of the proteasome lid subunit After recognition of the start codon, which might Rpn11(Pad1)49, human CSN5 (Ref. 26) and fission involve the small protein eIF1, GTP is hydrolysed by yeast pap1/AP-1 transcription factor homologs also eIF2 and eIF3 dissociates from the preinitiation cause drug resistance48. Given that overexpression of complex, which in turn permits the 60S subunit to other eIF3 subunits tested did not cause drug join the 40S subunit. The three activities of eIF3 are resistance, these results are exciting because they mediated by its interaction with other eIFs. A labile suggest a functional connection between the three supercomplex has been found recently33 that consists PCI complexes, eIF3 via eIF3e, the proteasome via of eIF1, eIF2, eIF3, eIF5 and tRNA–Met, which is Rpn11 and the CSN via CSN5. consistent with eIF3 having a central role. The subunit composition of eIF3 has been a matter Interactions among the PCI complexes and their of debate, with the composition often depending on subunits the method of purification. Mammalian eIF3 now An obvious question is whether the evolutionary appears to contain 11 subunits: eIF3a–eIF3k, in relationship among the PCI complexes signifies a descending size order from 170 kDa to 25 kDa34–38. functional relationship in regulating common Plant eIF3 closely resembles mammalian eIF3, pathways. having 10 of its 11 subunits in common, but it also contains a novel subunit, eIF3l, that is not present in CSN and the proteasome mammals34,39. The S. cerevisiae eIF3 core complex is Although the CSN and the lid resemble each other in smaller and consists of only five subunits, which are terms of subunit composition, they each have unique homologous to human eIF3a, eIF3b, eIF3c, eIF3g and structures5. However, there is now substantial yet eIF3i (Ref. 40), although other proteins remain circumstantial evidence hinting at a functional associated with the core complex when different relationship between the CSN and the proteasome purification conditions are used41–43. This has given lid. First, the CSN purifies with the proteasome in rise to the concept that eIF3 consists of a core of five mammalian cells20. Second, in Arabidopsis and subunits, conserved among all eukaryotes, and cauliflower, the PR500 complex, which might additional non-core subunits (Fig. 2). This idea agrees represent a lid complex detached from the with the finding that these five core subunits are proteasome base, depends on a functional involved in most of the known contacts between eIF3 signalosome15. This might indicate, among other and the other eIFs (Fig. 2). Exacerbated by the lack of possibilities, that the CSN functions as an mutants in higher eukaryotes, the role of the non-core alternative cofactor of the proteasome or that the eIF3 subunits (those missing in S. cerevisiae) has CSN modulates proteasome activity by altering the remained speculative. They might have regulatory activity of the PR500 lid complex. Third, consistent functions or provide structural support (e.g. eIF3e)44. with the idea of an interaction between lid and CSN, In wheat, the expression profiles of the eIF3 subunits Arabidopsis CSN1 interacted with Rpn6 in yeast, are not coordinated stoichiometrically, and this although any interaction in plants was too unstable includes both core and non-core subunits, which is or transient to be detected50. Arabidopsis Rpn6 suggestive of regulatory or accessory roles for at least also interacts with CSN7 (B. Karniol and some of the subunits45. D.A. Chamovitz, unpublished). At a more functional Mutational analysis in fission yeast, which level, it is now becoming clear that light signal contains most of the higher eukaryotic subunits, is transduction depends on the proteolysis of specific beginning to substantiate a regulatory role for at transcriptional regulators, for example the bZIP least one of the non-core subunits, eIF3e. A protein HY5. Two of the proteins required for dark- mutation in eIF3e of S. pombe causes a reduced dependent proteolysis of HY5 are the Ring-finger polysome:monosome ratio and slow growth, but is far protein COP1, postulated to function as an E3 less detrimental than a mutation of one of the core ubiquitin ligase, and the CSN (Ref. 11). In animal subunits, eIF3g, which is lethal46,47. Interestingly, the cells, the CSN has been implicated in regulating the eIF3e mutation results in different accumulation of phosphorylation or ubiquitin-mediated degradation cellular proteins48. In addition, it has discrete cellular of IRF-1, c-Jun and p53 (Refs 28,51,52). Collectively, phenotypes, including sporulation and chromosome these results lead us to think of the CSN and the segregation defects, and sensitivity to osmotic stress proteasome lid as complexes with a related and caffeine, suggesting that this subunit makes biochemical agenda. more than just a quantitative contribution to eIF3 Recent data suggest a mechanism for this ‘agenda’. activity. These results support the idea of a regulatory The CSN interacts with the E3 ubiquitin ligase role for the eIF3e subunit. Additional leads are now complex SCF in animals and plants, and affects SCF fueling speculation as to what this regulatory role activity by regulating the removal of the ubiquitin- might be. like protein NEDD8 (deneddylation) from the SCF Overexpression of eIF3e in fission yeast causes Cul1 subunit53,54. This result is particularly exciting drug resistance. Although the molecular basis of this because it suggests a mechanism linking light http://plants.trends.com 384 Review TRENDS in Plant Science Vol.6 No.8 August 2001

signaling through the CSN and auxin responses nuclear in roots, where the CSN is known to repress through the SCF and proteasome. light-inducible gene expression, these data amount to a positive correlation between the level of CSN and eIF3 activity of the CSN and the nuclear localization of Even though eIF3 is more distantly related to the PCI eIF3e (Ref. 56). complexes, there is also intriguing evidence that eIF3 Such a model is not too far fetched because, in and the CSN cooperate in some way. The first fission yeast as well as in mammalian cells, evidence evidence was biochemical: three eIF3 subunits is slowly accumulating to support a regulatory, purified with the CSN from cauliflower – the core rather than housekeeping, role for the non-core eIF3 subunit eIF3c and the non-core subunits eIF3e and subunits in the translation of subsets of transcripts. eIF3h (Refs 55,56). This result is probably significant A loss of polysome-associated mRNAs and their because direct interactions between eIF3e, eIF3c and conversion into the monosome fraction is seen in the CSN were detected in vitro, in yeast and in vivo, fission yeast mutants defective for non-core eIF3 whereas no such interaction was detected for another subunits47. In addition, eIF3e and another non-core eIF3 core subunit, eIF3b, and the CSN. Arabidopsis subunit, eIF3d, were identified as contributors to a eIF3c interacts with CSN1 and CSN8, whereas eIF3e RAS signaling pathway in fission yeast60. These interacts with CSN7. Because eIF3e and eIF3c results set one precedent for cross-talk between a interact with each other57, it seems likely that eIF3c, specific signaling pathway and eIF3. One caveat eIF3e and presumably eIF3h form a module that can remains, in that no direct interaction has been interact with either the rest of eIF3 or the CSN. detected between an eIF3 subunit and the CSN in The biochemical association between subunits of fission yeast. However, although the cytoplasmic the CSN and eIF3 seems to be well established, but location of eIF3 subunits and the nuclear location of the question of the biological role of this interaction the CSN might argue against this, the subcellular then arises, especially considering the role of the localization of certain eIF3 subunits in S. pombe is CSN in repressing light responses in plants. dynamic. For example, nuclear mislocalization of Clearly, additional data are needed to address this eIF3d occurs in the absence of eIF3e and vice versa60, point. However, it is clear that the light regulation suggesting that the two have intrinsic nuclear of development occurs at both the transcriptional localization potential yet tether each other in the and the translational levels. For instance, the pea cytoplasm. A regulatory role for eIF3e is also ferredoxin-1 (Fed-1) mRNA rapidly and specifically emerging from two lines of evidence in mammalian dissociates from the polysomal (i.e. translated) cells. Even before its identification as an eIF3 ribosome fraction within 20 min of shifting plants subunit, eIF3e had been identified as a preferred from light to darkness, followed by mRNA chromosomal integration site of mouse mammary destabilization. This process is reversible58,59. Other tumor virus61. It was found in mammalian cells that light-regulated RNAs, such as those for tobacco overexpression of the interferon-inducible P56 Fed-1 and Cab, are affected similarly, but not all protein represses translation via eIF3e (Ref. 62). photosynthesis-related messages are. Polysome These results suggest a model in which eIF3e, once assembly is probably regulated at the level of inserted into the eIF3 holocomplex, functions as a translation initiation. From a physiological point of receiver of cellular signals that impede or otherwise view, translational control of early genes in modulate eIF3 activity. photomorphogenesis ensures the rapid Although a regulatory role for eIF3e is now accumulation of protein soon after illumination. It supported by solid data, how can we interpret the is tempting to speculate that one of the roles of the interaction between this and other eIF3 subunits, CSN in regulating photomorphogenesis is to alter and the CSN? Especially, how could such an eIF3 activity such that these transcripts remain interaction occur in light of the partners’ untranslated in the absence of light signals, presumably distinct cellular locations (cytoplasmic perhaps by inhibiting the assembly of non-core for eIF3 and nuclear for the CSN)? The subcellular subunits into eIF3. distribution of eIF3e in animal systems has been This model then predicts that the subcellular controversial. Some investigators have only location of these eIF3 regulatory subunits should be detected cytoplasmic localization46,47,63,64 but others dynamic, allowing them to interact with eIF3 in the showed nuclear targeting, which might be cytoplasm or, alternatively, with the CSN in the regulated by eIF3e’s partner proteins such as the nucleus. At least for eIF3e, this appears to be above-mentioned eIF3d, P56, HTLV-1 Tax or the plausible. In Arabidopsis, immunofluorescence and human Ret finger protein57,60,62,65. Aside from eIF3a, green-fluorescent-protein fusion experiments eIF3e is the only plant eIF3 subunit with a indicated that eIF3e might associate with the predicted nuclear localization signal39. In addition, cytoplasmic eIF3 and the nuclear CSN (Ref. 56) in a mammalian eIF3e has one or more nuclear export cell-type-specific fashion, whereas the eIF3 core signals62 and the nuclear-export-signal residues are subunit eIF3b was, as expected, consistently conserved in the plant orthologs (T-H. Kim et al., excluded from the nucleus. Because eIF3e was unpublished). The presence of eIF3e in the

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nucleus appears well established but its role within these processes, it is logical that the coordination of the nucleus, if any, remains to be confirmed. It these processes is dependent on sophisticated remains to be determined whether the interaction cross-talk. Perhaps this cross-talk is mediated allows translational regulation by the CSN or through the PCI complexes and their interactions whether the activity of the CSN is modified by the with central kinase pathways. However, this eIF3 subunits. regulation does not end with these three complexes. What emerges from this diverse set of data is a In Arabidopsis, as in other organisms, there are a Acknowledgements Our research on the complex and often confusing picture. However, if few ‘orphan’ PCI- and MPN-containing proteins that interactions between the one considers that homeostasis and development have yet to be characterized (Table 1). It will be CSN and eIF3 was require the coordinated efforts of diverse cellular interesting to determine whether these proteins supported by a grant from processes, especially differential transcription and form a fourth complex, can replace subunits of the the USA–Israel Binational Science Foundations translation, and that regulated proteolysis now known complexes or interact with the known (96-00258). appears to be a central mechanism in regulating PCI complexes.

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