Conservation of the COP9/signalosome in budding yeast
© Wee et al. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. 2002
Received: 22 July 2002
Accepted: 20 August 2002
Published: 20 August 2002
The COP9/signalosome (CSN), a multiprotein complex consisting of eight subunits, is implicated in a wide variety of regulatory processes including cell cycle control, signal transduction, transcriptional activation, and plant photomorphogenesis. Some of these functions have been linked to CSN-associated enzymes, including kinases and an activity that removes the ubiquitin-like protein NEDD8/Rub1p from the cullin subunit of E3 ligases. CSN is highly conserved across species from fission yeast to humans, but sequence comparison has failed to identify the complex in budding yeast, except for a putative CSN5 subunit called Rri1p.
We show that disruption of four budding yeast genes, PCI8 and three previously uncharacterized ORFs, which encode proteins interacting with Rrr1p/Csn5p, each results in the accumulation of the cullin Cdc53p exclusively in the Rub1p-modified state. This phenotype, which resembles that of fission yeast csn mutants, is due to a biochemical defect in deneddylation that is complemented by wild-type cell lysate and by purified human CSN in vitro. Although three of the four genes encode proteins with PCI domains conserved in metazoan CSN proteins, their disruption does not confer the DNA damage sensitivity described in some fission yeast csn mutants.
Our studies present unexpected evidence for the conservation of a functional homologue of the metazoan CSN, which mediates control of cullin neddylation in budding yeast.
The COP9/signalosome (CSN) was first identified in Arabidopsis thaliana as an eight subunit complex involved in the suppression of light-dependent development . Subsequent studies have led to the identification of similar complexes in other plant species, Drosophila melanogaster, human cells, and fission yeast [2–7], thus indicating a high degree of structural conservation during evolution. Cloning of CSN subunits revealed their structural similarities to the eight subunits of the lid complex of the 26S proteasome [3, 8, 9, 10]. The similarity was most pronounced within the so-called MPN domains of CSN5 and 6 and the PCI domains of the remaining subunits .
CSN has been implicated in multiple biological processes, many involving ubiquitin-mediated proteolysis (reviewed in [12, 13]). For example, CSN is required for degradation of the plant transcription factor HY5 by the putative COP1 ubiquitin ligase . In addition, CSN is involved in auxin-induced turn-over of the transcriptional repressor AUX/IAA . This process is mediated by an ubiquitin ligase  related to SCF complexes first identified in budding yeast [16, 17]. All SCF complexes share the core subunits Cdc53p/cullin 1, SKP1, and the RING domain protein HRT1/RBX1/ROC1, which associate with different F-box proteins that mediate substrate specificity (reviewed in ). The discovery that CSN can interact with multiple cullins [7, 19] indicates that it may be a global regulator of cullin-based ubiquitin ligases.
Consistent with this possibility, CSN promotes removal of the ubiquitin-related protein NEDD8 from cullins , a modification that stimulates cullin ubiquitin ligase activity in vitro[20–23] and is required for cullin function in vivo. Fission yeast cells lacking CSN subunits accumulate the cullins Pcu1p and Pcu3p exclusively in the neddylated state [6, 7, 19]. Similarly, in human cell lysate, CSN is required to retain CUL1 in the deneddylated state . Remarkably, CSN purified from pig spleen can deneddylate Pcu1p in vitro. Recent studies strongly suggest that the deneddylation activity of CSN is intrinsic to a protease motif in subunit 5 .
Despite the remarkable degree of evolutionary conservation of CSN, sequence comparisons have failed to identify the complex in budding yeast with the exception of a putative CSN5 homologue named Rri1p . Similar to fission yeast csn5, disruption of RRI1/CSN5 in budding yeast leads to accumulation of the cullin Cdc53p in the Rub1p/Ned8p-modified state . Since mammalian CSN5 was implicated in numerous signaling pathways apparently independent of the CSN holocomplex (reviewed in ), it remained unclear whether budding yeast Rri1p/Csn5p executes its deneddylation function as a monomeric protein or whether it requires additional factors effectively constituting a homologue of the CSN holocomplex identified in higher eukaryotes.
In a recent large-scale survey of budding yeast protein complexes , five proteins co-purifying in a stable complex with Rri1p/Csn5p were identified by tandem mass spectrometry. However, the biological significance of these interactions remained unknown. Here we show that this protein complex has structural and functional similarities to metazoan CSN.
Results and discussion
Disruption of genes encoding Rri1p/Csn5p-associated proteins causes accumulation of Rub1p-modified Cdc53p
Budding yeast csn mutants have a biochemical defect in the removal of Rub1p
To determine whether budding yeast csn mutants exhibited a biochemical defect in the removal of Rub1p from Cdc53p similar to fission yeast csn mutants, we tested whether the readdition of cell lysate containing wild-type budding yeast CSN could rescue the Rub1p removal defect of csn deficient cell lysate. Total protein lysate from csn + and Δrri1/csn5 cells prepared in the absence of exogenous ATP was incubated at a ratio of 1:2 for up to 60 minutes at 30°C, and Cdc53p modification was assessed by immunoblotting. To exclude readdition of Rub1p to unmodified Cdc53p during the incubation, the csn + lysate was prepared from rub1 deletion strains as a source of functional CSN proteins. As previously described , Δrub1 cell lysate contained unmodified Cdc53p and no change in Cdc53p modification was observed upon incubation of this lysate for up to 60 minutes (Fig. 2A lanes 1–6). Likewise, incubation of Δrri1/csn5 lysate alone did not result in removal of Rub1p from Cdc53p over the 60 minute time course of the experiment (Fig. 2A, lanes 7–12).
When csn + Δrub1 and Δrri1/csn5 lysates were denatured in SDS sample buffer prior to mixing, the expected ratio of 1:2 was observed for Cdc53p/Cdc53p-Rub1p (Fig. 2A, lane 13, time point 0 minutes). In contrast, when native lysates were co-incubated at 30°C, the ratio reversed within 5 minutes, indicating complementation of the Δrri1/csn5 Rub1p removal defect by a functional CSN present in Δrub1 lysate (Fig. 2B, lanes). Identical complementation by Δrub1 lysate was obtained with several other Δcsn mutants (data not shown). These data show that, similar to fission yeast csn mutants, budding yeast Δcsn strains have a biochemical defect in the removal of Rub1p from Cdc53p, suggesting that the Rri1p/Csn5p, Csn9p, Rri2p/Csn10p, Pci8p/Csn11p, and Csi1p complex represents a budding yeast equivalent of the CSN complex described in fission yeast, plants, and human cells.
The Rub1p removal defect of csn mutants is complemented by purified CSN
To substantiate this conclusion, we tested whether human CSN could complement the Rub1p removal defect of csn mutant cell lysates. Human CSN highly purified from erythrocytes (Fig. 2C; ) was added to cell lysate prepared from all five csn mutants, and Rub1p modification of Cdc53p was assessed by immunoblotting. Addition of human CSN to csn deficient budding yeast cell lysate restored the ~1:1 ratio of modified to unmodified Cdc53p observed in wild-type cells, indicating efficient removal of Rub1p from Cdc53p in vitro (Fig. 2B). These results strengthen our conclusion that budding yeast csn mutants are deficient in a biochemical activity equivalent to human CSN.
While all five CSN proteins examined here are clearly involved in CSN-dependent cullin deneddylation, it is presently unclear whether they represent all subunits of the budding yeast CSN, in particular as all other known CSN complexes contain a minimum of eight subunits. It is equally unclear whether all five CSN proteins characterized here are part of the same complex. Although the budding yeast genome does not encode clearly discernible MPN or PCI domain proteins other than proteasome lid subunits and the CSN proteins characterized here (data not shown), it is possible that some of them dimerize to assemble a larger CSN complex. Alternatively, MPN and PCI domain containing proteasome lid subunits may substitute for authentic CSN subunits in budding yeast, thus giving rise to an eight-subunit complex. In support of this notion, Rpn5p/Nas5p, one of the non ATPase components of the 19S lid carrying a PCI domain that shares high homology with CSN4, was also found in the Rri1p/Csn5p complex isolated by tandem affinity purification . Similarly, Pci8p/Csn11p was previously shown to associate with eIF3 in budding yeast , suggesting a potentially complex pattern of CSN/eIF3/proteasome interactions. Further biochemical studies will be required to unravel this complexity and to reveal the exact stochiometry of the budding yeast CSN.
Budding yeast csn mutants are not sensitive to DNA damage
Our previous studies in fission yeast and the present studies in budding yeast showed that, apart from the S phase and DNA damage defects of fission yeast csn1 and csn2 mutants, the cellular phenotypes of yeast csn mutants are surprisingly unremarkable. In fact, no other gross growth or morphological phenotypes have been detected, despite the presence of maximally neddylated cullins in all yeast csn mutants examined (data not shown). Control of cullin deneddylation is therefore the only generally conserved biochemical function of CSN identified to date. In contrast, CSN has major developmental functions in multicellular eukaryotes, including D. melanogaster  and A. thaliana (reviewed in ), although it is presently unclear whether any of these are linked to CSN-mediated deneddylation. While no substrates of the Rub1p/Ned8p modification system other than cullins have been identified, fission yeast csn1 mutants expressing tagged Ned8p accumulate several other proteins reactive with anti-tag antibodies . No major cellular pathway of unicellular organisms seems to be disturbed by hyperneddylation of these substrates, but their existence extends the biological functions of CSN to a potentially wider array of cellular processes. The unexpected conservation of the budding yeast CSN revealed in this report will offer novel opportunities for deciphering these functions in a simple, genetically and biochemically tractable organism.
Our study provides evidence for a budding yeast CSN complex containing MPN and PCI domain proteins, which mediate cullin deneddylation, the primary conserved function of the metazoan CSN complex.
List of abbreviations used
SKP1/Cullin/F-box protein complex
We thank G. Cope, R. Deshaies, M. Glickman, and V. Maytal for sharing results prior to publication, and L. Samson for providing all budding yeast deletion strains used here. This study was funded by NIH grant GM59780 to D.A.W., by the NIEHS Center grant ES-00002, and by a grant from the German Israel Foundation to W. D.. S.W. is the recipient of a stipend from the NIH training grant ES-07155.
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