Role of the MPN Subunits in COP9 Signalosome Assembly and … · Role of the MPN Subunits in COP9 Signalosome Assembly and Activity, and Their Regulatory Interaction with Arabidopsis

Role of the MPN Subunits in COP9 Signalosome Assemblyand Activity, and Their Regulatory Interaction withArabidopsis Cullin3-Based E3 Ligases W

Giuliana Gusmaroli, Pablo Figueroa,1 Giovanna Serino,2 and Xing Wang Deng3

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8104

The COP9 signalosome (CSN) is an evolutionarily conserved multisubunit protein complex that regulates a variety of

biological processes. Among its eight subunits, CSN5 and CSN6 contain a characteristic MPN (for Mpr1p and Pad1p N-

terminal) domain and, in Arabidopsis thaliana, are each encoded by two genes: CSN5A, CSN5B and CSN6A, CSN6B,

respectively. We characterized both MPN subunits using a series of single and double mutants within each gene family. Our

results indicate that although CSN6A and CSN6B retain mostly redundant functions, CSN5A and CSN5B play unequal roles

in the regulation of plant development. Complete depletion of either of the two MPN members results in CSN instability and

the decay of various CSN components, along with the complete loss of CUL1, CUL3, and CUL4 derubylation. Furthermore,

we demonstrate that CSN interacts with CUL3, in addition to CUL1 and CUL4, and that the lack of CSN activity differentially

affects the stability of those three cullins. Interestingly, we also show that optimal CUL3 activity is required to maintain the

cellular pool of CSN5, through a posttranscriptional mechanism. Our data suggest the existence of reciprocal regulation

between CUL3 and CSN5 accumulation. This study thus completes the genetic analysis of all CSN subunits and confirms the

structural interdependence between PCI and MPN subunits in functional CSN complex formation.

INTRODUCTION

The COP9 signalosome (CSN) is a nucleus-enriched multisubunit

protein complex conserved throughout evolution (Wei et al.,

1994a, 1998; Chamovitz et al., 1996; Seeger et al., 1998; Freilich

et al., 1999; Mundt et al., 1999; Busch et al., 2003). Genetic and

molecular analyses indicate that the CSN is involved in the

regulation of a variety of signaling and developmental processes,

including embryogenesis, cell cycle, circadian rhythms, DNA

repair, and plant responses to light and hormones (reviewed in

Wei and Deng, 2003; Richardson and Zundel, 2005). The CSN

interacts with a large number of proteins, including several

components or regulators of the ubiquitin–proteasome system

(Smalle and Vierstra, 2004). Among the CSN’s eight subunits, six

contain a PCI domain (for Proteosome, COP9, Initiation factor 3)

and two (CSN5 and CSN6) contain a MPN/MOV34 domain (for

Mpr1p and Pad1p N-terminal) (Glickman et al., 1998; Hofmann

and Bucher, 1998; Wei et al., 1998).

A known biochemical activity of the CSN is the removal of the

ubiquitin-like protein RUB1/NEDD8 from the cullin subunit of the

Cullin-RING ligase (CRL) family of E3 complexes (Cope and

Deshaies, 2003; Wei and Deng, 2003; Harari-Steinberg and

Chamovitz, 2004). To date, it has been shown that the CSN

interacts with and promotes RUB1/NEDD8 deconjugation from

the Cullin1-containing SCF (for SKP1-CUL1/CDC53-F-box pro-

tein), Cullin3-containing BCR (for BTB/POZ domain-CUL3-

RING), and Cullin4-containing E3 ubiquitin ligases (Lyapina

et al., 2001; Schwechheimer et al., 2001; Liu et al., 2002, 2005;

Feng et al., 2003; Groisman et al., 2003; Higa et al., 2003; Pintard

et al., 2003; Wang et al., 2003). CSN also deconjugates RUB1/

NEDD8 from CUL2, a subunit of the VCB (for Von Hippel Lindau-

Elongin B-Elongin C) E3 complex (Yang et al., 2002). Like the

ubiquitin pathway, the RUB1/NEDD8 conjugation pathway is

catalyzed by an enzymatic cascade and is essential in Schizo-

saccharomyces pombe, Arabidopsis thaliana, Caenorhabditis

elegans, Drosophila, and mouse (Jones and Candido, 2000;

Osaka et al., 2000; Tateishi et al., 2001; Ou et al., 2002; Bostick

et al., 2004). In plants, rubylation is also important for several

physiological processes, including auxin and ethylene re-

sponses (del Pozo et al., 2002; Gray et al., 2002; Dharmasiri

et al., 2003; Bostick et al., 2004; Larsen and Cancel, 2004).

Conjugation of RUB1/NEDD8 to CUL1 increases the activity of

the SCF complexes in vitro and in vivo, probably by facilitating

substrate polyubiquitination and E2 recruitment to the E3 ligases

(Kawakami et al., 2001; Pan et al., 2004). In addition, it was dem-

onstrated recently that the increased availability of the F-box

subunit SKP2, and its substrate, promotes neddylation and the

assembly of SCFSKP2 complexes (Bornstein et al., 2006). At the

same time, CSN-mediated cullin derubylation is essential for in

vivo organized E3 functions (Wolf et al., 2003). In fact, CSN de-

rubylation and its associated UBP12-mediated deubiquitination

1 Current address: Delaware Biotechnology Institute, Department ofPlant and Soil Sciences, University of Delaware, Newark, DE 19711.2 Current address: Laboratorio per la Genomica Funzionale e Proteo-mica dei Sistemi Modello, Universita ‘‘La Sapienza,’’ 00185 Rome, Italy.3 To whom correspondence should be addressed. E-mail [email protected]; fax 203-432-5726.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Xing Wang Deng([email protected]).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.106.047571

The Plant Cell, Vol. 19: 564–581, February 2007, www.plantcell.org ª 2007 American Society of Plant Biologists

activities (Zhou et al., 2003) promote CRL E3 function by

counteracting the instability of the E3 components caused by

CRL autoubiquitination activity (He et al., 2005; Wee et al., 2005;

Cope and Deshaies, 2006; Wu et al., 2006). Together, these

findings demonstrate that cycles of rubylation/derubylation are

essential for maintaining an optimal pool of active Cullin-RING

ligases and explain the mechanism underlying substrate accu-

mulation in csn mutants (Lykke-Andersen et al., 2003; Cope and

Deshaies, 2006; Denti et al., 2006).

The CSN derubylation activity is located within the JAMM (for

JAB1/MPN/MOV34) or MPNþmotif of CSN5 (Cope et al., 2002),

which is embedded within its MPN domain. Interestingly, this

motif is absent in the other MPN domain subunit of CSN (CSN6),

whose function remains unknown.

The CSN was initially discovered during genetic screens for

constitutive photomorphogenic development in darkness, which

led to the identification of 10 COP/DET/FUS loci (reviewed in Wei

and Deng, 2003). Six of the nine molecularly characterized COP/

DET/FUS loci encode the six PCI subunits of the COP9 complex,

whereas the MPN/MOV34 subunits CSN5 and CSN6 are not

represented by any of those genes. In Arabidopsis, both MPN

subunits are encoded by two highly homologous genes: CSN5A

and CSN5B (Kwok et al., 1998) and CSN6A and CSN6B (Peng

et al., 2001a), respectively. In the case of CSN5, we have shown

previously that CSN5A and CSN5B incorporate into distinct CSN

(CSNCSN5A and CSNCSN5B) complexes in vivo (Gusmaroli et al.,

2004). The initial characterizations of mutant plants defective in

one (csn5a-1 and csn5a-2) or the other (csn5b-1) CSN5 subunit

have been reported (Gusmaroli et al., 2004; Dohmann et al.,

2005). Both studies showed that the two CSN5 isoforms play

unequal roles in plant development, with CSN5A exerting a more

prominent one.

In plants and mammals, loss of a PCI subunit normally triggers

the cellular depletion of the entire CSN (Kwok et al., 1998; Serino

et al., 1999; Peng et al., 2001a, 2001b; Wang et al., 2002; Lykke-

Andersen et al., 2003; Yan et al., 2003). In the case of the MPN

subunit CSN5, however, nonidentical effects have been ob-

served in different organisms. Whereas in mammals (Tomoda

et al., 2004; Denti et al., 2006) and fission yeast (Mundt et al.,

2002), CSN5 is necessary for CSN stability, in Drosophila csn5Null

mutants, CSN4 and CSN7 still form a large protein complex

similar to that of the wild type (Oron et al., 2002). In Arabidopsis,

the characterization of a double csn5 mutant line (csn5a-2

cns5b-1) led to the conclusion that loss of CSN5 results in the

formation of a stable CSN form lacking CSN5 only (Dohmann

et al., 2005). However, the csn5a-2 allele used in that study was a

partial loss-of-function allele, still producing reduced levels of a

functional CSN5A protein.

Here, we have undertaken a systematic characterization of

each member of the CSN5 and CSN6 gene families, using a

series of single and double null mutants within each gene family.

Together, our results reveal the specific role of each CSN5 and

CSN6 family member in CSN complex assembly and stability as

well as their contributions to the derubylation activity and CSN

functions in plant development. In addition, we demonstrate that

CSN interacts with CUL3-based E3 ubiquitin ligases both in vitro

and in vivo and that CSN derubylation activity is essential to

maintain the cellular pool of CUL3. Finally, we provide genetic

and biochemical evidence to show that CSN5 levels, in turn, can

be modulated by CUL3, revealing a reciprocal regulation be-

tween CSN5 and CUL3 accumulation.

RESULTS

Generation and Characterization of csn5a and csn5b Single

and Double Mutants Reveal That CSN5A and CSN5B Are

Differentially Expressed throughout Plant Development

To investigate whether the complete loss of CSN5 activity would

lead to the destabilization of the entire complex, we conducted a

detailed characterization of csn5a and csn5b single and double

mutants. We used two CSN5A T-DNA insertion lines and one

CSN5B T-DNA insertion line (see Methods for details) (Gusmaroli

et al., 2004; Dohmann et al., 2005). To determine whether the two

T-DNA insertions in CSN5A give rise to reduction or complete

loss of function, RT-PCR analyses were performed on RNA

extracted from each single mutant line (Figure 1). The positions of

the gene-specific primers used to selectively amplify the CSN5A

and CSN5B transcripts are shown in Figure 1A. The complete

absence of the CSN5A transcript in the csn5a-1 mutant (Figures

1B and 1C, third lanes) demonstrates that the T-DNA insertion in

the first exon of CSN5A gives rise to a null mutation. On the other

hand, we could amplify the CSN5A transcript in the csn5a-2

homozygous line using different sets of CSN5A gene-specific

primers (Figures 1B and 1C, second lanes). We detected wild-

type levels of CSN5A transcript using sets of primers located 59

of the T-DNA insertion (Figure 1B, second lane), whereas the

level of full-length CSN5A transcript was reduced considerably

but still detectable using different sets of primers spanning the

entire CSN5A coding region (Figure 1C, second lane). Together,

these results demonstrate that, in the csn5a-2 homozygous line,

the intron containing the T-DNA insertion can be correctly spliced

out, at low efficiency, producing full-length CSN5A transcript.

The removal of the T-DNA during pre-mRNA processing has

been observed previously in other T-DNA homozygous lines

when the T-DNA is inserted within an intron (our unpublished

data). In conclusion, the RT-PCR analyses demonstrated that

the csn5a-2 mutant still retains reduced levels of wild-type

CSN5A transcript and therefore should be considered a CSN5A

reduction-of-function line and not a null csn5a mutant. On the

other hand, the absence of CSN5B transcript in csn5b-1 seed-

lings demonstrated that the T-DNA insertion in CSN5B gives rise

to a null mutation (Figure 1D, first lane).

Protein gel blot analyses of the single csn5 mutants show that

CSN5A is expressed predominantly (Figure 1F, second lane),

whereas CSN5B is barely detectable (Figure 1F, fourth lane), in

Arabidopsis seedlings and throughout plant development (data

not shown). We did not observe a detectable dose effect of CSN5

function in heterozygous csn5a plants (csn5a-2/þ and csn5a-1/þ),

either at the biochemical (Figure 1F, last two lanes) or the

morphological (data not shown) level. The comparison of CSN5

level between csn5a-2 and csn5a-1 mutants (Figure 1F, third and

fourth lanes) indicates that csn5a-2 expresses reduced levels of

an intact CSN5A subunit (in addition to the CSN5B protein), as

anticipated by the presence of low levels of full-length CSN5A

transcript in the csn5a-2 genetic background.

Role of MPN Subunits in CSN Function 565

CSN5A and CSN5B Play Unequal Roles in Regulating

Photomorphogenesis, Root Elongation, Auxin Response,

and Vegetative and Reproductive Growth

During the initial days after germination (DAG) in white light, the

csn5a-1 and csn5a-2 seedlings exhibit a phenotype reminiscent

of the fusca-type phenotype of the cop/det/fus mutants, char-

acterized by purple cotyledons. However, both csn5a single

mutants are not lethal at the seedling stage, and at a few DAG the

purple cotyledons turn light green before the production of true

leaves (Figure 2A). The detailed characterization of the single

csn5 mutants at representative stages revealed that reduction or

loss of CSN5A results in shorter hypocotyls in the dark, shorter

roots, smaller flowers, a decreased number of root hairs in

response to jasmonic acid treatment, and altered light and auxin

responses (see Supplemental Figure 1 online).

At the vegetative stage, the csn5a plantlets are characterized

by small, light green, and curly rosettes (Figure 2B, 12 and 14

DAG), which are almost completely depleted of trichomes, in

contrast with wild-type and csn5b-1 plants (Figure 2C). At the

reproductive stage, csn5a-1 and csn5a-2 mutants exhibit severe

developmental defects that result in dwarf stature and the loss of

apical dominance (Figure 2B, 52 DAG).

It is worth noting that whereas csn5a-1 and csn5a-2 are

virtually indistinguishable from each other in the initial few DAG

(Figure 2A), starting from 8 DAG and throughout the vegetative

stage, the phenotype of csn5a-2 is less severe than that of

csn5a-1, in terms of both rosette size (Figure 2B) and trichome

density (Figure 2C). This difference becomes particularly evident

at the reproductive phase (Figure 2B, 52 DAG) and could be

explained by the residual activity of CSN5A in csn5a-2 mutants,

as shown below. By contrast, csn5b-1 mutants are virtually

indistinguishable from wild-type plants at all developmental

stages (Figures 2A and 2B).

Genetic and Biochemical Analyses of Two Different Double

Mutant Lines Indicate That csn5a-2 csn5b-1 Still Retains

Partial CSN5A Function

As a necessary step in the characterization of the contribution of

CSN5 to overall CSN function, we generated csn5a-1 csn5b-1

and csn5a-2 csn5b-1 double mutant lines. Because of low levels

of wild-type CSN5A transcript (Figure 1C, second lane) and

protein (Figure 1F, third lane) produced by the csn5a-2 allele,

we characterized both csn5a-1 csn5b-1 and csn5a-2 csn5b-1

double mutants. As shown in Figure 3A, during the first 2 to 3

Figure 1. Expression Analyses of csn5 Single Mutants.

(A) Structures of the Arabidopsis CSN5A (At1g22920) and CSN5B (At1g71230) loci and graphical representation of the T-DNA insertion mutants used in

this study. Arrows indicate the positions and orientations of the gene-specific primers used for the RT-PCR analyses.

(B) to (E) RT-PCR analyses of the wild type and csn5b-1, csn5a-2, and csn5a-1 homozygous mutants. Different sets of gene-specific primers were used

to selectively amplify the CSN5A 59 (B) and full-length (C) transcripts or the CSN5B transcript (D). The eEF-1A (E) (top panel) and Tsb1 (E) (bottom panel)

transcripts were used as positive controls for the RT-PCR analyses. Genomic DNA was used as a positive control for the PCR. Arrows indicate the PCR

products corresponding to mature CSN5A and CSN5B transcripts. The csn5a-2 mutants still express low levels of full-length CSN5A mRNA.

(F) Detection of CSN5 proteins from the wild type and null homozygous csn5b-1 and weak (csn5a-2) or null (csn5a-1) homozygous and heterozygous

csn5a mutants with anti (a)-CSN5 polyclonal antibodies, which recognize both CSN5A and CSN5B isoforms. Equal protein loading was confirmed using

a-RPN6 antibody. The csn5a-2 mutants express low levels of CSN5A protein in addition to traces of CSN5B.

566 The Plant Cell

DAG, both csn5a-1 csn5b-1 and csn5a-2 csn5b-1 double mutant

lines display the typical photomorphogenic phenotype of the

fusca mutants. At these time points, both lines are virtually

indistinguishable from each other. However, at 6 to 7 DAG, the

csn5a-2 csn5b-1 double mutants uniformly start to form the first

pair of true leaves (Figure 3A) and eventually develop into

impaired small plantlets, with dark red cotyledons and asym-

metrical leaves (Figure 3B). Similar defects also have been

described for other CSN reduction-of-function lines generated

by CSN6A antisense and cosuppression approaches (Peng

et al., 2001a). Interestingly, none of the csn5a-2 csn5b-1 double

mutants arrest at the seedling stage, and even though severely

compromised, csn5a-2 csn5b-1 plantlets survive to a mature

stage (Figure 3C). It is worth noting that the phenotype of the

csn5a-2 csn5b-1 double mutants shown in Figure 3 has been

homogeneously observed in 100% of the segregating csn5a-2

csn5b-1 F2 seedlings derived from several independent crosses

between the csn5a-2 and csn5b-1 homozygous parental lines.

On the other hand, csn5a-1 csn5b-1 double null homozygous

mutants invariably die at the seedling stage (Figure 3A), do not

express detectable CSN5 proteins (Figure 3D, third lane), and are

virtually identical to the null alleles of the cop/det/fus mutants.

The discrepancy between the severe pleiotropic phenotype of

csn5a-2 csn5b-1 mutants and the lethality of the csn5a-1 csn5b-1

seedlings can be explained by the residual CSN5A activity in the

csn5a-2 csn5b-1 background. In fact, these double mutants

express reduced levels of an intact CSN5A protein (Figure 3D,

second lane), which elutes almost exclusively in high molecular

mass fractions and cofractionates with other CSN subunits in

csn5a-2 csn5b-1 seedling extracts (Figure 3E). In agreement, we

observed a residual derubylation activity in csn5a-2 csn5b-1

seedlings, as shown below in Figure 7B.

Characterization of csn6a and csn6b Single and Double

Mutants Reveals That CSN6A and CSN6B Are Differentially

Expressed throughout Plant Development

Arabidopsis CSN6 is encoded by two conserved genes, CSN6A

and CSN6B (Peng et al., 2001a). The two genes encode proteins

that are 87% identical to each other and 47% identical to their

human counterpart. To evaluate their respective functional roles,

T-DNA insertion mutants in CSN6A and CSN6B loci were iden-

tified (Figures 4A to 4C) (see Methods). RT-PCR analyses con-

firmed that the T-DNA insertions in both CSN6 loci result in null

mutations (Figure 4D). We refer to the corresponding mutants as

csn6a-1 and csn6b-1. Protein gel blot analyses with polyclonal

antibody raised against a conserved CSN6A polypeptide sug-

gest that CSN6A is expressed predominantly (Figure 4E, third

lane), whereas CSN6B accumulates at a lower level (Figure 4E,

second lane) both in seedlings (Figure 4E) and in mature plants

(data not shown). These data are consistent with the expression

profiles of the two CSN6 transcripts obtained from Genevesti-

gator (http://www.genevestigator.ethz.ch), which collected hun-

dreds of microarray expression data sets. In fact, in all organs

Figure 2. Mutations in CSN5A but Not CSN5B Result in Multifaceted Developmental Defects at Vegetative and Reproductive Stages.

(A) and (B) Phenotypes of light-grown wild-type and csn5 single mutant plants at different DAG as indicated. At 14 DAG, the plantlet were transferred to

soil and grown in long-day conditions. At each time point, photographs were taken at the same magnification.

(C) Close-up images of wild type, csn5b-1, csn5a-2, and csn5a-1 new leaves at 14 DAG. For comparison, at each stage the photographs were taken at

the same magnification.


and conditions reported to date, CSN6A is expressed consis-

tently at a higher level than CSN6B.

CSN6A and CSN6B Subunits Are Functionally Redundant

and Essential for Plant Development

As shown in Figures 4G and 4H, both csn6 mutants display a mild

partial photomorphogenic phenotype in the dark and in blue

light, characterized by shorter hypocotyls with respect to wild-

type plants grown under the same conditions. In the dark, loss of

CSN6A seems to affect hypocotyl elongation slightly more than

loss of CSN6B; however, the absence of a characteristic fusca-

type phenotype in the single null lines clearly indicates that both

genes act in a largely redundant manner in the regulation of

photomorphogenesis. Despite a mild phenotype in the dark and

in blue light, csn6a-1 and csn6b-1 do not display any obvious

morphological defects in white light (Figure 4F) and after the

seedling stage (data not shown).

Figure 3. Comparison of the Developmental Defects of Two csn5 Double Mutants, Which Are Severely (csn5a-2 csn5b-1) or Completely (csn5a-1

csn5b-1) Defective in CSN5 Activity.

(A) Phenotypes of light-grown wild-type and csn5 double mutant seedlings at different developmental stages.

(B) Phenotype of csn5a-2 csn5b-1 homozygous mutants at 14 DAG.

(C) Phenotype of 9-week-old csn5a-2 csn5b-1 double mutants, grown in long-day conditions.

(D) First five panels, PCR-based genotype analyses of wild-type, csn5a-2 csn5b-1, and csn5a-1 csn5b-1 double mutant lines. The positions of the

CSN5A and CSN5B gene-specific primers are indicated in Figure 1A. Primers B1/B2 and B2/LBb1 were used to selectively amplify the CSN5B wild-

type and csn5b-1 T-DNA insertion alleles, respectively. Primers A2/A5 were used to selectively amplify the CSN5A wild-type allele. Primers A5/LBb1

were used to selectively amplify the csn5a-2 (lane 2) and csn5a-1 (lane 3) T-DNA insertion alleles. The genomic sequence of EF-1A was used as a

positive control for the PCR. Last two panels, protein blot analyses of wild-type and csn5 double mutant lines, as indicated. Equal amounts of total

protein extracts were subjected to SDS-PAGE and immunoblot analyses with anti (a)-CSN5 antibody. The a-RPN6 antibody was used as a loading

control.

(E) Immunoblot analyses of Superose 6 gel filtration fractions obtained from 13-d-old light-grown csn5a-2 csn5b-1 seedlings. Column fractions were

subjected to SDS-PAGE and immunoblot analyses with a-CSN3, a-CSN5, and a-CSN6 polyclonal antibodies. Fraction numbers are indicated. Lane T

contains the total unfractionated extracts. The csn5a-2 csn5b-1 double mutant possesses reduced levels of an intact COP9 complex containing a

functional CSN5A subunit.

568 The Plant Cell

Figure 4. The MPN Subunits CSN6A and CSN6B of CSN Are Essential and Functionally Redundant.

(A) Structures of Arabidopsis CSN6A (At5g56280) and CSN6B (At4g26430) loci and graphical representation of the corresponding T-DNA insertion lines

used in this study. Arrows represent the positions and orientations of the gene-specific primers and the left border T-DNA primer (LBb1) used for the

PCR-based genotype (6A.1, 6A.2, 6B.1, and 6B.2) and RT-PCR (6A.3, 6A.4, 6B.2, and 6B.3) analyses.

(B) and (C) PCR-based genotype analyses of Arabidopsis wild type and csn6a-1 (B) or wild type and csn6b-1 (C) heterozygous and homozygous

mutants. The genomic sequence of the EF-1A locus was used as a positive control for the PCR.

(D) RT-PCR analyses of the wild type and csn6a-1 and csn6b-1 mutants. Primers 6A.3/6A.4 and 6B.2/6B.3 were used to selectively amplify the CSN6A

(top left panel) or CSN6B (bottom left panel) mature transcripts, as indicated. Sets of primers specific for histone H1 (top right panel) and ACT2 (bottom

right panel) were used as positive controls for cDNA calibration.

(E) Detection of CSN6 proteins from the wild type and single and double null homozygous mutants by SDS-PAGE and immunoblot analyses with anti

(a)-CSN6 polyclonal antibodies, which recognize both CSN6A and CSN6B subunits. Equal protein loading was confirmed using a-RPN6 antibody.

(F) Phenotypes of 5-d-old wild-type, csn6b-1, csn6a-1, and csn6a-1 csn6b-1 seedlings grown in continuous white light.

(G) and (H) Hypocotyl elongation (G) and phenotypes (H) of wild-type and csn6 mutant seedlings grown for 5 d in darkness or different light qualities, as

indicated (see Methods). Values shown in (G) represent means of 25 seedlings 6 SD. The colored boxes shown in (H) correspond to the genotypes

indicated in (G).


We further investigated the role of CSN6 in plant development

by generating csn6a-1 csn6b-1 double null mutant plants. In the

dark and under all light conditions tested, loss of function for both

CSN6 proteins results in lethality at the seedling stage and in a

photomorphogenic phenotype indistinguishable from the fusca-

type phenotype of the null cop/det/fus mutants described pre-

viously for the other CSN subunits (Figures 4F to 4H).

Loss of Either CSN5 or CSN6 Results in a Comparable

Destabilization of Other CSN Components

In plants and mammals, it has been shown that several CSN

subunits are unstable in an unbound form (Wei and Deng, 2003).

Therefore, a dramatic reduction in their accumulation generally

reflects the absence of an intact CSN complex and has been

commonly used as a marker of CSN ablation. To define what

happens to the Arabidopsis CSN complex in the absence of one

of its MPN subunits, we compared the stability of all CSN

subunits in the single and double csn5 and csn6 mutants. We

also included in our analyses two known PCI subunit mutants,

csn7 (fus5-1) (Karniol et al., 1999) and csn8 (cop9-1) (Wei et al.,

1994a), for comparison.

As shown in Figure 5A (third and fourth lanes), in csn5a single

mutants, the cellular pools of CSN1, CSN3, CSN5, CSN6, CSN7,

and CSN8 are reduced drastically compared with those in wild-

type and csn5b-1 seedlings, whereas no significant changes are

observed for CSN2 and CSN4. On the other hand, loss of

function of CSN5B does not trigger any detectable changes in

the cellular pools of other CSN subunits, including CSN5 (Figure

5A, second lane). Together, the results presented in Figure 5A

suggest that when CSN5A or CSN5B is reduced or lost, their

corresponding complexes (CSNCSN5A or CSNCSN5B) become

unstable. Because CSNCSN5A is much more abundant, this will

affect more dramatically the cellular pools of other CSN subunits

than the loss of CSNCSN5B. This is different from what is observed

for csn6a-1 and csn6b-1 single mutants (Figure 5B). In fact, lack

of one or the other member of the CSN6 family does not affect the

stability of the other CSN subunits (Figure 5B, second and third

lanes).

Complete loss of function of CSN5, in the csn5a-1 csn5b-1

double null mutant (Figure 5A, last lane), results in the significant

reduction of CSN1, CSN6, CSN7, and CSN8 and in a very slight

reduction of CSN4. In the case of CSN3, the complete loss of

function of CSN5 results in the accumulation of a larger and

possibly modified CSN3 form, whereas the normal (smaller)

CSN3 form is present predominantly in wild-type and csn5b-1

extracts. The only subunit that does not appear to be affected is

CSN2. Effects comparable to that caused by CSN5 depletion on

CSN subunit stability have also been observed in the case of

csn6a-1 csn6b-1 double null mutants (Figure 5B, last lane). In

fact, complete loss of function of CSN6 (csn6a-1 csn6b-1) results

in the significant reduction of CSN1, CSN3, CSN4, CSN7, and

CSN8. The only subunits not affected are CSN2 and CSN5.

Analogous effects on the stability of other CSN components

have also been observed in the absence of the PCI subunits

CSN7 and CSN8. In fact, complete loss of function of CSN7

(Figure 5C, last lane) results in CSN1, CSN3, CSN4, CSN6, and

CSN8 instability, accompanied by a mild reduction of CSN5.

Likewise, complete loss of function of CSN8 (Figure 5D, last lane)

triggers the instability of CSN1, CSN3, CSN5, CSN6, and CSN7.

Once again, the only subunit that does not seem do be signif-

icantly affected is CSN2 and, in the case of the csn8 mutant, also

Figure 5. Loss of Function of CSN5, CSN6, CSN7, or CSN8 Triggers the Destabilization of Several Other CSN Subunits.

Protein blot analyses of CSN subunits in the wild type and csn5 single and double mutants (A), the wild type and csn6 single and double null mutants (B),

the wild type and the csn7 (fus5-1) mutant (C), or the wild type and the csn8 (cop9-1) mutant (D). Equal amounts of total protein extracts from 13-d-old

light-grown seedlings were subjected to SDS-PAGE and immunoblot analyses with anti (a)-CSN1, a-CSN2, a-CSN3, a-CSN4, a-CSN5, a-CSN6,

a-CSN7, and a-CSN8 polyclonal antibodies. The a-RPN6 antibody was used as a loading control. The lanes indicated by asterisks correspond to

complete loss-of-function mutants.

570 The Plant Cell

CSN4. Together, these results confirm that whereas most CSN

subunits are unstable in the absence of an intact CSN, CSN2 and

CSN4 cannot be reliably used as markers of CSN ablation,

because their stability is differentially affected in different csn null

mutants.

The MPN Subunits CSN5 and CSN6 Are Essential for the

Structural Integrity of the CSN Holocomplex

In wild-type Arabidopsis seedling extracts, CSN subunits cofrac-

tionate in high molecular mass fractions in gel filtration chroma-

tography (Figure 6A). The elution peak is centered between 450

and 550 kD and corresponds to the CSN complex. Besides the

CSN holocomplex, some CSN subunits can be found in smaller

molecular mass forms (ranging from 50 to 300 kD) in several

organisms, including mammals (Tomoda et al., 2002; Fukumoto

et al., 2005), Drosophila (Oron et al., 2002), fission yeast (Zhou

et al., 2001; Mundt et al., 2002), and Arabidopsis (Wei and Deng,

2003).

To investigate the fate of the CSN holocomplex in the complete

absence of both CSN5 subunits, we analyzed the gel filtration

profile of csn5a-1 csn5b-1 double null mutants. In agreement

with the severe reduction of several CSN subunits (Figure 5A, last

lane), longer exposure times were required to detect their

corresponding signals after gel filtration chromatography. Pro-

tein blot analyses with antibodies against each CSN subunit

Figure 6. Both MPN Subunits CSN5 and CSN6 Are Essential for Maintaining the Structural Integrity of the CSN Complex.

(A), (B), and (D) Immunoblot analyses of Superose 6 gel filtration chromatography fractions obtained from 13-d-old light-grown wild-type (A), CSN5

complete loss-of-function mutant (csn5a-1 csn5b-1) (B), or CSN6 complete loss-of-function mutant (csn6a-1 csn6b-1) (D) seedlings. Column fractions

were subjected to SDS-PAGE and immunoblot analyses with anti (a)-CSN1, a-CSN4, a-CSN5, and a-CSN6 (A) or a-CSN1, a-CSN2, a-CSN3, a-CSN4,

a-CSN5, a-CSN6, a-CSN7, and a-CSN8 ([B] and [D]) polyclonal antibodies. Fraction numbers are indicated. Lane T contains the total unfractionated

extracts. The asterisks indicate total unfractionated wild-type extract. Loss of function of CSN5 (B) or CSN6 (D) in csn5a-1 csn5b-1 or csn6a-1 csn6b-1

double null backgrounds results in the disassembly of CSN subunits into CSN-independent subcomplex forms.

(C) CSN4 associates with CSN1 and CSN3 in csn5a-1 csn5b-1 double null mutant seedlings. Total soluble protein extracts (input) from 13-d-old wild-

type and csn5a-1 csn5b-1 double mutant seedlings were incubated with CSN4 antibody coupled to protein A-agarose (see Methods). The

immunoprecipitates (IP) were then separated by SDS-PAGE and immunoblotted with antibodies against CSN1, CSN3, CSN4, CSN6, TBP, and histone

H3, as indicated.


confirmed the absence of a CSN5-free large CSN complex in

csn5a-1 csn5b-1 double null seedlings (Figure 6B). In fact, CSN2,

CSN3, CSN4, CSN6, and traces of CSN7 exclusively or mostly

fractionate in low molecular mass fractions, ranging from 250 to

60 kD. Only the residual CSN1 subunit (whose accumulation is

reduced dramatically), traces of CSN3, and the first peak of

CSN4 elute in high molecular mass fractions that do not contain

CSN2, CSN5, CSN6, CSN7, and CSN8, possibly as a result of an

association with unknown proteins or self-aggregation. An iden-

tical situation has been observed in the case of csn8 (cop9) null

mutants. In fact, in csn8 mutants, CSN1 and CSN4 (plus traces of

CSN3) still elute in high molecular mass fraction that do not

contain any other CSN subunits (our unpublished data). To test

whether in csn5a-1 csn5b-1 double mutants CSN1, CSN3, and

CSN4 can still associate in the formation of a partial CSN com-

plex, we performed coimmunoprecipitation analyses using total

protein extracts obtained from the csn5a-1 csn5b-1 seedlings.

As shown in Figure 6C, CSN4 can efficiently pull down CSN1 and

CSN3, but not the negative control proteins, from the csn5a-1

csn5b-1 extract, indicating that CSN1, CSN3, and CSN4 asso-

ciate with each other in the absence of the other CSN subunits.

In the case of csn6a-1 csn6b-1 double null mutants (Figure

6D), whereas only residual CSN1 elutes in high molecular mass

fractions, CSN2, CSN3, CSN4, and CSN5 fractionate exclusively

in low molecular mass fractions, ranging from 250 to 60 kD. In the

case of CSN7 and CSN8, we have been unable to detect their

corresponding signals, in agreement with a dramatic reduction in

their accumulation levels in this mutant (Figure 5B, last lane).

All CSN Complete Loss-of-Function Mutants Share a Similar

Impairment in Cullin Derubylation

The six PCI subunit mutants identified by genetic screens for

constitutive photomorphogenic phenotype are virtually indistin-

guishable from the csn5 and csn6 complete loss-of-function

double mutant lines described in this study (Figure 7A). On this

basis, we investigated whether their identical phenotype re-

flected an identical impairment in cullin derubylation, the major

known biochemical function of CSN. We were also interested in

establishing the functional contribution of each CSN5 and CSN6

family member to the overall CSN biochemical activity. To this

end, we compared the rubylated/derubylated ratio of CUL1,

CUL3, and CUL4 in the single and double csn mutants. As shown

in Figure 7B, in the case of the CSN5 gene family (left panel),

CSN5A and CSN5B differentially contribute to the derubylation

activity observed in wild-type plants. In fact, whereas loss of

Figure 7. The Essential Role of the CSN in Cullin Derubylation as Well as the Maintenance of the Cellular Pools of CUL3 and CUL4.

(A) Phenotypes of 13-d-old white light–grown wild-type (left) and the set of eight csn complete loss-of-function mutant (right) seedlings, corresponding

to each CSN subunit. The six PCI subunit mutants shown correspond to cop/det/fus mutants identified by genetic screens (see Methods), as indicated

below the asterisks.

(B) Protein blot analyses of the wild type and csn5 single and double mutants (left panel), the wild type and csn6 single and double mutants (middle

panel), or the wild type and the six PCI subunit mutants shown in (A) (right panel), as indicated. Equal amounts of total proteins extracted from 13-d-old

white light–grown seedlings were subjected to SDS-PAGE and immunoblot analyses with anti (a)-CUL1, a-CUL4, and a-CUL3 polyclonal antibodies.

a-CUL3 antibody recognizes both CUL3A and CUL3B isoforms. The a-RPN6 antibody was used as a loading control. Top arrows indicate rubylated

CUL1, CUL4, and CUL3, respectively, and bottom arrows indicate the un-rubylated forms. The asterisks indicate the complete loss-of-function

mutants.

572 The Plant Cell

CSN5B has no effects (Figure 7B, left panel, second lane),

reduction or loss of CSN5A results in significantly increased

levels of rubylated CUL1, CUL3, and CUL4 in seedlings (Figure

7B, left panel, third and fourth lanes). In the case of the CSN6

gene family, however, loss of either CSN6A or CSN6B does not

affect the derubylation of cullins (Figure 7B, middle panel, sec-

ond and third lanes).

As expected, in csn5a-2 csn5b-1 seedlings, the residual activ-

ity of CSNCSN5A results in compromised, but not abolished, CUL1,

CUL3, and CUL4 derubylation (Figure 7B, left panel, fifth lane).

Thiscorrelateswith theseverebutnot lethal phenotypeof csn5a-2

csn5b-1 double mutants, which can survive to the adult stage.

The total depletion of CSN5 (csn5a-1 csn5b-1) (Figure 7B, left

panel, last lane) or CSN6 (csn6a-1 csn6b-1) (Figure 7B, middle

panel, last lane), as well as each one of the six PCI subunits of the

CSN (Figure 7B, right panel), results in complete loss of deruby-

lation activity and the accumulation of CUL1, CUL3, and CUL4

exclusively in the rubylated forms. These data confirm that CSN

possesses the only activity able to deconjugate RUB/NEDD8

from cullins in Arabidopsis seedlings.

The Cellular Levels of CUL1, CUL3, and CUL4 Are

Differentially Affected by CSN Depletion

It is interesting that in seedling extracts, the total cellular level of

CUL1 remains almost unchanged and loss of a PCI or an MPN

subunit only induces a redistribution of CUL1 from the unmod-

ified to the modified form (Figure 7B). On the contrary, in the case

of CUL3, the lack of CSN activity results in a drastic decrease in

the total cellular pool of this cullin. In fact, in csn complete loss-

of-function mutants (Figure 7B, lanes indicated by asterisks),

whereas unmodified CUL3 is completely absent, RUB-CUL3 is

barely detectable. Interestingly, we observed an opposite effect

of CSN on the stability of CUL4. In fact, the absence of CSN

activity triggers a significant increase in the total cellular level of

CUL4 (Figure 7B, lanes indicated by asterisks) compared with

the level of CUL4 in the wild type.

Arabidopsis CUL3-Based E3 Ubiquitin Ligases Associate

with CSN and the Proteasome in Vivo

In Arabidopsis, it has been shown that CSN associates with and

regulates the activity of several SCF complexes (Schwechheimer

et al., 2001; Wang et al., 2003; Feng et al., 2003). Similarly,

Arabidopsis CSN interacts with a CUL4-RBX1-CDD E3 ubiquitin

ligase in vivo (Chen et al., 2006). Recently, it was shown that

Arabidopsis CUL3 associates directly with RBX1 and BTB pro-

teins to form multisubunit E3 ubiquitin ligases (Figueroa et al.,

2005; Gingerich et al., 2005; Weber et al., 2005). However, an in

vivo biochemical interaction between CSN and CUL3-RBX1–

based E3 complexes has not been reported. To test whether

CSN interacts physically with CUL3-based E3 ubiquitin ligases,

we first used a yeast two-hybrid assay (Figure 8A). Positive

interactions in the plate assay (Figure 8A) were tested again with

Figure 8. The in Vivo Association of CSN, CUL3-Based E3 Ligases, and the 26 Proteasome.

(A) In vitro interaction between Arabidopsis CUL3A, RBX1, and CSN subunits by yeast two-hybrid assay. Blue colonies indicate LacZ production and

thus protein interaction.

(B) Colonies that tested positive for interactions in (A) were subjected to liquid quantitative assays to verify the strength of the binding. Numbers shown

on the y axis are means of two experiments. For each experiment, the numbers on the y axis were calculated as mean values of at least six independent

transformants. Error bars represent SD.

(C) CSN specifically associates with the CUL3A-RBX1 E3 module in vivo. Total soluble protein extracts (input) from wild-type and FLAG-CSN1

transgenic seedlings were incubated with monoclonal anti-FLAG–conjugated agarose (a-FLAG; see Methods), and the immunoprecipitates (IP) were

separated by SDS-PAGE and immunoblotted with antibodies against CSN2, CSN3, CSN5, CSN6, CUL3, and RBX1. The control represents a protein

band recognized by a-CSN2 antibody.

(D) CUL3 specifically associates with CSN and the proteasome in vivo. Total soluble protein extracts (input) from wild-type seedlings were incubated

with CUL3 preimmune serum (preI) or with CUL3 and CUL1 antibodies coupled to protein A-agarose (see Methods), as indicated. The immunopre-

cipitates (IP) were then separated by SDS-PAGE and immunoblotted with antibodies against CUL3, RBX1, CSN4, CSN5, RPT5, RPN6, and TBP.


a liquid b-galactosidase activity assay (Figure 8B). As shown in

Figures 8A and 8B, CUL3A interacts with CSN2 and CSN4. In

addition, CUL3 binds to RBX1 (the conserved catalytic RING

subunit common to all classes of cullin-based E3s) but not to

CUL1 or COP1 (components of other classes of E3s ligases).

These results are consistent with the previously reported in vitro

interaction between CSN2 and CUL3A (Serino et al., 2003).

The specific in vivo association between CSN and BTB-CUL3-

RBX1 (BCR) E3 ubiquitin ligases was then investigated by

immunoprecipitation in a stable transgenic line expressing a

FLAG-tagged version of CSN1 (FLAG-CSN1) (Wang et al., 2002).

As shown in Figure 8C, FLAG-CSN1 can efficiently pull down the

CSN complex and consistently coimmunoprecipitate CUL3 and

RBX1. We further immunoprecipitated CUL3 and, as a positive

control, CUL1 from wild-type seedling protein extracts. As

predicted, CUL3, like CUL1, was capable of interacting specif-

ically with RBX1 and coimmunoprecipitating several CSN sub-

units, clearly demonstrating that CSN and BCR E3 ubiquitin

ligases can associate with each other in vivo.

It has been suggested that the E3 ligases could make physical

contact with the proteasome as part of their function in the

protein degradation process (Peng et al., 2003). Based on this

consideration, we tested whether CUL3 was able to coimmuno-

precipitate representative subunits of the 26S proteasome in

vivo. As shown in Figure 8D, CUL3, like CUL1, could efficiently

pull down a lid subunit (RPN6) and a base subunit (RPT5) of the

19S regulatory particles, indicating a strong physical association

between CUL1- and CUL3-based E3 ligase complexes, CSN,

and the proteasome.

Mutations in CUL3A or CUL3B Suppress the Pleiotropic

Phenotype of csn5a Mutants

Arabidopsis has two largely redundant CUL3 genes (CUL3A and

CUL3B), which together are essential for embryo development

(Figueroa et al., 2005; Gingerich et al., 2005; Tohmann et al.,

2005). However, when grown under normal conditions, the single

cul3a and cul3b mutants are virtually indistinguishable from their

wild-type siblings throughout their entire life cycles (Figueroa

et al., 2005). To gain new insight into possible developmental

pathways regulated by CUL3, we crossed the single cul3a and

cul3b mutants with the csn5a mutant, which exhibits impaired

CUL3 derubylation. Considering possible further analyses, we

used the weak csn5a-2 mutant, which is not lethal when com-

bined with csn5b. We generated two double homozygous mu-

tant lines: csn5a-2 cul3a and csn5a-2 cul3b. As a control, we

crossed the csn5b-1 mutant with cul3a and cul3b, generating

two double homozygous mutant lines, csn5b-1 cul3a and csn5b-1

cul3b. For each cross, to eliminate or reduce possible effects

attributable to the variability between different parental lines,

which might differ significantly in terms of allelic composition at

any given locus, we reisolated all relevant single and double

homozygous lines (including the wild type) from the same seg-

regating F2 population for comparative analysis.

Surprisingly, loss of either CUL3A or CUL3B results in the

suppression of the pleiotropic phenotype of csn5a-2 mutants

(Figures 9A to 9C). In fact, whereas the csn5a-2 mutant displayed

the characteristic phenotype shown in Figure 2, the double

csn5a-2 cul3a and csn5a-2 cul3b mutants are virtually indistin-

guishable from wild-type siblings in the dark and in all light

conditions, both at seedling (Figures 9A and 9B) and mature

(Figure 9C) stages. On the other hand, we could not detect any

genetic interaction between CSN5B and CUL3A or CUL3B. In

fact, the single csn5b-1, cul3a, and cul3b mutants and the double

csn5b-1 cul3a and csn5b-1 cul3b mutants did not show any

observable defects throughout their life cycle (Figure 9D; data

not shown).

Loss of Either CUL3A or CUL3B Function Results in the

Accumulation of Higher Levels of CSN5 Proteins

As shown in Figure 9D, protein gel blot analyses of double csn5b-1

cul3a and csn5b-1 cul3b mutants indicate that loss of CUL3A or

CUL3B does not affect CSN5A expression. On the contrary, in

the csn5a-2 cul3a and csn5a-2 cul3b backgrounds, loss of

function of CUL3A or CUL3B results in a significant increase in

the total amount of CSN5 with respect to the CSN5 level

observed in the csn5a-2 single mutant (Figure 10A, lanes indi-

cated by asterisks). Using an image-analyses software (http://

rsb.info.nih.gov/ij/), we estimated that loss of cul3a or cul3b

results in an increase of approximately fivefold to sixfold in the

cellular pool of CSN5.

Protein gel blot analyses of seedling extracts with antibody

against CUL3 confirmed that the loss of one (cul3a) or the other

(cul3b) functional CUL3 loci triggers a measurable reduction in

the cellular pool of CUL3, confirming that both genes contribute

to the maintenance of optimal CUL3 levels, even though muta-

tion in CUL3A seems to affect the pool slightly more at this

examined developmental stage (Figure 10A, middle panel, last

two lanes).

Not surprisingly, the increase in the cellular pool of CSN5 in the

csn5a-2 cul3a and csn5a-2 cul3b double mutants resulted in

improved derubylation of all three cullins (Figure 10A, middle

panel). In fact, the ratios of rubylated/un-rubylated CUL1, CUL3,

and CUL4 are lower in the csn5a-2 cul3a and csn5a-2 cul3b

mutants with respect to csn5a-2 (Figure 10A, middle panel, lanes

indicated by asterisks), particularly in the case of CUL1 and

CUL4. Thus, the increased accumulation of CSN5 is responsible

for ameliorating the csn5a-2 deficiency in cullin derubylation,

suppressing the csn5a-2 developmental defects at all stages.

Loss of CUL3A or CUL3B Does Not Significantly Affect

CSN5A and CSN5B Expression at the Transcriptional Level

To distinguish whether transcriptional or posttranscriptional

mechanisms were responsible for the observed changes in

CSN5 abundance, we performed semiquantitative RT-PCR anal-

yses on RNA extracted from wild-type and single and double

csn5 and cul3 lines (Figures 10B and 10C). In the case of CSN5A

transcript, csn5a-1 null mutants were used as negative controls

for the RT-PCR analysis (Figure 10B, fourth lane). We first used

a set of CSN5A-specific primers spanning the region 59 to the

T-DNA insertion (Figure 10B, top panel). We could not detect any

major change in the total level of CSN5A transcript between

csn5a-2 and csn5a-2 cul3a or csn5a-2 cul3b (lanes indicated by

asterisks). Subsequently, we used a set of primers spanning the

574 The Plant Cell

entire CSN5A coding sequence (Figure 10B, bottom panel). As

shown in Figure 1, the transcript of the csn5a-2 allele (which is

transcribed at wild-type levels) is correctly spliced into a full-

length CSN5A mRNA at low efficiency. Therefore, in the csn5a-2

background, the full-length CSN5A transcript is detectable only

after 35 cycles of PCR amplification (Figure 10B, bottom panel,

third lane). Once again, however, we observed no significant

difference in CSN5A expression levels between the csn5a-2

mutant and the csn5a-2 cul3a or csn5a-2 cul3b double mutant

lines (Figure 10B, bottom panel, lanes indicated by asterisks). As

shown in Figure 10C, similar results were obtained in the case of

the CSN5B transcript (lanes indicated by asterisks). A semi-

quantitative RT-PCR analysis (at 28 cycles or less) confirmed the

absence of significant changes in CSN5B expression and a very

minor change (;50%) in CSN5A transcript between the single

csn5a-2 and the double csn5a-2 cul3a or csn5a-2cul3b mutants.

Even though we cannot completely exclude the possibility that

subtle changes (below our detectable threshold) in CSN5 mRNA

expression might, at least in part, contribute to CSN5 accumu-

lation, the results shown in Figure 10 suggest that CUL3 affects

CSN5 abundance most likely at the posttranscriptional level.

DISCUSSION

CSN5 and CSN6 Subunits Are Essential for Plant Viability

and Development but with Distinct Functional

Diversification between Their Isoforms

In Arabidopsis, the two CSN MPN subunits, CSN5 and CSN6,

have escaped the genetic screenings that led to the identification

of the six PCI subunits. Here, we have undertaken a reverse

genetic approach to systematically identify and characterize

single and double null mutants in each of those two-gene

families. Our results demonstrate that, despite some degree of

divergence in their coding sequences, CSN6A and CSN6B

proteins are functionally equivalent in the regulation of plant

development at all stages (Figure 4). In fact, both in white light

and long-day conditions (16 h of light/8 h of dark), the two CSN6

subunits are completely redundant and the expression of only

one of the two genes is sufficient for full function. The CSN5 gene

family is somewhat different. In fact, CSN5A and CSN5B have

largely diverged in their ability to regulate plant development. In

agreement with their different expression levels (Figure 1F), we

Figure 9. CUL3A or CUL3B Loss of Function Results in the Suppression of the Pleiotropic Developmental Defects of the csn5a-2 Mutant.

(A) and (B) Genetic interaction between CSN5 and CUL3. Phenotypes (A) and hypocotyl elongation (B) of 5-d-old wild-type, csn5a-2, cul3a, and cul3b

single and double mutant seedlings grown in darkness or in different light qualities, as indicated (see Methods). Values shown represent means of 25

seedlings 6 SD. The colored boxes shown in (A) correspond to the genotypes indicated in (B).

(C) Loss of CUL3A or CUL3B suppresses csn5a developmental defects at all stages. Phenotypes of 8-week-old wild-type, csn5a-2, and cul3a single

and double mutant plants (left) or wild-type, csn5a-2, and cul3b single and double mutant plants (right), as indicated, grown in long-day conditions.

(D) Top panels, phenotypes of 4-d-old wild-type, csn5b-1, and cul3a single and double mutant plants (left) or wild-type, csn5b-1, and cul3b single and

double mutant plants (right) grown for 4 d in white light. Bottom panels, detection of CSN5 proteins from wild-type, csn5b-1, cul3a, and cul3b single and

double mutant plants, as indicated. Total protein extracts from 6-d-old white light–grown seedlings were subjected to SDS-PAGE and immunoblot

analyses with anti (a)-CSN5 polyclonal antibodies. Equal protein loading was confirmed using a-RPN6 antibody.


have shown that lack of CSN5B does not cause any obvious

defects in all conditions tested, whereas loss of CSN5A triggers

pleiotropic defects in multiple developmental processes at all

stages (Figure 2; see Supplemental Figure 1 online). Remarkably,

the pleiotropic phenotypes of csn5a-1 and csn5a-2 are reminis-

cent of the severe developmental defects caused by the overex-

pression of catalytically inactive CSN5A derivatives (Gusmaroli

et al., 2004).

Our early and current studies suggest that many aspects of the

csn5a mutant phenotypes are caused by a partial (csn5a-2) or

complete (csn5a-1) loss of CSN5A-mediated cullin derubylation,

likely resulting in a certain level of impairment in the ubiquitination

activity of multiple E3 ubiquitin ligases, such as SCFTIR1 (Gray

et al., 1999), SCFCOI1 (Xu et al., 2002), or CUL4-RBX1-E3 com-

plexes (Bernhardt et al., 2006; Chen et al., 2006), all of which

interact with CSN in the regulation of hormone- or light-mediated

responses (Schwechheimer et al., 2001; Feng et al., 2003; Chen

et al., 2006).

Nevertheless, the multifaceted developmental defects of

csn5a mutants are less severe than the fusca-type phenotype

of csn5a-1 csn5b-1 double null mutants (Figure 3). Indeed, the

residual derubylation activity observed in csn5a null mutants

(Figure 7B, left panel, fourth lane) reveals a minor role for low-

abundance CSN5B (and its CSNCSN5B complex) in cullin deru-

bylation. This finding implies that CSN5A is the predominantly

functional subunit, whereas CSN5B contributes to CSN5 func-

tion to a lesser extent, suggesting that the two CSN5 genes might

have experienced a dissimilar reduction of functions with respect

to the ancestor gene. Finally, we showed that the complete loss

of function of CSN5 or CSN6 results in severe abnormalities,

which lead to postembryonic lethality at the seedling stage

(csn5a-1 csn5b-1 [Figure 3A] and csn6a-1 csn6b-1 [Figure 4F]).

These findings demonstrate that, despite the fact that only CSN5

is catalytically active in cullin derubylation, both MPN subunits

are essential for plant viability and development.

Arabidopsis MPN Subunits Are Essential for CSN Assembly

or Stability

It was reported recently that, in Arabidopsis, a lethal csn5 double

mutant line (csn5a-2 csn5b-1) retains a CSN complex lacking

only its subunit 5 (Dohmann et al., 2005). However, we showed

here that the csn5a-2 allele used in that study is not a null allele

(Figures 1B and 1C, second lanes, and 1F, third lane). In fact, the

csn5a-2 csn5b-1 double mutants are not lethal at the seedling

stage (Figures 3A to 3C) and possess reduced levels of an intact

CSN complex containing a functional CSN5A subunit (Figure 3E).

To unambiguously determine the contributions of CSN5 and

CSN6 to the structure and function of the CSN complex, we

generated csn5 and csn6 double null mutant lines. We demon-

strated that the complete depletion of CSN5 or CSN6 results in

the loss of the CSN holocomplex and the redistribution of some

CSN subunits into subcomplex forms (Figures 6B and 6D),

accompanied by the instability of several subunits, including

CSN1, CSN3, CSN6, CSN7, and CSN8 (Figures 5A and 5B, last

lanes). Therefore, in Arabidopsis, the CSN complex is unstable in

the absence of one of its MPN subunits, even though residual

CSN1, CSN3, and CSN4 still associate with each other in a

complex (Figures 6B and 6C). This association of CSN1, CSN3,

and CSN4 in the csn5 double null mutants likely has an altered

stoichiometry or includes other proteins.

Together, our data are in agreement with previous findings that

CSN1 (FUS6), CSN6, and CSN8 (COP9) are unstable as free

forms and that their accumulation depends upon the formation of

Figure 10. Loss of CUL3A or CUL3B Activity Results in a Significant Increase in the Cellular Pool of CSN5.

(A) Protein blot analyses of the wild type and csn5 and cul3 single and double mutants, as indicated. Equal amounts of total proteins from 6-d-old white

light–grown seedlings were subjected to immunoblot analyses with anti (a)-CSN5, a-CUL1, a-CUL4, and a-CUL3 polyclonal antibodies. The a-RPN6

antibody was used as a loading control. Loss of either CUL3A or CUL3B results in CSN5 accumulation.

(B) and (C) Semiquantitative RT-PCR analyses of the wild type and csn5 and cul3 single and double mutants, as indicated. The positions of the gene-

specific primers used to selectively amplify the CSN5A (B) or CSN5B (C) transcripts are shown in Figure 1. Primers A1/A2 selectively amplify the full-

length CSN5A cDNA. A set of primers specific for ACT2 was used for sample calibration. Genomic DNA obtained from wild-type seedlings was used as

a positive control for PCR. The asterisks indicate the relevant genotypes for comparison.

576 The Plant Cell

an intact complex (Staub et al., 1996; Peng et al., 2001a, 2001b;

Wang et al., 2002). Similarly, it has been demonstrated that a re-

duction of CSN5 in CSN5 antisense/cosuppression plants leads

to a reduction in the level of the entire CSN (Schwechheimer

et al., 2001). Null csn1 (Wang et al., 2002), csn5, and csn6 mu-

tants display slightly different gel filtration patterns for CSN4 and

CSN7, which suggests that, although all three subunits are

required for the structural integrity of the CSN holocomplex, their

specific roles in complex assembly are not identical.

Because the gel filtration profiles of several CSN subcom-

plexes do not overlap completely (Figures 6B and 6D), it is

possible that other proteins associate with some of the CSN

subunits. In fact, the existence of small complexes containing

only one subunit of CSN, in association with other unknown

proteins, has been described for mammalian CSN5, CSN7, and

CSN8 (Fukumoto et al., 2005). In addition, several reports dem-

onstrated that specific CSN subunits, particularly CSN5, could

bind to interacting proteins in a CSN-independent manner (Wei

and Deng, 2003).

The Complete Collection of csn Mutants Highlights That

CSN Regulates All Major Classes of Known Cullin-Based

E3 Uqibuitin Ligases

With this study, we concluded the collection and analysis of

complete loss-of-function mutants in each one of the eight CSN

subunits and established the specific contribution of each CSN5

and CSN6 family member to the structure and function of the

complex as a whole. Our biochemical analyses demonstrated

that PCI and MPN subunits are structurally interdependent

during the formation of the COP9 complex, explaining why all

csn loss-of-function mutants share an identical phenotype (Fig-

ure 7A). Our data confirm that CSN function is essential for cullin

derubylation and to sustain the optimal activity of CUL1-, CUL3-,

and CUL4-based E3 complexes and suggest that the lethality

of csn mutants is triggered by the pleiotropic deregulation of

multiple ubiquitin-mediated pathways.

At this point, it would be interesting to establish what additional

CSN functions might contribute to the lethal phenotype of csn

loss-of-function mutants. In fact, several lines of evidence in

Arabidopsis (Wang et al., 2002) and yeast (Zhou et al., 2001;

Mundt et al., 2002) have demonstrated that there are other

essential functions of CSN independent from cullin derubylation.

However, because the loss of each CSN subunit results in the

loss of the entire complex, this important issue still remains

unanswered.

CSN5 and CUL3 Interact in Vivo and Reciprocally Regulate

Each Other’s Abundance

In this study, we have demonstrated that CSN interacts both in

vitro and in vivo with CUL3-RBX1–based E3 ubiquitin ligases

(Figure 8). We have also shown that CUL3-RBX1–based E3

complexes associate with the proteasome 19S regulatory par-

ticle in vivo, consistent with their function in substrate polyubi-

quitination and proteasome-mediated degradation (Figure 8D).

Interestingly, the complete lack of CSN activity results in a

drastic depletion of CUL3 in seedlings (Figure 7B, lanes indicated

by asterisks). In agreement with our findings, a recent report

showed that rubylated CUL1 and CUL3 are destabilized in

csn5Null and csn5D148N Drosophila mutant larvae, demonstrating

that CSN activity is required to prevent the degradation of

rubylated CUL3 (Wu et al., 2005). These results suggest that

CSN protects CUL1 and CUL3 from degradation only when it

possesses a reliable derubylation activity and highlight the idea

that RUB1 might function as a degradation signal for cullins.

However, our results showed that, in Arabidopsis seedlings, only

rubylated CUL3 is unstable whereas rubylated CUL1 is relatively

stable. Notably, CUL1 appears to be unstable in the flower tissue

of an Arabidopsis csn1 partial mutant (Wang et al., 2002),

suggesting that the stability of rubylated cullins may be regulated

in a tissue-specific manner. Moreover, the observation that loss

of function of CSN has an opposite effect on the stability of CUL4

emphasizes that cullin stability is regulated by specific mecha-

nisms and is not simply triggered by RUB/NEDD8 modification.

In the process of investigating possible physiological re-

sponses regulated by CUL3 during plant development, we

revealed a genetic interaction between CSN5 and CUL3 (Figures

9A to 9C). We have shown that a suboptimal dose of active CUL3

(attributable to either cul3a or cul3b loss of function) causes

CSN5 upregulation, most likely through a posttranscriptional

mechanism (Figure 10, lanes indicated by asterisks). Therefore,

whereas CSN activity is essential to positively regulate CUL3

function by protecting CUL3 from degradation, a robust CUL3

activity is required to negatively regulate CSN5 accumulation.

Because CSN5A is expressed at very high levels in wild-type

plants and in the single cul3a and cul3b mutants, the effect of

CUL3 reduction on CSN5 expression becomes evident only

when CSN5A is reduced dramatically (e.g., in csn5a-2 cul3a and

csn5a-2 cul3b backgrounds). Interestingly, CSN5A and CSN5B

possess a certain degree of divergence in their coding sequences

(Kwok et al., 1998) and incorporate into distinct CSN complexes

(Gusmaroli et al., 2004), which are present at very different levels

throughout plant development. Therefore, it is plausible to spec-

ulate that, in normal growth conditions, the activity of both cullins

is required to differentially regulate CSN5A and CSN5B expres-

sion. Even though we cannot rule out a possible indirect effect

on the control of CSN5 mRNA translation, an intriguing possibility

is that CUL3, as a component of E3 ubiquitin ligases, controls

CSN5 stability by targeting this subunit for degradation.

METHODS

Plant Materials and Growth Conditions

The wild-type Arabidopsis thaliana plants used in this study are the

Columbia-0 and Landsberg erecta ecotypes. The CSN5 and CSN6

T-DNA insertion lines SALK_063436 (csn5a-1), SALK_027705 (csn5a-2),

SALK_007134 (csn5b-1), SALK_146926 (csn6a-1), and SALK_036965

(csn6b-1) were identified in the SIGnAL database (Alonso et al., 2003).

The csn5a-1 and csn5a-2 T-DNA insertion mutants have lost the resis-

tance markers associated with their corresponding T-DNA insertions.

Both lines were crossed to wild-type plants before further investigations.

The phenotypes of csn5a-2 and csn5a-1 homozygous mutants cosegre-

gate in 100% of cases with the homozygous T-DNA insertion at the

CSN5A locus. In the case of csn5b-1, heterozygous T1 plants carrying

the csn5b-1 T-DNA insertion allele show a segregation ratio of 3:1


kanamycin-resistant:kanamycin-sensitive in T2 progeny, indicative of a

single T-DNA insertion (Gusmaroli et al., 2004). The T-DNA insertion lines

in CUL3A (cul3a mutant) and CUL3B (cul3b mutant) have been described

previously (Figueroa et al., 2005). The cop/det/fus mutants used in this

study were fus6-1 (Wang et al., 2002), fus12-U228 (Serino et al., 2003),

fus11-U203 (Peng et al., 2001b), cop8-1 (Wei et al., 1994b), fus5-1 (Karniol

et al., 1999), and cop9-1 (Wei et al., 1994a).

Arabidopsis seedlings were surface-sterilized, cold-treated at 48C for 3

to 5 d, and then germinated on solid Murashige and Skoog medium with

Gamborg’s vitamins (Sigma-Aldrich) supplemented with 1% sucrose

(GM) in continuous white light (150 mmol�m�2�s�1) at 228C. To obtain adult

plants, 8- to 16-d-old seedlings were transferred to soil and grown under

long-day conditions (16 h of light/8 h of dark) in a controlled-environment

chamber at 228C.

Light and Hormone Treatments

For the hypocotyl elongation experiments shown in Figure 4 and Sup-

plemental Figure 1 online, 4-d cold-treated seeds were exposed to white

light for 12 h (150 mmol�m�2�s�1) and then transferred to continuous

darkness or white (150 mmol�m�2�s�1), far-red (4 mmol�m�2�s�1), red

(80 mmol�m�2�s�1), or blue (11 mmol�m�2�s�1) light. For the other analyses

shown in Supplemental Figure 1 online, growth conditions were as

follows. For root length measurements, 4-d cold-treated seeds were

germinated in continuous white light (150 mmol�m�2�s�1) on GM vertical

plates and root length was measured after 5 d. For auxin and jasmonic

acid treatments, 4-d cold-treated seeds were germinated in continuous

white light (150 mmol�m�2�s�1) on vertical Murashige and Skoog plates for

4 d and then transferred to vertical GM plates supplemented with 2,4-D as

indicated or with 2 mM jasmonic acid. Root length and number of lateral

roots were measured 4 d after transfer to the auxin-containing medium.

Root hairs were photographed at 4 d after transfer to jasmonic acid–

containing medium. In all cases, seedlings were grown in controlled

chambers at 228C.

PCR-Based Genotyping

DNA of the Arabidopsis T-DNA lines and their progeny was extracted and

screened for T-DNA insertions at the CSN5A, CSN5B, CSN6A, CSN6B,

CUL3A, and CUL3B loci. In each case, sets of forward and reverse gene-

specific primers, in combination with the T-DNA left border–specific

primer (Alonso et al., 2003), were used to selectively amplify the wild-type

or T-DNA insertion alleles at the locus of interest. In the case of csn5a-1,

csn5a-2, csn5b-1, csn6a-1, and csn6b-1 mutants isolated in this study,

the identity of PCR fragments was confirmed by sequencing. All crosses

using csn5a-1, csn5a-2, csn5b-1, csn6a-1, csn6b-1, cul3a, and cul3b

alleles were confirmed in F1 by PCR-based genotyping. In all cases, the

genotypes of the segregating F2 single and double homozygous mutants

(csn5a-1, csn5a-2, csn5b-1, csn6a-1, csn6b-1, cul3a, cul3b, csn5a-1

csn5b-1, csn5a-2 csn5b-1, csn6a-1 csn6b-1, csn5a-2 cul3a, csn5a-2

cul3b, csn5b-1 cul3a, and csn5b-1 cul3b) and their progeny were

confirmed by PCR-based genotyping.

RT-PCR Analyses

Total RNA was extracted from the wild type, the single csn5a-1, csn5a-2,

csn5b-1, csn6a-1, csn6b-1, cul3a, and cul3b mutants, and the double

csn5a-2 cul3a and csn5a-2 cul3b homozygous mutant lines using the

RNeasy Plant Mini Kit (Qiagen). For each genotype, 100 mg of fresh tissue

obtained from 10-d-old light-grown seedlings was used as starting

material. For semiquantitative RT-PCR analyses, before retrotranscrip-

tion, total RNAs were treated with DNase I (Roche) to eliminate possi-

ble traces of contaminant DNA. The DNase I treatments and RT-PCR

were performed as described previously (Gusmaroli et al., 2004). Sets of

primers specific for eEF-1A (gene identifier 837307) and Tsb1 (gene

identifier 835571) were used as positive controls for RT-PCR. For semi-

quantitative RT-PCR analyses, the cDNAs were first calibrated using

ACT2 (gene identifier 821411). Different sets of gene-specific primers

were used to selectively amplify the cDNA corresponding to each partic-

ular mRNA transcript. RT-PCR was performed at 25, 28, 35, and 38 cycles.

Protein Extraction, Immunoblot Analyses, and

Gel Filtration Chromatography

Protein extractions and immunoblot analyses were performed as de-

scribed previously (Gusmaroli et al., 2004). The protein concentration in

the supernatants was determined by Bradford assay (Bio-Rad). For gel

filtration analyses, 300 mg of total proteins was fractionated onto a

Superose 6 (HR10/30) protein liquid chromatography column (Amersham

Biosciences) and fractions were concentrated using Strataclean resin

(Stratagene), as described previously (Gusmaroli et al., 2004). In all cases

in which equal loading of the samples was required, the same samples or

the blots were probed with a-RPN6 to confirm equal loading. The

antibodies used in this study are as follows: CSN1 (Staub et al., 1996),

CSN2 (Serino et al., 2003), CSN3 (Peng et al., 2001a), CSN4 (Serino et al.,

1999), CSN5 (Kwok et al., 1998), CSN6 (Peng et al., 2001b), CSN7 (Karniol

et al., 1999), CSN8 (Wei et al., 1994a), RPN6 and RPT5 (Kwok et al., 1999),

TBP (Schwechheimer et al., 2001), RBX1 (Schwechheimer et al., 2002),

CUL1 (Wang et al., 2002), CUL3 (Figueroa et al., 2005), CUL4 (Chen et al.,

2006), and histone H3 (Upstate).

Yeast Two-Hybrid Assay

The full-length cDNA clones of RBX1 and COP1 were translationally fused

to the transcription activation domain of pJG4-5 (Origene Technologies).

The two-hybrid constructs of the CSN and CUL1 subunits fused to the

pJG4-5 activation domain and the CUL3A construct fused to the pEG202

LexA binding domain have been described previously (Schwechheimer

et al., 2001; Serino et al., 2003). The CUL3A-LexA fusion construct was

transformed into yeast strain EGY48 (Invitrogen). Activation domain

fusion constructs were transformed into yeast strain L40 (Invitrogen),

which contains a b-galactosidase reporter gene. The plate and liquid

assays were performed as described previously (Serino et al., 1999).

In Vivo Coimmunoprecipitation Analyses

For pull-down assays, Arabidopsis tissues were ground in liquid nitrogen

and subsequently homogenized in cold immunoprecipitation buffer

(25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5% glycerol, 0.05% Nonidet P-40,

2.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 13 complete

cocktail of protease inhibitors). The total protein extracts were centri-

fuged twice at 13,000g for 15 min at 48C, and the supernatants were

filtered through 0.22-mm filters for use in immunoprecipitation assays. For

coimmunoprecipitation, 1.4 mg of total proteins was incubated for 4 h at

48C with 30 mL of monoclonal anti-FLAG antibody immobilized onto

agarose beads (Sigma-Aldrich) or with 30 mL of polyclonal CUL1, CUL3,

and CSN4 antibodies coupled to protein A-agarose beads (Sigma-

Aldrich). The antigene-antibody-protein A-agarose conjugates were

washed three times at 48C for 15 min in immunoprecipitation buffer and

centrifuged at 48C for 5 min at 3000 rpm between washes. The immu-

noprecipitates were subsequently released by boiling for 5 min in 23 SDS

sample buffer. Protein blot analyses were performed as described

previously (Gusmaroli et al., 2004).

Accession Numbers

Arabidopsis Genome Initiative locus identifiers for the genes mentioned in

this article are as follows: At3g18780 (ACT2); At3g61140 (CSN1);

578 The Plant Cell

At2g26990 (CSN2); At5g14250 (CSN3); At5g42970 (CSN4); At1g22920

(CSN5A); At1g71230 (CSN5B); At5g56280 (CSN6A); At4g26430 (CSN6B);

At1g02090 (CSN7); At4g14110 (CSN8); At4g02570 (CUL1); At1g26230

(CUL3A); At1g69670 (CUL3B); At5g46210 (CUL4); At5g60390 (EF-1A);

At1g06760 (histone H1); At3g42830 (RBX1); At1g29150 (RPN6);

At3g05530 (RPT5); At3g13445 (TBP); and At5g54810 (TSB1).

Supplemental Data

The following material is available in the online version of this article.

Supplemental Figure 1. Characterization of the Single csn5 Mutants

in Different Growth Conditions at Representative Developmental

Stages.

ACKNOWLEDGMENTS

We are grateful to Ning Wei and Sinead Drea for critical reading of the

manuscript. We also thank the Salk Institute Genomic Analyses Labo-

ratory for generating the sequence-indexed Arabidopsis T-DNA inser-

tion mutants and the Nottingham and Ohio Arabidopsis stock centers for

providing seeds of the corresponding lines. This research was sup-

ported by National Science Foundation 2010 Grant MCB-0519970 and

by National Institutes of Health Grant GM-47850 to X.W.D. G.G. was ini-

tially supported by a fellowship from the Universita’ degli Studi di Milano.

Received September 21, 2006; revised January 15, 2007; accepted

January 30, 2007; published February 16, 2007.

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DOI 10.1105/tpc.106.047571; originally published online February 16, 2007; 2007;19;564-581Plant Cell

Giuliana Gusmaroli, Pablo Figueroa, Giovanna Serino and Xing Wang Deng Cullin3-Based E3 LigasesArabidopsisInteraction with

Role of the MPN Subunits in COP9 Signalosome Assembly and Activity, and Their Regulatory

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Supplemental Data /content/suppl/2007/03/15/tpc.106.047571.DC1.html

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Role of the MPN Subunits in COP9 Signalosome Assembly and … · Role of the MPN Subunits in COP9 Signalosome Assembly and Activity, and Their Regulatory Interaction with Arabidopsis