Resubmission (including cover letter) - Sanford-Burnham Medical ...

Elsevier Editorial System(tm) for Molecular Cell
Manuscript Draft
Manuscript Number: MC-D-08-00448R1
Title: F-Box-directed CRL complex assembly and regulation by the CSN and CAND1
Article Type: Research Manuscript
Keywords: Cop9 signalosome; Cullin-RING ubiquitin ligase; CAND1; F-box protein; Fission yeast
Corresponding Author: Dr. Dieter A. Wolf, M.D.
Corresponding Author's Institution: Burnham Institute for Medical Research
First Author: Michael W Schmidt, Ph.D.
Order of Authors: Michael W Schmidt, Ph.D.; Philip R McQuary; Susan Wee, Ph.D.; Kay Hofmann,
Ph.D.; Dieter A. Wolf, M.D.
Abstract: The COP9 signalosome (CSN) is thought to maintain the stability of cullin-RING ubiquitin
ligases (CRL) by limiting the autocatalytic destruction of substrate adapters such as F-box proteins
(FBPs). CAND1, a protein associated with unneddylated CUL1, was proposed to assist in this role
in an as yet unclear fashion. We found that only a subset of S. pombe FBPs, which feature a critical
F-box proline that promotes their interaction with CUL1, required CSN for stability. Unlike the CRL3
adapter Btb3p, none of the CSN-sensitive FBPs were affected by deletion of ubp12. Contrary to
current models, CAND1 does not control adapter stability, but maintains the cellular balance of
CRL1 complexes by preventing rare FBPs from being out-competed for binding to CUL1 by more
ample adapters. These findings were integrated into a new model of CRL control where substrate
availability toggles CRLs between independent CSN and CAND1 cycles.
Suggested Reviewers:
Opposed Reviewers:

A) Cover Letter
Dieter A. Wolf, M.D.
Professor and Director
NCI Cancer Center Proteomics Facility
Dr. Mark Landis
Associate Editor
Molecular Cell
600 Technology Square, 5th floor
Cambridge, Massachusetts 02139
USA
June 24, 2009
Dear Dr. Landis,
We are re-submitting our manuscript entitled “F-Box-Directed CRL Complex Assembly and
Regulation by the CSN and CAND1” for your consideration for publication in Molecular Cell.
We apologize that the revision has required more time than we had hoped. In order to
experimentally address the reviewers’ comments, we had to create several new strains,
including a knock-in allele of a particular F-box protein point mutant. Since I had recruited my
entire lab anew when I moved to the Burnham Institute, there was a significant training period
for my co-workers. However, the wait was well worth it, because we can now include several
new experiments that we believe should address the major concerns of the reviewers.
Specifically, we are addressing three major points:
(i.) Reviewers 1 and 2 wondered whether the degradation of FBPs in csn5 mutants is mediated
by the proteasome. We are now including this data for the unstable FBPs Pof1p, Pof9p, and
Pof10p.
(ii.) Reviewers 2 and 3 were curious whether insertion of a proline residue into the CSNindependent
FBP, Pof12p, would render its stability dependent on csn5. In order to do this
experiment, we had to create a strain in which endogenous Pof12p is replaced by the Pof12p-
SP point mutant. The experiments showed that Pof12p-SP, expressed at endogenous levels, is
indeed ectopically targeted into a CRL complex. However, this does not render the protein
unstable in csn5 mutants. This indicates that this artificial protein is not subject to autocatalytic
ubiquitylation and degradation, probably because it does not display lysine residues in positions
that are amenable to modification with ubiquitin. Nevertheless, this experiment adds further
proof that the F-box proline is required and sufficient for CRL complex formation.
(iii.) Reviewer 2 asked whether we had tested the effect of knd1 deletion on other FBPs in
addition to Pof1p. We have now done so, and the results confirm our model of a CAND1 cycle,
which prevents rare adapters from being outcompeted for binding to Cul1p by abundant
adapters. Next to the F-box proline, this is the main novel finding reported in this manuscript.
We have also addressed all other points of the reviewers as indicated in the included rebuttal
letter. We hope that you will find our revised manuscript acceptable for publication in Molecular
Cell.
Sincerely,
10901 North Torrey Pines Road. │ La Jolla, California 92037 │ 858.646.3117 │ 858.646.3172 fax │ www.burnham.org

* B) Response to reviewers
We wish to sincerely thank all reviewers for their insightful comments, which we feel
have considerably strengthened the manuscript. In the following, we are addressing the
reviewers’ comments point-by-point.
Reviewer 1
1. The reviewer wonders whether “cullin 1 binding FBPs are ubiquitinated and
subsequently degraded by the 26S proteasome? It would be nice to show selected
cycloheximide chases in the presence of MG132 or other appropriate proteasome
inhibitors”.
This is an interesting question that we have addressed with several new experiments.
Since proteasome inhibitors do not work well in fission yeast probably due to inefficient
passage through the cell wall, we have created strains containing tagged FBPs (Pof1p,
Pof9p, and Pof10p) in the mts3-1 proteasome mutant background. These strains as well
as the corresponding proteasome wild-type strains were also deleted for csn5 to induce
FBP instability. The cells were maintained at the restrictive temperature of 36.5 C for 90
min to inactivate proteasome function. Immunoblotting revealed a dramatic increase in
the steady-state levels of the three FBPs in mts3-1 mutants (data included as revised
Supplementary Figure 3). In addition, Pof1p and Pof10p showed an accumulation of
high molecular weight species, which we believe to be ubiquitylated forms. CHX chase
experiments demonstrated complete stabilization of all three FBPs in csn5 mutants upon
inhibition of the proteasome (see revised Fig. 2C, D). These data strongly suggest that
FBPs that are destabilized in csn5 mutants are degraded by the proteasome. The new
data is included in the revised manuscript as Fig. 2C and D.
2. With respect to our model in Fig. 7, the reviewer asks us to hypothesize on how “the
substrate can induce its own ubiquitination without inducing the ubiquitination of the
FBPs. Any hypothesis?”
This is indeed a difficult question that remains open to speculation until the enzymatic
mechanism of CRLs is entirely clarified. An additional level of uncertainty is added by the
possibility that different CRLs may have different mechanisms. However, we can offer
the following hypothesis, which we believe is consistent with the majority of observations
in the field. We will divide the reviewer’s question into two parts: (i.) How is a true CRL
substrate different from a substrate adaptor (FBPs and other CRL adaptors)? (ii.) Why
does a substrate, but not an adaptor, induce its own ubiquitylation?
As for the first part, we proposed in a 2003 Perspective for Nature Cell Biology that
substrate adaptors may bear lysine residues in regions that are accessible by E2s
cooperating with CRLs (Wolf et al., 2003). Thus, by mere proximity these adaptors may
be ubiquitylated by their cognate CRL core modules. In this sense, adaptors could also
be viewed as substrates. However, the specific positioning of lysine residues of true
substrates appears to enable more efficient ubiquitin transfer, since substrates are
generally turned over more quickly than their corresponding adaptors. It is already well
established that some CRLs can use a variety of different substrate lysines, albeit with
different efficiencies (Petroski and Deshaies, 2003). It is also possible that substrate
binding shields adaptor lysine residues, which may become only accessible, once the
substrate has been ubiquitylated and degraded. We believe that differential accessibility
of substrate and adaptor lysine residues may function as a clock that times the
successive ubiquitylation and destruction of first the substrates and then the adaptors.
As for the second part, we believe the different abilities of substrates and adaptors to
induce their own degradation may be due to their different abilities to stimulate cullin
neddylation. Two reports have clearly demonstrated that substrate is required to induce

cullin neddylation (Bornstein et al., 2006; Chew and Hagen, 2007). Importantly, an
adaptor that binds to cullin, but not to substrate fails to promote neddylation (Chew and
Hagen, 2007).
Since we have discussed some of these ideas previously (Wee et al., 2005; Wolf et
al., 2003) but our new findings do not shed new light on this particular question of the
reviewer, we decided not to persevere on these speculations beyond what is stated in
the description of our model in Fig. 7. If the reviewer believes, however, that our
manuscript would gain from including these ideas, we’d be happy to do so in a final
version.
3. “Is the labeling of Cul1p-Nedd8p and Cul1p in Fig. 5B, middle panel, really correct?”
We apologize, the labeling was off. It is fixed in the revised figure, which was moved into
the supplement (Supplementary Fig. 5), because another panel was added to revised
Fig. 5.
4. “In supplementary Fig. 4 the lanes +/- Myc-Knd1p expression should be labeled and
the antibody used in the lower panel should be indicated.”
The missing labels were included in revised Suppl. Fig. 7 (former Suppl. Fig. 4).
Reviewer 2
All spelling and wording suggestions of the reviewer were incorporated into the revised
version of the manuscript. Below, we are only discussing comments about the
experiments and conclusions.
1) The reviewer asks us to discuss the following apparent discrepancy: “Previous reports
hypothesize that neddylation is the driving force of CAND1-Cullin/RBX1 dissociation.
However, based on the data from this manuscript, it seems unlikely that neddylation
plays such a role in the proposed rapid CAND1 cycle. This point and the possible driving
force for CAND1-Cullin/RBX1 dissociation in CAND1 cycle should be discussed.”
The reviewer’s comment points to one of the big mysteries of the interplay between
CAND1 and CRLs. Using a system completely reconstituted from bacterially expressed
proteins, Goldenberg et al. demonstrated that neither the NEDD8 E3, Ubc12, nor Skp1
in a complex with the F-box of Skp2 can dissociate CAND1 from CUL1 (Goldenberg et
al., 2004). The authors speculated that there must be an additional factor that induces a
conformational change in either CAND1 or CUL1 to cause their dissociation. A
subsequent study by Bornstein et al. (Bornstein et al., 2006) showed data that
suggested that a complex of “Skp2–Skp1 promotes the dissociation of Cul1 from
CAND1, whereas substrate increases the neddylation of Cul1 by a different mechanism.”
The CAND1 cycle shown in our model in Fig. 7 is entirely consistent with these findings.
Exactly how SKP2, and by extension other FBPs, drive the displacement of CAND1
remains to be established, but it is conceivable that proline-dependent binding of SKP1-
FBP dimers causes a conformational change in CUL1 that leads to the displacement of
CAND1. The matter is now included on page 17 of the revised discussion.
2. “Performing MG132 treatment in the protein stability tests (both steady state and time
course) is a necessary control to show that the observed protein level decrease is
indeed due to proteasomal degradation.”
We have addressed this with several new experiments. Since proteasome inhibitors do
not work well in fission yeast probably due to inefficient passage through the cell wall,
we have created strains containing tagged FBPs (Pof1p, Pof9p, and Pof10p) in the

mts3-1 proteasome mutant background. These strains as well as the corresponding
proteasome wild-type strains were also deleted for csn5 to induce FBP instability. The
cells were maintained at the restrictive temperature of 36.5 C for 90 min to inactivate
proteasome function. Immunoblotting revealed a dramatic increase in the steady-state
levels of the three FBPs in mts3-1 mutants (data included as revised Supplementary
Figure 3). In addition, Pof1p and Pof10p showed an accumulation of high molecular
weight species, which we believe to be ubiquitylated forms. CHX chase experiments
demonstrated complete stabilization of all three FBPs in csn5 mutants upon inhibition of
the proteasome (see revised Fig. 2C, D). These data strongly suggest that FBPs that are
destabilized in csn5 mutants are degraded by the proteasome. The new data is included
in the revised manuscript as Fig. 2C and D.
3. “The authors have shown that Pof12p(SP) has enhanced Cul1p binding. This is
interesting, but more work should be performed in this line. At least the stability of this
mutant protein should be checked to show if incorporation into CRL could trigger
degradation.”
We have performed this experiment, which was also requested by reviewer 3. In order to
do so, we had to place the Pof12p-S15P mutation into the genomic pof12 locus (as we
did for Pof10p-P35S), because we have previously found that overexpressed FBPs are
not properly controlled by autocatalytic degradation (data not shown). In revised Fig. 5E,
we are now showing that unlike wildtype Pof12p, Pof12p-S15P expressed from its
endogenous promoter gained the ability to interact with Cul1p, albeit at a low level.
Whereas, knock-in of the F-box proline was sufficient to target Pof12p into a CRL1
complex, the mutant protein was neither downregulated nor destabilized in csn5 mutants
(Fig. 5E; Supplementary Fig. 6). This might be due to the low level of recruitment to
Cul1p. In addition, the mutant protein, which is not normally destined for a CRL1
complex, may not bear lysine residues accessible to autocatalytic modification by its
associated Cul1p core complex. In conclusion, the proline is sufficient to target at least a
portion of Pof12p into CRL1 complexes, but this does apparently not result in
autocatalytic instability, and hence there is no effect of CSN.
4. “Do the authors have any example that certain FBPs are less efficiently incorporated
into CRL in knd1 mutant? Based on their CAND1 cycle model, it is expected that, while
some FBPs are interacting with more Cul1p in the absence of Knd1p, some of the less
abundant FBPs may not be able to compete and thus have reduced interaction with
Cul1p in the absence of Knd1p.”
The reviewer’s question gets to the heart of our model, and we have therefore
addressed this with additional experiments. In addition to Pof1p, we have now tested two
other FBPs for interaction with Cul1p in knd1 mutants. For this, we chose Pof3p and
Pof7p, because both are substantially less abundant than Pof1p. While Pof7p binding to
Cul1p was unchanged in the absence of Knd1p, Pof3p showed a stark decrease
(revised Fig. 6E). In addition, knd1 mutants carrying epitope-tagged Pof3p exhibit a pof3
deletion phenotype with slow growth and cell elongation, suggesting that these cells
have a defect in SCF Pof3p function (revised Fig. 6F). This phenotype was neither
observed with wildtype cells carrying tagged Pof3p nor with knd1 mutants containing
other tagged FBPs. This finding suggests that the tagged Pof3p protein is partially
defective and therefore requires an intact CAND1 cycle to maintain a sufficient level of
activity. In conclusion, these findings show that loss of CAND1 differentially affects the
composition of CRL1 complexes, presumably by allowing increased recruitment of some
abundant FBPs at the expense of less abundant adapters as predicted by our model.
These new data should provide the confirmation the reviewer has asked for.

5. “It looks like that the exposure time or protein loading amount in the left panels and
the right panels in Figure 2A must be very different, so that the starting protein
abundance for CSN-sensitive FBPs in WT and csn5 mutant appear to be the same. The
authors should mention this in the legends; otherwise it is quite misleading.”
This adjustment was done in order to enable a better comparison of FBP stability in
wildtype versus csn5 mutant cells. It is now explained in the figure legend.
6. “More than half of the lower panel in Figure 6B is not displayed.”
This is unfortunate, but not our fault. We have checked the integrity of all display items
on the uploaded version of the manuscript. If the problem persists in the revised version
of the manuscript, the reviewer may want to contact the editorial office.
7. “According to Figure 6C, the abundance of Btb3p seems to be more reduced in knd1
mutant than in WT at the 120-minute time point. Is it reproducible?”
A quantification of Btb3p levels in wildtype and mutant cells showed that there is indeed
a 1.4-fold difference in Btb3p levels in the 120 minute time point in this blot. The
difference is not reproducible, but has little bearing on the conclusion we draw from this
figure. Our claim is that the half-live of Btb3p is not affected in a major way by deleting
knd1. The half-live measurement relies on all 5 lanes shown and showed only a 1.18-
fold difference (107.7 versus 90.6 min). We therefore believe that the figure fully
supports our conclusion.
8. “According to Figure S4, the pull-down of Myc-Knd1p is not efficient. A better pulldown
experiment is needed here.”
The reviewer is correct that the amount of overexpressed Myc-Knd1p retrieved by Mycantibody
beads is less than one would hope. However, we have repeated this
experiment several times and always found that overexpressed Myc-Knd1p is apparently
degraded during the immunoprecipitation. However, the retrieved amount is sufficient to
demonstrate binding to unneddylated Cul1p. In addition, we show the same interaction
with proteins expressed at the endogenous levels (Fig. 6A) and the finding is therefore
sound. We have not repeated this experiment but we have included the missing figure
labels the reviewer pointed out.
9. “To make the story more complete, Skp1 blot should be included in the IP assays in
Figure 5A and 5B.”
We are already showing the amount of Skp1p copurifying with FBPs and point mutant
Pof10p at the level of the endogenous proteins at several places (Fig. 4B, C; Fig. 5D). In
addition, we include a figure with overexpressed Pof1p (Suppl. Fig. 4). We therefore do
not believe that our study is incomplete without showing Skp1p levels in Figs. 5A and B.
Reviewer 3
In the general comments, the reviewer raised a number of issues that are clarified first.
“While the observation that the interaction and thus CSN-dependent regulation of F-box
proteins requires the presence of a conserved proline residue is interesting, similar noncullin
associated Skp1-F-box complexes have been described before, and the authors
fail to show an in vivo function for these complexes.”
The reviewer is right that, as we pointed out in the discussion, several FBPs have been

implicated in functions that are apparently independent of CRLs (reviewed in (Hermand,
2006)). However, our goal was not to study these functions, but to understand why only
a subset of FBPs forms CRL complexes. We have successfully pinpointed the
discriminating feature as a single conserved lysine residue in the F-box motif. This is a
significant advance, since it enables a rapid distinction of FBPs that are engaged in CRL
complexes versus those that are not. In addition, our data suggest a mechanism for FBP
recruitment into CRL complex that depends on a conformational change in the N-
terminus of CUL1. Lastly, our findings may enable the design of small molecule
compounds that can specifically interfere with the proline-dependent recruitment of FBPs
into CRL complexes.
“Also, the role of Cand1/knd1 for cullin ligase regulation is not clear, especially as no
effects of knd1 deletions on cullin ligase activity/fidelity are presented in the manuscript.
This contradicts observations in other systems, and it is thus not justified to draw general
conclusions and models from a setting where no effects on cullin activity by Cand1 are
observed.”
To address this comment, we have performed new experiments that clearly show that
CAND1 differentially regulates the disposition of cellular CRL1 complexes according to
the model we have proposed. In the original submission, we had shown increased
recruitment of the abundant FBP, Pof1p, into CRL complexes in knd1 mutants (revised
Fig. 6D). Based on this finding, we predicted that some rare FBPs may be out-competed
thus leading to a defect in CRL1 functions. Our new experiments are confirming this
critical prediction of our model.
We examined the interaction of two other FBPs with Cul1p in knd1 mutants. For this,
we chose Pof3p and Pof7p, because both are substantially less abundant than Pof1p.
While Pof7p binding to Cul1p was unchanged in the absence of Knd1p, Pof3p showed a
stark decrease (revised Fig. 6E). In addition, knd1 mutants carrying epitope-tagged
Pof3p exhibit a pof3 deletion phenotype with slow growth and cell elongation (revised
Fig. 6F), suggesting that these cells have a defect in SCF Pof3p function. This phenotype
was neither observed with wildtype cells carrying tagged Pof3p nor with knd1 mutants
containing other tagged FBPs. This finding suggests that the tagged Pof3p protein is
partially defective and therefore requires an intact CAND1 cycle to maintain a sufficient
level of activity. In conclusion, these findings show that loss of CAND1 differentially
affects the composition of CRL1 complexes, presumably by allowing increased
recruitment of some abundant FBPs at the expense of less abundant adapters as
predicted by our model. These new data also provide the link between Knd1p deficiency
and CRL activity the reviewer has asked for.
1. “What is the function of the non-cullin associated Skp1/F-box complexes?”
This question is addressed in a very nice review (Hermand, 2006), which we are
referencing in the revised version of the manuscript. As pointed out above, the main goal
of our study was to understand why only a subset of FBPs is engaged in CRL
complexes. Determining the function of those FBPs that are targeted into other
complexes, perhaps through Skp1p, will be reserved to future studies by this and other
labs.
2. “S15P mutation of Pof12 established binding to Cul1 - based on the author's model,
this F-box protein should now be subject to regulation by the CSN, but the authors fail to
demonstrate this.”
We have performed this experiment, which was also requested by reviewer 2. In order to
do so, we had to place the Pof12p-S15P mutation into the genomic pof12 locus (as we

did for Pof10p-P35S), because we have previously found that overexpressed FBPs are
not properly controlled by autocatalytic degradation (data not shown). In revised Fig. 5E,
we are now showing that unlike wildtype Pof12p, Pof12p-S15P expressed from its
endogenous promoter gained the ability to interact with Cul1p, albeit at a low level.
Whereas, knock-in of the F-box proline was sufficient to target Pof12p into a CRL1
complex, the mutant protein was neither downregulated nor destabilized in csn5 mutants
(Fig. 5E; Supplementary Fig. 6). This might be due to the low level of recruitment to
Cul1p. In addition, the mutant protein, which is not normally destined for a CRL1
complex, may not bear lysine residues accessible to autocatalytic modification by its
associated Cul1p core complex. In conclusion, the proline is sufficient to target at least a
portion of Pof12p into CRL1 complexes, but this does apparently not result in
autocatalytic instability, and hence there is no effect of CSN.
3. “Why and how is Pof13 regulated by the CSN, but not on the protein level (unlike the
other cullin-associated F-boxes)? What is the mechanism of regulation in this case?”
We have no definitive answer to this question other than the one offered on page 6 of
our manuscript where we describe the observation that Pof13p drops slightly in csn5
mutants, although the protein is not destabilized. We have repeated this experiment
several times with the same result. We also show that pof13 mRNA is not
downregulated in csn5 mutants. By way of elimination, this leaves an effect of CSN
deficiency on pof13 mRNA translation as the most likely explanation. For example, a
protein, which regulates pof13 mRNA translation may be a substrate of a CRL and
hence be dysregulated in csn5 mutants. We plead with the reviewer to see our inability
to pinpoint the exact defect in Pof13p synthesis in the context of our entire work, which
clearly demonstrates that the proline residue in the F-box of several FBPs is required
and sufficient for regulation of FBP stability by the CSN.
4. “While Ubp12 and Ubp1 show no effect on CRL1 adaptor stability, are there other
Ubps that do? If not, why not? Does Ubp12/1 not associate with CSN when it is in a
complex with Cul1?”
We have previously shown that the interaction of cullins with Ubp12p depends on the
CSN (Wee et al., 2005; Zhou et al., 2003). In contrast, the CSN-Ubp12 interaction is
independent of cullins. The CSN complex purified via multiple chromatographic steps will
copurify Ubp12 but not cullins (Zhou et al., 2003). No other DUB was identified in this
purification. We therefore do not believe that other DUBs contribute to CSN-mediated
adaptor stability. The differential effect of Ubp12p on Btb3p versus FPBs is highly
reproducible. A possible explanation is that CUL3 adaptors are regulated differently than
CUL1 adaptors for reasons that remain to be established, perhaps because there are
fewer of them (three BTB domain proteins versus ~16 FBPs). These ideas were already
discussed in the last paragraph of the discussion section.
References
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deneddylation of cullin1 in SCFSkp2 ubiquitin ligase by F-box protein and substrate.
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Hermand, D. (2006). F-box proteins: more than baits for the SCF? Cell Div 1, 30.
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* C) Manuscript
Click here to view linked References
F-Box-directed CRL complex assembly and
regulation by the CSN and CAND1
Michael W. Schmidt 1,6 , Philip R. McQuary 2,6 , Susan Wee 1,4 , Kay Hofmann 3 ,
Dieter A. Wolf 1,2,5
Address:
1 Department of Genetics and Complex Diseases, Harvard School of Public Health, 665
Huntington Avenue, Boston, Massachusetts, 02115, USA
2 Burnham Institute for Medical Research, Signal Transduction Program, 10901 North
Torrey Pines Road, La Jolla, CA 92037
3 Milteny Biotec GmbH, D-50829 Koeln, Germany;
Present Address: 4 Bristol-Myers Squibb, Bristol-Myers Squibb, Route 206 and
Provinceline Road, Princeton, NJ.
5 Corresponding author
6 These authors contributed equally
Running title: CRL control by CSN and CAND1

Summary
The COP9 signalosome (CSN) is thought to maintain the stability of cullin-RING
ubiquitin ligases (CRL) by limiting the autocatalytic destruction of substrate
adapters such as F-box proteins (FBPs). CAND1, a protein associated with
unneddylated CUL1, was proposed to assist in this role in an as yet unclear fashion.
We found that only a subset of S. pombe FBPs, which feature a critical F-box
proline that promotes their interaction with CUL1, required CSN for stability.
Unlike the CRL3 adapter Btb3p, none of the CSN-sensitive FBPs were affected by
deletion of ubp12. Contrary to current models, CAND1 does not control adapter
stability, but maintains the cellular balance of CRL1 complexes by preventing rare
FBPs from being out-competed for binding to CUL1 by more ample adapters. These
findings were integrated into a new model of CRL control where substrate
availability toggles CRLs between independent CSN and CAND1 cycles.
2

Introduction
CRLs represent an extensive class of multisubunit E3 ubiquitin ligases each consisting of
a core module containing a member of the cullin family and the RING domain protein
RBX1 (= HRT1, ROC1), which recruits E2 ubiquitin conjugating enzymes to the ligases
(reviewed in (Bosu and Kipreos, 2008; Petroski and Deshaies, 2005)). This core is joined
by one of several hundred adapter proteins each of which appears to target a distinct array
of substrates for ubiquitylation and proteasomal degradation. Whereas FBPs are tethered
to the CUL1 core through the linker protein SKP1 to form SCF (or CRL1) complexes
(Deshaies, 1999), CUL3 adapters are recruited into CRL3 complexes via their inherent
BTB domains (Furukawa et al., 2003; Geyer et al., 2003; Pintard et al., 2003; Xu et al.,
2003). Several members of both adapter families are unstable proteins, due to
autoubiquitylation by the intrinsic ubiquitin ligase activity of their associated core
modules (Galan and Peter, 1999; Geyer et al., 2003; Luke-Glaser et al., 2007; Rouillon et
al., 2000; Wirbelauer et al., 2000; Zhou and Howley, 1998) .
CRLs are stimulated through modification of cullins with the ubiquitin-related
peptide NEDD8 (reviewed in (Pan et al., 2004). Cullin neddylation is reversed by the
COP9 signalosome (CSN), a highly conserved protein complex that binds cullins
(Lyapina et al., 2001; Schwechheimer et al., 2001; Wee et al., 2002; Zhou et al., 2001)
and thereby exposes them to a deneddylating activity intrinsic to subunit 5 of the CSN
(Cope et al., 2002). Consistent with neddylation being a stimulatory modification is the
observation that purified CSN inhibits CRL activity in vitro. In addition, the CSN-
3

associated deubiquitylating enzyme (DUB) Ubp12/USP15 acts to neutralize CRL activity
when assayed in vitro (Groisman et al., 2003; Zhou et al., 2003).
These biochemical findings conflicted with genetic evidence that CSN is required for
efficient CRL-dependent substrate degradation in vivo (Reviewed in (Cope and Deshaies,
2003; von Arnim, 2003; Wolf et al., 2003)). This so-called CSN paradox was resolved by
the demonstration that CSN’s inhibitory enzymatic activities revealed in vitro prevent the
autocatalytic degradation of CRL substrate adapters in vivo thus promoting CRL activity.
For example, the Cul3p adapter Btb3p is destabilized in fission yeast csn and ubp12
mutants (Wee et al., 2005). In addition, several FBPs are unstable in csn mutants of S.
pombe (Zhou et al., 2003) and N. crassa (He et al., 2005), and in human CSN knockdown
cells (Cope and Deshaies, 2006; Denti et al., 2006).
CAND1 is a highly conserved protein that binds to the unneddylated form of human
and plant CUL1 and inhibits complex formation with SKP1-FBP modules (Hwang et al.,
2003; Liu et al., 2002; Min et al., 2003; Oshikawa et al., 2003; Zheng et al., 2002a). By
virtue of these properties, CAND1 inhibits CRL activity in vitro. However, CAND1 was
also shown to be required for efficient CRL function in vivo (Chuang et al., 2004; Feng et
al., 2004; Zheng et al., 2002a). This contradictory pattern reiterates the CSN paradox, and
it was therefore suggested that CAND1 and CSN participate in the same pathway of CRL
adapter stabilization (Cope and Deshaies, 2003; He et al., 2005; Min et al., 2005),
although experimental proof is still outstanding. It also remained unclear whether the
model is broadly applicable to all FBPs. In the present report, we address these questions
by determining the regulation by CSN and CAND1 of a panel of eight FBPs from fission
yeast.
4

Results
Differential effect of CSN on FBP levels
In previous studies we demonstrated that the stability of the CRL1 adapter Pop1p and the
CRL3 adapter Btb3p is promoted by the CSN in vivo (Wee et al., 2005; Zhou et al.,
2003). In addition, genetic interaction studies suggested that FBPs other than Pop1p are
also subject to regulation by the CSN (Wee et al., 2005). The S. pombe genome encodes a
minimum of 16 FBPs (named Pofs and Pops), but whether all are targets for regulation by
the CSN is unknown. We compared the steady-state protein levels of eight randomly
chosen FBPs in wild-type and csn5 mutant cells, utilizing a panel of strains harbouring
FBPs modified at their endogenous genomic loci with C-terminal Myc-epitope tags
(Lehmann et al., 2004). Pof1p, 3p, 7p, 9p, 10p, and 13p steady-state levels were reduced
in csn5 mutants, whereas Pof8p and Pof12p levels were largely unaffected (Fig. 1A).
The levels of the CSN-regulated Pof1p and Pof10p were also diminished in csn3 and
csn4 mutants (Fig. 1B, Supplementary Fig. 1). Conversely, downregulation of Pof10p in
csn5 mutants was complemented by providing wild-type csn5 from a plasmid (Fig. 1B).
This rescue failed in csn4 csn5 double mutants (Fig. 1B). In addition, efficient rescue
depended on the enzymatic function of the deneddylating enzyme Csn5p, since point
mutants in the catalytic JAMM motif were unable to maintain Pof10p levels in csn5
mutants (Fig, 1C). These results suggested that the cullin deneddylation function of the
entire CSN complex is required to maintain the steady-state expression levels of CSNsensitive
FBPs.
5

CSN regulates FBP protein stability
The levels of the mRNAs encoding Pof1p and Pof10p were unchanged in csn5 mutants as
determined by semiquantitative RT-PCR (Supplementary Fig. 2A), suggesting that CSNsensitive
FBPs might be regulated at the level of protein stability. Cycloheximide (CHX)
chase experiments confirmed that the CSN-sensitive FBPs Pof1p, Pof3p, Pof7p, Pof9p,
and Pof10p were considerably destabilized in csn5 mutants (Fig. 2A, B). Destabilization
of Pof1p, Pof9p, and Pof10p did not occur in csn5 mutants that carried the temperaturesensitive
mts3-1 allele (Gordon et al., 1996), which encodes a mutant version of the
proteasome subunit Rpn12p (Fig. 2C, D). Likewise, the downregulation of these FBPs in
csn5 mutants was rescued in csn5 mts3-1 double mutants, indicating that proteasome
activity is required for FBP destabilization in csn5 mutants (Supplementary Fig. 3). No
destabilization was observed for Pof8p and Pof12p. Pof13p stability was also unaffected
in csn5 mutants (Fig. 2A, B) despite the decrease in Pof13p steady-state levels apparent
in Fig. 1A. Since pof13 mRNA was not downregulated in csn5 mutants as determined by
quantitative real time PCR (Supplementary Fig. 2B), decreased Pof13p protein levels in
csn5 mutants may reflect an unidentified role of CSN in facilitating Pof13p protein
synthesis. It is well established that some CSN components have dual functions in
proteolysis and protein synthesis (Luke-Glaser et al., 2007).
The DUB Ubp12p maintains the stability of the CRL3 adapter Btb3p but not
FBPs
We previously showed that the level of the Cul3p adapter Btb3p is strongly reduced in
cells lacking CSN deneddylation activity (Wee et al., 2005). An even more pronounced
6

downregulation was observed in cells deficient of the CSN-associated DUB Ubp12p, and
both effects were attributed to increased autocatalytic destruction of Btb3p by its
associated CRL3 core module (Wee et al., 2005). Unlike with Btb3p, the steady-state
levels of the CSN-regulated FBPs Pof1p, Pof3p, Pof9p, and Pof10p were only minimally
altered in ubp12 mutants when compared to csn5 mutants (Fig. 3A), indicating that
Ubp12p is not a major regulator of these adapters.
To illustrate the differential effect of Ubp12p on Btb3p and FBPs more rigorously, we
generated a ubp12 deletion strain coexpressing protein A-tagged Btb3p and Myc-tagged
Pof1p from their endogenous promoters. A CHX chase experiment revealed that Btb3p
was drastically destabilized in ubp12 mutants as described (Wee et al., 2005), whereas
Pof1p was entirely stable in the same cells (Fig. 3B).
To exclude the possibility that Ubp1p, a DUB sharing 48% amino acid similarity
(32% identity) with Ubp12p and an overall identical domain structure and length (data
not shown), maintained FBP stability in ubp12 mutants, we compared Pof1p levels in
ubp1 and ubp12 single mutants and in ubp1 ubp12 double mutants. Pof1p levels were
unchanged in either mutant (Fig. 3C). These data suggested that, unlike with Btb3p, the
steady-state levels of CSN-sensitive FBPs were not affected by lack of CSN-associated
DUB activity, although we cannot entirely exclude minor destabilization in ubp12
mutants as previously detected for Pop1p (Zhou et al., 2003).
CSN-insensitive FBPs are deficient in forming canonical CRL1 complexes
To understand why the stability of only five of the eight FBPs tested was regulated by the
CSN, we searched for structural features setting the two groups apart. Since the F-box is
7

the only motif shared by all of these proteins, we performed a ClustalW alignment of the
F-boxes of all 16 fission yeast FBPs. Human SKP2 and -TRCP1 as well as budding
yeast Cdc4p were included for reference. The alignment revealed that all CSN-insensitive
FBPs missed a conserved proline residue at the beginning of the F-box motif (Fig. 4A).
None of the other signature residues of the F-box motif segregated consistently with CSN
regulation. Since the proline is one of the most highly conserved amino acids of the F-
box motif across all species, we reasoned that it might be important for CRL complex
formation.
To test this, Myc-tagged FBPs were immunoprecipitated with Myc antibodies,
followed by immunoblotting with Cul1p and Skp1p antisera. Binding of Cul1p was only
detected for the CSN-regulated FBPs Pof1p (Fig. 4B). In contrast, Pof8p, 12p, and 13p
showed no detectable interaction with Cul1p (Fig. 4B). Skp1p exhibited a binding pattern
closely resembling that of Cul1p, although a low level of Skp1p was also retrieved in
immunoprecipitates of Pof8p, 12p, and 13p (Fig. 4B), suggesting that these FBPs are
principally capable of interacting with Skp1p. In fact, Skp1p immunoprecipitates
prepared from the same lysates efficiently retrieved all eight FBPs (Fig. 4C). These
results suggested that CSN-insensitive FBPs lacking the conserved proline residue in
their F-boxes are impaired in binding Cul1p, and thus, in forming canonical CRL
complexes.
The conserved proline residue determines CRL1 complex formation
To directly address the role of the proline residue, we constructed a point mutant of the
CSN-regulated Pof1p, exchanging proline 114 for serine (= Pof1p-P114S), which is
8

found in the corresponding position in the F-box of the CSN-independent Pof12p (Fig.
4A). Similarly, we mutated the conserved proline 35 in Pof10p to serine (Pof10p-P35S).
Myc-tagged wild-type and proline mutant FBPs were expressed from pRep81 plasmids
driven by the low strength nmt1 promoter, and binding to endogenous Cul1p was
determined by co-immunoprecipitation. The Pof1p-P114S and Pof10p-P35S mutants
interacted with Cul1p much less efficiently than the respective wild-type proteins (Fig.
5A). For Pof1p-P114S, this deficiency was verified in both wildtype and csn5 mutant
backgrounds, whereas binding of Skp1p was not affected by the proline mutation
(Supplementary Fig. 3).
We next asked whether the proline was sufficient to target an FBP into a CRL1
complex. To this end, we changed serine 15 of the Pof12p F-box to proline and
determined binding to Cul1p. As with endogenous Pof12p (see Fig. 4B), plasmid-derived
wildtype Pof12p was inefficient in binding Cul1p (Fig. 5A). In contrast, prolinecontaining
Pof12p bound Cul1p (Fig. 5A). Thus, the F-box proline residue appears to be
both required and sufficient to target FBPs into canonical CRL1 complexes.
The instability of Pof9p in csn5 mutants (Fig. 2A) and the binding of Pof9p to Cul1p
(Fig. 4B) suggested that its F-box also contains the conserved proline. Indeed, under the
parameters used, the ClustalW algorithm aligned proline 5, a residue near the beginning
of the Pof9p F-box, with the conserved proline of other FBPs (Fig. 4A). The alignment
also highlighted a short insertion following the proline that is shared by human -TRCP
but not by other S. pombe FBPs (Fig. 4A). Remarkably, unlike with Pof1p and Pof10p,
mutation of proline 5 to serine did not reduce the binding of Pof9p to Cul1p but enhanced
it instead (Fig. 5A). Manual editing of the sequence alignment revealed that Pof9p can
9

also be aligned such that it features a serine in the position of the conserved proline, a
maneuver that would place Pof9p into the group of CSN-independent FBPs (designated
Pof9p* at the bottom of Fig. 4A).
To reconcile this apparent paradox, we considered the possibility that Pof9p may be
targeted into CRL1 complexes independently of its F-box motif. Precedence for such a
scenario was previously provided by the S. pombe FBP Pop2p, which can be recruited
into functional CRL1 complexes in an F-box independent manner through dimerization
with another FBP, Pop1p (Seibert et al., 2002). Truncated Pof9p lacking the N-terminal
F-box retained efficient interaction with Cul1p similar in extent to the wildtype and the
proline mutant proteins (Supplementary Fig. 5). In summary, these findings indicated that
Pof9p, unlike most other FBPs, is targeted into CRL1 complexes and subjected to
stability control by the CSN independently of the integrity of its F-box. Nevertheless,
insertion of a serine upstream further enhances Pof9p binding to Cul1p for reasons that
are presently unclear (Fig. 5A).
Proline-dependent regulation of Pof10p stability by the CSN
Our data revealed a strict correlation between the ability of FBPs to form CRL1
complexes and their requirement for CSN to maintain stability. From this correlation, we
predicted that proline mutant FBPs are no longer targets for stability control by CSN. To
critically test this prediction, we replaced the endogenous copy of the non-essential pof10
gene with the proline mutant Pof10p-P35S in both wildtype as well as csn5 mutants, and
determined its stability by CHX chase. Whereas wild-type Pof10p was downregulated
and destabilized in csn5 mutants, Pof10p-P35S was as stable in the mutant as in wild-
10

type cells (Fig. 5B,C). Like plasmid expressed Pof10p-P35S (Fig. 5A), the point mutant
expressed from its endogenous genomic locus in csn5 mutants, was completely deficient
in binding Cul1p, but maintained wild-type levels of interaction with Skp1p (Fig. 5D).
These data establish that proline-dependent recruitment of FBPs into CRL1 complexes
destines FBPs for stabilization by the CSN pathway.
We also created a proline knock-in mutation in the endogenous pof12 gene to
generate a strain that expressed Pof12p-S15P in either a wildtype or csn5 mutant
background. Unlike wildtype Pof12p, Pof12p-S15P expressed from its endogenous
promoter gained the ability to interact with Cul1p, albeit at a low level (Fig. 5E).
Whereas, as with overexpressed Pof12p-S15P (Fig. 5A), knock-in of the F-box proline
was sufficient to target Pof12p into a CRL1 complex, the mutant protein was neither
downregulated nor destabilized in csn5 mutants (Fig. 5E; Supplementary Fig. 6). This
might be due to the low level of recruitment to Cul1p. In addition, the mutant protein,
which is not normally destined for a CRL1 complex, may not bear lysine residues
accessible to autocatalytic modification by its associated Cul1p core complex.
CAND1 is not required for maintaining the stability of CSN-regulated FBPs
CAND1 was proposed to participate in the same process of CRL adapter stabilization as
the CSN (Cope and Deshaies, 2003; He et al., 2005; Min et al., 2005), although no
supporting experimental evidence was provided. To address this proposition in fission
yeast, we turned our attention to the uncharacterized open reading frame SPAC1565.07c,
which we named knd1. This gene encodes a protein with an overall similarity of 43%
(22% identity) with human CAND1 over its entire length of 1220 amino acids. In
11

addition, 25 of the 27 HEAT repeats found in human CAND1, which are responsible for
its hallmark solenoid structure (Goldenberg et al., 2004), are readily predicted from the
primary sequence of S. pombe Knd1p (data not shown). These considerations suggested
that Knd1p is the homolog of CAND1 identified in higher eukaryotes.
This was supported by the finding that Knd1 expressed from a plasmid specifically
interacted with the unneddylated form of Cul1p but not with neddylated Cul1p present in
csn5 mutants (Supplementary Fig. 4). Likewise, Knd1p modified with a single N-
terminal protein A tag at the endogenous genomic locus co-immunoprecipitated the
unneddylated form of Cul1p (Fig. 6A). Importantly, whereas >50% of ProA-Knd1p was
depleted from the cell lysate upon absorption to IgG resin, the bulk of Cul1p was retained
in the lysate, indicating that only a minor fraction of Cul1p was in a stable complex with
Knd1p, at least under steady-state conditions (Fig. 6A).
Like most csn and ubp12 deletions strains (Mundt et al., 2002; Zhou et al., 2001;
Zhou et al., 2003), haploid cells lacking knd1 were viable and did not exhibit any gross
morphological or growth phenotypes (data not shown). Unlike csn mutants, however,
knd1 mutants did not display accumulation of Cul1p in the neddylated state (Fig. 6D).
Nevertheless, since neddylated Cul1p can not interact with CAND1 (Hwang et al., 2003;
Liu et al., 2002; Min et al., 2003; Oshikawa et al., 2003; Zheng et al., 2002a), and since
Cul1p is fully neddylated in csn5 mutants, CSN deficiency might mimic CAND1
deficiency with respect to CRL1 activity
To test this idea, we performed a genetic assay using a strain containing a
temperature-sensitive allele of skp1, which is specifically impaired in binding of FBPs
(Lehmann et al., 2004), but not in binding of Cul1p (Wee et al., 2005). In this strain
12

ackground, we previously demonstrated that CSN becomes essential for viability when
adapter recruitment to CRL core complexes is compromised. Whereas skp1-ts csn5
mutants lost viability upon shift to the restrictive temperature as demonstrated before
(Wee et al., 2005), skp1-ts knd1 double mutants were fully viable at 36.5 0 C (Fig. 6B).
Thus, unlike CSN, Knd1p is dispensable for viability, even when adapter recruitment to
CRL1 core complexes is compromised.
This finding suggested that knd1 mutants do not suffer the same adapter instability as
csn mutants. In fact, downregulation of Pof1p, Pof3p, and Pof7p steady state levels
occurring in csn5 mutants was not observed in knd1 mutants (Fig. 6D, E). In addition, the
stabilities of the CSN-regulated FBPs, Pof1p and Pof10p, as well as the CRL3 adapter
Btb3p were unaffected (Fig. 6C). These findings indicated that CSN maintains CRL
adapter stability independently of CAND1.
CAND1 controls the composition of cellular CRL1 complexes
Since, CAND1 deficiency did not phenocopy CSN deficiency with regards to the control
of FBP levels and stability, we wondered whether it affected the recruitment of FBPs to
Cul1p. Co-immunoprecipitation experiments revealed that binding of Pof1p to Cul1p was
strongly enhanced in a knd1 mutants (Fig. 6D). Consistent with CAND being displaced
from fully neddylated Cul1p, this interaction was also enhanced in a csn5 deletion strain,
in particular when accounting for the low steady-state levels of Pof1p present in this
mutant (Fig. 6D). Whereas the Pof7p-Cul1p interaction was unaffected by the absence of
Knd1p, the Pof3p-Cul1p interaction was substantially decreased (Fig. 6E). Knd1 mutants
carrying C-terminally tagged Pof3p showed the same slow growth and cell elongation
13

phenotype as described for pof3 deletion strains (Katayama et al., 2002), indicating that
they have a defect in SCF Pof3p function (Fig. 6F). These phenotypes were neither
observed in wildtype cells carrying tagged Pof3p nor in knd1 mutants harbouring tagged
Pof7p (Fig. 6F), suggesting that the pof3-13myc allele is partially defective and therefore
requires CAND1 to maintain a sufficient level of activity. Taken together, these findings
show that loss of CAND1 differentially affects the composition of CRL1 complexes,
presumably by allowing increased recruitment of some abundant FBPs such as Pof1p at
the expense of less abundant adapters such as Pof3p.
14

Discussion
F-box directed CRL assembly and control by the CSN
The results of this study add further credence to our model that CSN’s cullin
deneddylation activity revealed in vitro serves to maintain the stability of CRL adapters
thus promoting CRL activity in vivo (Wolf et al., 2003; Zhou et al., 2003). However, not
all FBPs are subject to this mode of regulation, a surprising finding, considering that all
FBPs examined here share a canonical F-box motif and interact with Skp1p (Lehmann et
al., 2004). Yet, the CSN-insensitive FBPs are not efficiently incorporated into CRL1
complexes, because they lack a critical proline residue in their F-boxes, which is required
for binding of Cul1p, but not Skp1p (Fig. 4). This proline is also missing from budding
yeast Rcy1p, its fission yeast ortholog Pof6p, and from human Emi1, all of which were
previously found to bind Skp1p, but not Cul1p (Galan et al., 2001; Hermand, 2006;
Hermand et al., 2003; Seol et al., 2001); and Peter K. Jackson, personal communication).
These findings can be rationalized by the crystal structure of the human CUL1-
RBX1-SKP1-SKP2 complex, which showed that the proline is the only conserved F-box
residue within a cluster of three contiguous amino acids that makes side chain contacts
with CUL1 (Zheng et al., 2002b). Despite its extensive interface with both CUL1 and the
F-box, SKP1 is insufficient to recruit FBPs into CRL complexes; instead, this process is
specified by proline-dependent F-box-CUL1 interactions. An interesting possibility is
that recruitment of FBP-SKP1 dimers to the CRL1 core complex requires the induction
of proline-dependent conformational changes within the N-terminus of CUL1.
15

Based on these data, we propose that all FBPs that lack the critical proline residue are
not engaged in canonical CRL1 complexes and are hence not subject to stabilization by
the CSN. Consistent with this interpretation is our demonstration that mutation of the
conserved proline circumvents the CSN requirement for maintaining Pof10p stability
(Fig. 5C). The same scenario would apply to at least 11 human FBPs that lack the
proline. Conversely, CSN-mediated cullin deneddylation would assist in the assembly of
proline-containing FBPs into CRL1 complexes by shielding them from autocatalytic
inactivation. This mechanism provides a safe environment for the de novo assembly and
maintenance of CRL complexes with labile FBPs (Wolf et al., 2003).
Distinct CAND1 and CSN cycles
CAND1 inhibits CRL activity in vitro (Hwang et al., 2003; Liu et al., 2002; Min et al.,
2003; Oshikawa et al., 2003; Zheng et al., 2002a), but is required for full CRL activity in
vivo (Chuang et al., 2004; Feng et al., 2004; Zheng et al., 2002a). This pattern reiterates
the CSN paradox, and similar resolutions involving adapter stabilization were invoked for
both the CSN and CAND1 paradoxes (Cope and Deshaies, 2003, 2006; He et al., 2005;
Zheng et al., 2002a). This seemed attractive considering that inactivation of CSN is
expected to phenocopy deletion of CAND1.
The results presented here contradict several key prediction of a model of adapter
stabilization involving CAND1: First, only a miniscule fraction of unneddylated Cul1p
was in a stable complex with Knd1p, although the vast majority of Cul1p was in the
unneddylated state (Fig. 6A). Conversely, unneddylated Cul1p readily interacted with
Skp1p and FBPs (Fig. 4B). Both findings are inconsistent with quantitative sequestration
16

of unneddylated Cul1p into inert CAND1 complexes. Secondly, deletion of knd1 did not
interfere with adapter stability (Fig. 6C). Likewise, the FBP COL1 is not stabilized in
plant CAND1 mutants (Feng et al., 2004). Finally, unlike CSN, Knd1p was not rendered
essential when adapter recruitment to CRL1 core complexes was compromised (Fig. 6D).
Based on these findings, we conclude that CSN-dependent adapter stabilization
principally functions independently of CAND1. CSN is sufficient to sustain this mode of
regulation, apparently because the autocatalytic activity of CRL1 complexes is
dominantly suppressed by CUL1 deneddylation.
If CAND1 does not maintain adapter stability, what might its function be in
promoting CRL activity? Our findings suggest a model according to which transient
interaction of CAND1 with unneddylated CUL1-RBX1 core complexes promotes a
continuous cycle of adapter recruitment and displacement (CAND1 cycle, Fig. 7). This
cycle would assure rapid and constitutive turnover of a given CRL complex in the
absence of its substrate. In support of this mechanism are biochemical studies that
demonstrated that human SKP2 can displace CAND1 from CUL1 (Bornstein et al.,
2006). If substrate becomes available, the corresponding CRL-adapter complex would be
removed from the CAND1 cycle by substrate driven CUL1 neddylation and transferred
into an independent CSN cycle (Fig. 7). Once the substrate is consumed, the CRL will be
deneddylated and redirected into the CAND1 cycle.
This model can readily reconcile the biochemical phenotypes of csn and knd1
mutants: Although only a small fraction of Cul1p was engaged in a stable complex with
Knd1p, a situation that is reiterated in A. thaliana (Feng et al., 2004), lack of Knd1p,
nevertheless, caused a substantial increase in the recruitment of the abundant FBP, Pof1p,
17

to Cul1p (Fig. 6B). This constellation can only be explained by a continuous and rapid
adapter exchange cycle driven by transient Knd1p-Cul1p interactions. In the absence of
CAND1, slow exchange would competitively disadvantage relatively rare adapters such
as Pof3p thus disturbing the disposition of a subset of cellular CRL complexes. Indeed, in
A. thaliana, an organism with an extensive FBP repertoire, CRL-dependent auxin
response and photomorphogenesis are compromised in the absence of CAND1, albeit to a
lower extent than in CSN mutants, which are presumed to be broadly deficient in all
CRLs (Chuang et al., 2004; Feng et al., 2004). Likewise, in S. pombe, loss of knd1 does
not show the same synthetic effects with skp1-ts as loss of csn5 (Fig. 6E), again
suggesting that only a subset of CRLs is compromised in knd1 mutants.
The proposition that substrate availability transfers CRLs from the CAND1 to the
CSN cycle is corroborated by the finding that the CRL substrate p27 can drive CUL1
neddylation and CAND1 displacement from SCF SKP2 in vitro (Bornstein et al., 2006).
Likewise, CUL1 and CUL2 neddylation is stimulated by substrate in vivo (Chew and
Hagen, 2007). Substrate-dependent toggling of CRLs between the CAND1 and CSN
cycle thus provides a ready means of driving both the selection of corresponding CRLs
and their recycling.
Role of Ubp12p in CRL regulation
Like Knd1p, the CSN-associated deubiquitylating enzyme Ubp12p was dispensable for
the stability of the FBPs examined here. However, the Cul3p adapter Btb3p is strongly
destabilized in the same ubp12 deleted cells, in which FBPs are largely stable ((Wee et
al., 2005) and Fig. 3). Since we were unable to detect an interaction of Cul3p with Knd1p
18

(data not shown) under conditions at which Knd1p bound Cul1p, CRL3 complexes may
not be subject to the CAND1 cycle. The much reduced adapter repertoire for Cul3p –
only three BTB adapters exist in fission yeast (Geyer et al., 2003) – may alleviate the
need for constitutive exchange. Instead, BTB adapters stably associated with neddylated
Cul3p appear to be protected from autocatalytic turn-over by CSN-bound Ubp12p.
Additionally, Ubp12p may stabilize CRL core components as described for some
organisms (He et al., 2005; Hetfeld et al., 2005; Wu et al., 2005), although we have yet to
detect conditions under which similar regulation would occur in fission yeast.
19

Materials and methods
Yeast strains and plasmids
The knd1 and ubp1 deletion strains and the epitope-tagged strains were constructed by
one-step gene replacement using PCR-generated fragments containing ura4 or
kanamycin cassettes (Bahler et al., 1998). The ubp1 ubp12 double mutant was obtained
by mating and confirmed by PCR. Strains containing Myc-tagged FBPs were provided by
T. Toda and crossed into our wild-type background and into csn5 mutants. The skp1-ts
knd1 mutant was created by mating, followed by verification of the recombinants by
colony PCR. The strain was assayed for synthetic phenotypes by spotting serial dilutions
exactly as described (Wee et al., 2005). N-terminally protein A tagged knd1 was
constructed following a published protocol (Werler et al., 2003).
A pof10 disruptant was constructed by one-step gene replacement using a PCRgenerated
fragment containing the ura4 cassette and flanking regions thus inserting the
ura4 cassette between codon 31 and 636 of the pof10 open reading frame. The pof10
disruptant was transformed with a PCR-generated fragment consisting of the coding
sequence of full length pof10 carrying a mutation of serine 35 to proline. Transformants
were grown in 10 ml liquid YES at 30 o C for 22 hours. Ten 10-fold serial dilutions of the
liquid culture with fresh YES were prepared and 100µl of each dilution was plated on
YES plates containing 0.1% 5-fluororotic acid (5-FOA). After 3-5 days, colonies growing
on YES + 5-FOA were confirmed as containing the pof10-P35S mutation by colony-PCR
and sequencing. Subsequently, the point mutant pof10 locus was modified with a 13Myc
20

tag. The pof10-P35S Δcsn5 mutant was created by mating. The pof12-S15P and pof12-
S15P csn5 mutants were created in an identical manner.
The plasmids expressing Csn5p JAMM mutants were described previously (Wee et
al., 2005). FBP plasmids were prepared by amplifying the respective ORFs from
Schizosaccharomyces pombe complementary DNA. PCR products were sequenced and
then cloned into pRep3 plasmids, which drive the expression of amino-terminally Mycepitope-tagged
proteins from the thiamine-repressible nmt1 promoter. FBP point mutants
were constructed with the QuickChange site-directed mutagenesis method (Stratagene)
and verified by sequencing.
Immunological methods
Epitope-tagged proteins were detected by the monoclonal anti-Myc antibody 9E10 or
with monoclonal anti-Protein A antibodies (Sigma). Cell lysates for immunoprecipitation
were prepared as described in a buffer containing 50 mM Tris, pH 7.4, 50 mM NaCl, and
0.5% Triton X-100 (Zhou et al., 2001). Lysates were cleared by centrifugation, and
proteins were precipitated with the respective antisera. Immunocomplexes were collected
by binding to protein A beads, washed, and analyzed by immunoblotting as described
(Zhou et al., 2001). Protein A-tagged proteins were precipitated using whole rabbit
immunoglobulin absorbed to Dynabeads. Affinity-purified rabbit antisera against Cul1p,
Skp1p, and Rbx1p were described before (Geyer et al., 2003; Seibert et al., 2002). For
loading controls Cdc2 (PSTAIR, Santa Cruz) antibodies were used at a dilution of 1:500.
21

Cycloheximide chase experiments
To measure the stability of adapter proteins, strains containing epitope-tagged versions of
FBPs and Btb3p were grown to an OD 600 of 1.0 in 50 ml YES media. 100 ug/ml
cycloheximide (CHX) was added, and cultures were incubated at 25 o C. 10 ml aliquots
removed after the times indicated in the figures. NaN3 was added to a final concentration
of 1 mM, in order to instantly kill the cells. Cell were harvested by centrifugation and
frozen in liquid nitrogen. Once all samples of the chase period were obtained, cell lysates
were prepared by standard bead lysis, and equal amounts of proteins were analyzed by
immunoblotting.
CHX chase experiments with strains containing the temperature-sensitive mts3-1
allele were performed in the same way, except that strains were maintained at 37 o C for 90
minutes prior to the addition of CHX.
RT/PCR
Total cellular RNA was isolated by cell lysis in hot RNAzol (TelTest, Inc.) and three
cycles each of vortexing in the presence of beads for 5 minutes and heating to 65 o C for 5
minutes. The RNA was extracted and precipitated according to the recommendations of
the manufacturer. 2µg RNA was used in each RT/PCR reaction. Primer design and RT-
PCR conditions were according to the manual supplied with the Platinum Quantitative
RT-PCR Thermoscript kit (Invitrogen). The primer concentration for actin amplification
was 0.25 µM. Other primers were used at 1 µM. Pof13 expression was assayed using a
iQ Multiplex Powermix enzyme kit (BioRad) following the supplied protocol. Triplicate
reactions were analyzed with a iCycler iQ PCR detection system (BioRad).
22

Acknowledgements
We are grateful to T. Toda for providing strains expressing tagged FBPs and to R. J.
Deshaies for JAMM mutants. We wish to thank C. Hoffman for help with the creation of
the pof1-P35S mutant. M.W.S is grateful to D. H. Wolf (University of Stuttgart) for
support. We thank M. Petroski for helpful comments on the manuscript. This study was
funded by NIH grant GM59780 to D.A.W.
23

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27

Figure legends
Fig. 1 Differential effect of CSN on FBP levels
(A) Steady-state levels of Myc-tagged FBPs in csn5 deletion strains. Cdc2p and Cul1p
are shown for reference in the lower panels. The mobility differences of Cul1p reflect the
loss of deneddylation activity in csn5 mutants.
(B) Complementation of Pof10p-Myc levels. The indicated csn4 and csn5 deletion strains
were transformed with plasmids driving the expression of Myc-tagged Csn4p or Csn5p,
and Pof10p-Myc levels were determined by immunoblotting. Two independently derived
strains are shown in the right panel.
(C) Dependence of the rescue of Pof10p-Myc levels on the JAMM motif of Csn5p. Myctagged
wild-type Csn5p or the JAMM point mutant Csn5p-H118A were expressed in
csn5 deletion strains, and the level of Pof10p-Myc was determined by immunoblotting.
Two independently derived strains are shown.
Fig. 2 Stability of FBPs in csn5 mutants
(A). Strains expressing the indicated Myc-tagged FBPs in a wild-type or csn5 mutant
background were employed in cycloheximide (CHX) chase experiments to determine
their stability (see Materials and methods). Note that exposure times were adjusted such
that comparable base line signals of the FBPs in wildtype and csn5 mutant cells were
obtained for improved comparison.
(B) The levels of FBPs remaining in wild-type and csn5 mutants upon CHX chase were
quantified. Quantification was done using IMAGEJ v1.40f analysis software. Mean
28

intensities were background subtracted and normalized to the loading control Cdc2 (blots
not shown).
(C) Strains expressing the indicated Myc-tagged FBPs in a csn5 or csn5 mts3-1 mutant
background were employed in cycloheximide (CHX) chase experiments. Note that
loading and exposure times were adjusted such that comparable base line signals of the
FBPs in csn5 and csn5 mts3-1 mutant cells were obtained for improved comparison.
(D) The levels of FBPs remaining in csn5 and csn5 mts3-1 mutants upon CHX chase
were quantified. Quantification was done using IMAGEJ analysis software. Mean
intensities were background subtracted and normalized to the loading control Cdc2 (blots
not shown).
Fig. 3 Ubp12p maintains the stability of the CRL3 adapter Btb3p but not
FBPs
(A) Steady-state levels of the indicated CSN-sensitive FBPs in csn5 and ubp12 mutants.
(B) A strain coexpressing protein A-tagged Btb3p and Myc-tagged Pof1p from their
respective endogenous genomic loci was employed in a CHX chase experiment to
determine the stability of the adapters.
(C) Pof1p-Myc levels were determined in ubp1 and ubp12 single mutants, and in ubp1
ubp12 double mutants.
29

Fig. 4 CSN-insensitive FBPs are less efficient in forming CRL complexes
(A) CLUSTALW alignment of the F-boxes of the indicated proteins. The conserved
proline residue is highlighted by an arrow. Two different alignments are shown for Pof9p
(stippled green and red boxes). Pof9p* denotes an alignment according to which the
Pof9p F-box does not contain the conserved proline. The proline containing FBPs
examined in this study are highlighted in a green box, the proline-less FBPs are in a red
box.
(B) Interaction of FBPs with Cul1p and Skp1p. The indicated Myc-tagged FBPs were
immunoprecipitated with Myc antibodies, followed by immunoblotting with Cul1p and
Skp1 sera. The negative control (denoted “--“) represents cell lysate from a strain not
expressing any Myc-tagged proteins. Total cell lysates are shown in the bottom panel.
(C) The same lysates as in (B) were subjected to immunoprecipitation with Skp1p
antisera, followed by immunoblotting with Myc antibodies to detect co-purification of
Myc-tagged FBPs. Total cell lysates are shown in the bottom panel.
Fig. 5 The conserved proline residue determines FBP binding to Cul1p and
regulation by the CSN
(A) Interaction of plasmid derived wildtype and proline mutant FBPs with Cul1p. Myctagged
FBPs were immunoprecipitated and copurification of Cul1p was assayed by
immunoblotting. “C” denotes a specificity control (lysate from untransformed cells).
(B) Steady state levels of endogenously Myc-tagged wildtype Pof10p-Myc and Pof10p-
P35S-Myc in csn5 mutants and in wildtype cells. Note that, in contrast to wildtype
30

Pof10p, Pof10p-P35S is not downregulated in csn5 mutants. Cul1p and Cdc2p are shown
for reference. The asterisk denotes an unspecific cross reactivity of the Cul1p sera.
(C) The stabilities of Pof10p and Pof10p-P35S in wildtype (top panel) csn5 mutants
(bottom panel) were determined by CHX chase.
(D) Interaction of Pof10p and Pof10p-P35S with Cul1p and Skp1p. Lysates from
wildtype and csn5 mutant cells harbouring endogenously Myc-tagged Pof10p and
Pof10p-P35S were immunoprecipitated with Myc antibodies followed by
immunoblotting with Cul1p, Skp1p, and Myc antisera. Whereas Pof10p-P35S is deficient
in binding of Cul1p, its interaction with Skp1p is maintained.
(E) Interaction of Pof12p and Pof12p-S15P with Cul1p. Lysates from wildtype and csn5
mutant cells harbouring endogenously Myc-tagged Pof12p and Pof12p-S15P were
immunoprecipitated with Myc antibodies followed by immunoblotting with Cul1p and
Myc antisera. Whereas wildtype Pof12p is deficient in binding of Cul1p, the mutant
Pof12p-S15P shows binding (strong exposure, second panel from top). The Pof10p-
Cul1p interaction is shown for reference (weak and strong exposures, top two panels).
The asterisk denotes an unspecific cross reactivity of the Cul1p sera.
Fig. 6 CAND1/Knd1p does not maintain FBP stability but regulates cellular
CRL composition
(A) Lysate from a strain expressing Knd1p modified with a single N-terminal protein A
tag at the endogenous genomic locus was absorbed to IgG beads, followed by
immunoblotting with Cul1p sera. Total cell lysates before and after chromatography on
IgG resin are shown on the left to indicate the extent of the Knd1p-Cul1p interaction.
31

(B) Genetic interactions of knd1 and csn5 with the temperature-sensitive allele skp1-A7.
Serial dilutions of the indicated strains were spotted onto YES plates and incubated at the
indicated temperatures. Whereas csn5 shows synthetic interaction with skp1-A7, knd1
does not.
(C) The stability of Pof1p-Myc, Pof10p-Myc, and Btb3p-proA in knd1 mutants was
determined by CHX chase. Cdc2p is shown for reference.
(D) Binding of Pof1p to Cul1p in csn5 and knd1 mutants. Lysates from csn5 or knd1
mutant cells harbouring endogenously Myc-tagged Pof1p were immunoprecipitated with
Myc antibodies followed by immunoblotting with Cul1l antibodies. Duplicate
experiments are shown. Csn5 and knd1 mutants not containing Pof1p-Myc are shown as
negative controls. The asterisk in the Cul1p blot denotes an unspecific band.
(E) Binding of Pof3p and Pof7p to Cul1p in knd1 mutants. The experiment was
performed as described in (D). Pof10p was included as reference for an abundant FBP.
(F) Cell elongation phenotype of pof3-13myc knd1 cells. The indicated strains were
grown in liquid YES, fixed with formaldehyde, and visualized by digital interference
contrast microscopy. The table shows the doubling times (T d ) determined in mid log
phase.
Fig. 7 Model for toggling of CRL complexes between the CAND1 and CSN
cycles
In the absence of substrate, CRL complexes undergo continuous and rapid adapter
exchange in the CAND1 cycle (left). Upon substrate-induced neddylation, CRLs
32

transition into the CSN cycle (right). Once substrate is consumed, CSN-mediated
deneddylation toggles CRLs back into the CAND1 cycle for maintenance.
33

Figure 1
A
Pof 1 3 7 8 9 10 12 13
∆csn5 + + + + + + + +
wt + + + + + + + +
FBPs
kDa
124
84
Cul1p-Ned8p
Cul1p
Cdc2p
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
84
26
B
Pof10p
wt
∆csn4
∆csn5
∆csn4 csn5
∆csn4
pRep3.myc-csn5
∆csn5
∆csn4
csn5
kDa
124
C
wt ∆csn5
pREP3.myc-csn5 + +
pREP3.myc-csn5-H118A + +
Pof10p
Myc-Csn5p
kDa
124
52
Myc-Csn5p
Cdc2p
52
27
Cul1p-Ned8p
Cul1p
Cdc2p
84
27

Figure 2
A
CHX (min)
Pof1p
Pof3p
Pof7p
Pof8p
Pof9p
Pof10p
wt
∆csn5
0 20 40 60 90 0 20 40 60 90
B
1.5
1
0.5
0
1.5
1
0.5
0
1.5
1
0.5
0
Pof1p
0 50 100
Pof7p
0 50 100
Pof9p
0 50 100
1.5
1
0.5
0
1.5
1
0.5
0
1.5
1
0.5
0
Pof3p
0 50 100
Pof8p
0 50 100
Pof10p
0 50 100
Pof12p
Pof13p
2
1.5
1
0.5
0
Pof12p
0 50 100
1.5
1
0.5
0
Pof13p
0 50 100
min
C
CHX (min)
Pof1p
∆csn5
∆csn5 mts3-1
0 20 40 60 90 0 20 40 60 90
1.2
1
0.8
0.6
0.4
0.2
0
Pof1p
0 50 100
1.2
1
0.8
0.6
0.4
0.2
0
wt
Pof9p
∆csn5
0 50 100
Pof9p
Pof10p
1.2
1
0.8
0.6
0.4
0.2
0
Pof10p
0 50 100
∆csn5
∆csn5 mts3-1

Figure 3
A
wt
∆csn5
∆ubp12
B
CHX (min): 0 30 60 90 120 0 30 60 90 120
Btb3p
wt
∆ubp12
kDa
83
Pof1p
Pof1p
83
Pof3p
Cdc2p
Pof9p
Pof10p
C
∆ubp12
∆ubp1
wt
Cul1p- Ned8p
Cul1p
Pof1p
Cdc2p
Cdc2p

Figure 4
A
Pof1p
Pof3p
Pof7p
Pof10p
Pof9p
Pof2p
Pof5p
Pof11p
Pop1p
Pop2p
APB1A10
Fdh1p
Skp2
bTRCP
Cdc4p
Pof8p
Pof12p
Pof13p
Pof6p
Pof9p*
LPV-----EISFRILS-FL----DARSLCQAAQVSKHWKE-LA-DDDVIWHRM
LPR-----EVLLCILQ-QL----NFKSIVQCMQVCKHWRDCIK-KEPSLFCCL
LPD-----EVLLVILENCIRDLHDLRYLSSIALTCKHFAKALR-ADS-LYRSF
LPK-----EILIIIFS-FL----DPRSLLSAQCTCKYWKK-LL-SDDLSWRTA
SPFLELSYDILLEIST-YL----DYKDIVHLSETCKSLSYVFD--DKTIWHRF
VPN-----EVCFNILS-YL----EADELRCKSTVCTSWRNFII---PTLWEKV
FPP-----EIWHHIFDHLIS--FDKFEAKNFGGLLRICRSSYVGGLHAIYYFP
FPE-----EVSLRVFS-YL----DQLDLCKCKLMSKRWKR-LL-EDPGIWKAL
FPA-----EITNLVLT-HL----DAPSLCAVSQVSHHWYKLVS-SNEELWKSL
LPF-----SIVQSILL-NL----DIHSFLSCRLVSPTWNRILD-VHTSYWKHM
LPV-----EVIDSVMQ-YL----PAHDVIQSSFASYPLTL-IA--NKIIRARL
LPL-----EIIPLICR-FL----SVQDIQSFIRVFPSFQTILDSSNDLFWKK-
LPD-----ELLLGIFS-CL----CLPELLKVSGVCKRWYR-LA-SDESLWQTL
LPARGL-DHIAENILS-YL----DAKSLCAAELVCKEWYR-VT-SDGMLWKKL
LPF-----EISLKIFN-YL----QFEDIINSLGVSQNWNKIIR-KSTSLWKKL
LNP-----DFLSAVDS-IL----EIYFHRERQKEKVHLAFLIQ--QDDFWKGI
FSH-----ETLLHVLN-DL----SAHDLAALERVSRSWNSIVR--RSSVWHNL
LNR-----DIWSLIIN-YL----DAFDILRLMHSSRQFYYWLR--KSAVDECC
LTI-----NIYLKIFT-LI----STPDLCNCRLVCRKFQQLCD--YNSIYVKK
LSY-----DILLEIST-YL----DYKDIVHLSETCKSLSYVFD--DKTIWHRF
B
IP: α-Myc
Pof 1 3 7 8 9 10 12 13 --
kDa
185
C
Pof
Skp1p
IP: α-Skp1
1 3 7 8 9 10 12 13
kDa
18
FBPs
124
84
185
Cul1p-Ned8p
Cul1p
Skp1p
84
18
FBPs
124
84
Cul1p-Ned8p
Cul1p
Skp1p
Lysate
84
18
FBPs
Lysate
124
84
IB: myc

Figure 5
A
IP: α-Myc
Myc-Pof
1 9 12 10
Cul1p
Pof1p
Pof9p
PS
PS
SP
PS
C
Pof10p
IgH
Pof12p
B
pof10-P35S-13myc
pof10-13myc
Pof10p
Cul1p-Ned8p
Cul1p
wt
∆csn5
kDa
130
95
130
95
D
pof10-P35S-13myc
pof10-13myc
IP: α-Myc
Pof10p 130
Cul1p-Ned8p
Cul1p
wt
∆csn5
kDa
95
Lysate
Cul1p
Cdc2p
34
Skp1p
17
C
wt
CHX (min)
Pof10p
Cdc2p
pof10-13myc
pof10-P35S-13myc
0 20 40 60 90 0 20 40 60 90
kDa
130
34
E
wt
∆csn5
Cul1p-Ned8p
Cul1p
pof12-13myc
pof12-S15P-13myc
pof10-13myc
pof10-P35S-13myc
kDa
95
72
short
∆csn5
Pof10p
130
IP: α-Myc
Cul1p-Ned8p
Cul1p
Pof10p
Pof12p
95
72
130
95
72
long
Cdc2p
34
Lysate
Cul1p-Ned8p
Cul1p
95
72

Figure 6
A
C
post IP
pre IP
Knd1p
Cul1p-Ned8p
Cul1p
CHX (min)
Btb3p
Pof1p
knd1-proA
Lysate
wt
No tag
IP:
α-ProA
csn5
knd1-proA
No tag
Lysate
∆knd1
wt
kDa
182
124
80
0 20 40 60 120 0 20 40 60 120 kDA
84
84
B
D
∆csn5
∆knd1
skp1-A7
skp1-A7 ∆csn5
skp1-A7 ∆knd1
IP: α-Myc
∆knd1
∆csn5
wt
Pof1p
Cul1p-Ned8p
Cul1p
25 ºC 36.5 ºC
pof1-13myc
No tag
Pof10p
Cdc2p
124
25
Lysate
Pof1p
Cul1p-Ned8p
Cul1p
E F wt pof3-13myc
IP:
α-Myc
∆knd1
Cul1p-Ned8p
Cul1p
Pof10p
Pof3p
Pof7p
wt
pof3-13myc
pof7-13myc
pof10-13myc
kDa
170
130
95
72
170
130
95
72
55
Strain
wt
pof3-13myc
pof3-13myc knd1
pof7-13myc
pof7-13myc knd1
T d (min)
129
146
175
146
144
pof7-13myc
pof3-13myc
∆knd1
pof7-13myc
∆knd1

Figure 7
S
Rbx1
FBP
Skp1 P
FBP
Skp1 P
Rbx1
N8
S
FBP
Skp1 P N8Rbx1
UUUUU
Cul1
Cul1
Cul1
CSN
CAND1 Cycle
CSN Cycle

F) Supplemental Text and Figures
Supplement to:
F-Box-directed CRL complex assembly and regulation by the CSN and
CAND1
Michael W. Schmidt, Philip R. McQuary, Susan Wee, Kay Hofmann,
Dieter A. Wolf
Supplementary Figure 1
Effect of csn deletions on Pof1p and Pof10p levels
The indicated wild-type and csn deletion strains harboring Myc-tagged Pof1p or Pof10p
were examined for FBP expression by immunoblotting with Myc antibodies. Two
independently derived strains are shown for each mutant. Note that Pof1p is known to
occasionally resolve into duplet bands on SDS gels (Harrison et al., 2005). The exact
nature of these forms remains to be determined.
wt
∆csn3 ∆csn4 ∆csn5
Pof1p
kDA
84kDa
Cdc2p
27kDa
Pof10p
124kDa
Cdc2p
1
2 3 4 5 6 7 8
27kDa

Supplementary Figure 2
Steady-state mRNA levels of (A) pof1 and pof10, and (B) pof13 in wildtype cells and in
csn5 mutants. mRNA levels were determined by semiquantitaive RT-PCR (A) or by
quantitative real time RT-PCR (B).
A
wt wt ∆csn5
# cycles: 30 30 24 26 28 30 24 26 28 30
Actin
pof1
Actin
pof10
B
100%
% pof13 mRNA
80%
60%
40%
20%
0%
wt
?csn5

Supplementary Figure 3
Accumulation of Myc-tagged FBPs in the mts3-1 proteasome mutant strain. Csn5 and
csn5 mts3-1 strains harboring Myc-tagged alleles of Pof1p, Pof9p, or Pof10p were
maintained at 37 o C for 90 min. The expression of FBPs was determined by
immunoblotting with Myc antibodies. Only 1/10 of cell lysate was loaded for mts3-1
strains. The high molecular weight smears likely represent polyubiquitylated FBP
species.
∆csn5
mts3-1
Pof10p
Pof9p
kDa
170
130
Pof1p
72
55

Supplementary Figure 4
Interaction of plasmid derived wild-type and proline mutant Pof1p with Cul1p and Skp1p
in wildtype cells and in csn5 mutants. Myc-tagged FBPs were immunoprecipitated and
copurification of Cul1p and Skp1p was assayed by immunoblotting. As noted in the
legend to Supplementary Fig. 1, Pof1p occasionally resolves into a duplet, probably due
to unknown posttranslational modification. “C” denotes a specificity control (cell lysate
from untransformed cells).
pREP81.pof1
Pof1p
wt ∆csn5
wt ∆csn5
wt PS wt PS C wt PS wt PS C
Cul1p-Ned8p
Cul1p
Skp1p
Lysate
IP

Supplementary Figure 5
The indicated variants of Pof9p were expressed as Myc-tagged proteins from pRep81
plasmids and binding to Cul1p was assessed by immunoprecipitation with Rbx1p and
Myc antibodies. “C1, C2, C3” denote specificity controls (cell lysate from untransformed
cells). The asterisk denotes an unspecific band.
Pof9p
Pof9p-PS
Pof9p-∆F
Cul1p-Ned8p
Cul1p
Lysate
Pof9
Pof9-PS
Pof9-∆F
IP:Myc
Pof9
Pof9-PS
Pof9-∆F
IP:Rbx1p
C1 C2 C3
Pof9
Pof9-PS
Pof9-∆F
Pof9p variants
1 2 3 4 5 6 7 8 9 10 11 12

Supplementary Figure 6
Myc epitope-tagged alleles of Pof12p and Pof12p-S15P were created by homologous
recombination and crossed into a csn5 mutant background. The stability of Pof12p and
Pof12p-S15P in wildtype (wt) csn5 mutants were determined by CHX chase. Cul1p and
Cdc2p blots are shown for reference. The asterisk denotes an unspecific band.
pof12-13myc
pof12-S15P-13myc
CHX (min)
Pof12p
Cul1p-Ned8p
Cul1p
Cdc2p
wt ∆csn5 wt ∆csn5
0 20 40 60 90 0 20 40 60 90
0 20 40 60 90 0 20 40 60 90
kDa
95
72
95
72
36
28

Supplementary Figure 7
Interaction of Knd1p with Cul1p. Myc-tagged Knd1p was expressed in wildtype cells
(wt) and in csn5 mutants from a thiamine repressible pRep81 plasmid. Cells were kept in
the presence (promoter off) or absence (promoter on) of thiamine for 20 h followed by
preparation of cell lysate. Cell lysates were subjected to immunoprecipitation with Myc
antibodies, and co-purifying Cul1p was revealed by immunoblotting with Cul1p antisera.
Lysates prepared from cells not expressing Myc-Knd1p (promoter off) are shown as
negative controls. Note than only the unneddylated form of Cul1p present in wildtype
cells bound to Knd1p. The asterisk denotes an unspecific band.
Lysate IP: α-Myc
wt ∆csn5 wt ∆csn5
pRep81.myc-knd1 off on off on off on off on
Cul1p-Ned8p
Cul1p
Myc-Knd1p
References
Harrison, C., Katayama, S., Dhut, S., Chen, D., Jones, N., Bahler, J., and Toda, T. (2005).
SCF(Pof1)-ubiquitin and its target Zip1 transcription factor mediate cadmium response in
fission yeast. Embo J 24, 599-610.