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 ().
Differential effect of CSN on FBP levels
The levels of the CSN-regulated Pof1p and Pof10p were also diminished in csn3
mutants (, Supplementary Fig. 1
). Conversely, downregulation of Pof10p in csn5
mutants was complemented by providing wild-type csn5
from a plasmid (). This rescue failed in csn4 csn5
double mutants (). 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 (). These results suggested that the cullin deneddylation function of the entire CSN complex is required to maintain the steady-state expression levels of CSN-sensitive FBPs.
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 CSN-sensitive 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 (). Destabilization of Pof1p, Pof9p, and Pof10p did not occur in csn5
mutants that carried the temperature-sensitive mts3-1
allele (Gordon et al., 1996
), which encodes a mutant version of the proteasome subunit Rpn12p (). 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 () despite the decrease in Pof13p steady-state levels apparent in . 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
Stability of FBPs in csn5 mutants
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 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 (), indicating that Ubp12p is not a major regulator of these adapters.
Ubp12p maintains the stability of the CRL3 adapter Btb3p but not FBPs
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 ().
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
single mutants and in ubp1 ubp12
double mutants. Pof1p levels were unchanged in either mutant (). 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 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 β-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 (). 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.
CSN-insensitive FBPs are less efficient in forming CRL complexes
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 (). In contrast, Pof8p, 12p, and 13p showed no detectable interaction with Cul1p (). 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 (), 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 (). 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 found in the corresponding position in the F-box of the CSN-independent Pof12p (). 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 (). 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
The conserved proline residue determines FBP binding to Cul1p and regulation by the CSN
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 ), plasmid-derived wildtype Pof12p was inefficient in binding Cul1p (). In contrast, proline-containing Pof12p-S15P bound Cul1p (). 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 () and the binding of Pof9p to Cul1p () 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 (). The alignment also highlighted a short insertion following the proline that is shared by human β-TRCP but not by other S. pombe FBPs (). 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 (). Manual editing of the sequence alignment revealed that Pof9p can 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 ).
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 of the F-box further enhances Pof9p binding to Cul1p for reasons that are presently unclear ().
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-type cells (). Like plasmid expressed Pof10p-P35S (), 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 (). 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 (). Whereas, as with overexpressed Pof12p-S15P (), 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 (; 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 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 (). 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 ().
CAND1/Knd1p does not maintain FBP stability but regulates cellular CRL composition
Like most csn
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 (). 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 background, 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 °C (). 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 (). In addition, the stabilities of the CSN-regulated FBPs, Pof1p and Pof10p, as well as the CRL3 adapter Btb3p were unaffected (). 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 (). Consistent with CAND1 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 (). Whereas the Pof7p-Cul1p interaction was unaffected by the absence of Knd1p, the Pof3p-Cul1p interaction was substantially decreased (). Knd1
mutants carrying C-terminally tagged Pof3p showed the same slow growth and cell elongation phenotype as described for pof3
deletion strains (Katayama et al., 2002
), indicating that they have a defect in SCFPof3p
function (). These phenotypes were neither observed in wildtype cells carrying tagged Pof3p nor in knd1
mutants harbouring tagged Pof7p (), 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.