APC/C functions by assembling K11-linked ubiquitin chains
To determine the topology of the ubiquitin chains that mediate functions of the human APC/C, we tested recombinant ubiquitin mutants in in vitro assays recapitulating APC/C-activity. We employed mutants that had a single lysine replaced with arginine, such as ubiquitin-K48R (ubi-R48). Alternatively, all lysine residues were mutated except for one, as in ubiquitin with K48 as its only lysine (ubi-K48). Together, these mutants allowed us to assess whether chains of a specific topology are required or sufficient for APC/C-functions.
We first assayed the ubiquitin mutants for their capacity to support the degradation of a mitotic APC/C-substrate, cyclin B1. Addition of UbcH10 and p31comet
to extracts of mitotic cells with an activated spindle checkpoint (CP-extracts) triggers the APC/C-dependent disassembly of Cdc20/Mad2 complexes (Reddy et al., 2007
; Stegmeier et al., 2007
). This leads to full activation of APC/CCdc20
and, consequently, cyclin B1 ubiquitination and degradation. As reported previously, cyclin B1 is efficiently degraded in UbcH10/p31comet
-treated CP-extracts containing wt-ubiquitin (). Strikingly, cyclin B1 is also turned over in a proteasome-dependent manner, when CP-extracts are supplemented with a ubiquitin mutant that has K11 as its only lysine (ubi-K11; ). By contrast, mutation of K11 of ubiquitin (ubi-R11) interferes with cyclin B1-degradation, and also with disassembly of Cdc20/Mad2-complexes (, Supp. Fig. 1A
). No single-lysine ubiquitin mutant other than ubi-K11, including ubi-K48, supports degradation of cyclin B1, while no mutation other than that of K11 stabilizes cyclin B1. These results suggest that in CP-extracts APC/CCdc20
achieves cyclin B1-degradation by decorating it with K11-linked chains.
K11-linked ubiquitin chains mediate APC/C-functions
From anaphase until late in G1, Cdc20 is replaced by a homologous co-activator, Cdh1 (Peters, 2006
). To determine whether the co-activator or cell cycle stage influence the topology of APC/C-dependent chains, we tested our ubiquitin mutants in degradation assays using extracts with active APC/CCdh1
. Consistent with our experiments in mitotic extracts, the APC/C-substrate securin is rapidly degraded by the 26S proteasome in G1-extracts supplemented with ubi-K11, but it is stabilized if K11 of ubiquitin is absent, such as in ubi-R11 or methyl-ubiquitin (; Supp. Fig. 1B
). No single-lysine mutant other than ubi-K11 fully supports the degradation of securin in G1-extracts. Ubi-K11 allows the degradation of multiple APC/C-substrates (Supp. Fig. 1C
) in extracts prepared from cells in G1 or in quiescence, when APC/CCdh1
is also active (Supp. Fig. 1D
). By contrast, inhibiting the formation of K11-linked chains does not impair the ubiquitination or degradation of the SCF-substrate Emi1 (Supp. Fig. 1E
). These findings provide evidence that in extracts both APC/CCdc20
function by decorating substrates with K11-linked chains.
To determine the importance of K11-linked chains in mediating APC/C-functions in vivo, we overexpressed ubi-R11 in human cells or injected recombinant ubi-R11 into Xenopus tropicalis embryos at the two cell stage. The overexpression of ubi-R11 in human 293T cells impedes the Cdh1-dependent degradation of the APC/C-substrates geminin, Plk1, and securinΔD (). Moreover, injection of ubi-R11 into X. tropicalis embryos delays early cell divisions and results in death of injected embryos before gastrulation (). These phenotypes are less dramatic, but similar to those observed after injection of a dominant-negative mutant of the APC/C-specific E2, UbcH10C114S. By contrast, overexpression or injection of wild-type ubiquitin does not affect the degradation of APC/C-substrates, progression through the cell cycle, or development of embryos. Thus, interfering with the formation of K11-linked chains stabilizes APC/C-substrates and impairs cell cycle progression and development in vivo, attesting to the importance of K11-linked chains for APC/C-activity.
UbcH10 provides specificity for the assembly of K11-linked chains
E2s often contribute to the specificity of ubiquitin chain formation (Dye and Schulman, 2007
). Human APC/C has been reported to cooperate with three E2s, the specific UbcH10 and the more promiscuous UbcH5 and E2-25K. To dissect the mechanism underlying the formation of K11-linked chains, we purified these E2s and tested their specificity in APC/CCdh1
-dependent chain assembly. Strikingly, APC/CCdh1
and its specific E2 UbcH10 form long ubiquitin chains only in the presence of ubi-K11, but not with other single-lysine mutants. The same strong preference for formation of K11-linked chains is observed with the distributive substrate cyclin A (), the processive substrate securin (), and for UbcH10-autoubiquitination (). The mutation of K11 in ubiquitin delays chain formation by APC/CCdh1
and UbcH10 (). Furthermore, as shown below, ubiquitin chains assembled by APC/CCdh1
and UbcH10 using ubi-R11 are not efficiently recognized by proteasomal receptors. These results indicate that UbcH10 endows the APC/C with specificity for assembling functional K11-linked chains.
APC/CCdh1 and UbcH10 preferentially assemble K11-linked chains in vitro
In contrast to UbcH10, UbcH5a and UbcH5c can use ubi-K11, ubi-K48, and ubi-K63 to catalyze the ubiquitination of APC/CCdh1
-substrates (; data not shown), and thus, allow the formation of chains linked through lysine residues other than K11. E2-25K assembles chains very inefficiently, and earlier experiments indicated that these chains are linked through K48 (Supp. Fig. 2A, B
; Rodrigo-Brenni and Morgan, 2007
). Consistent with the importance of K11-linked chains for APC/C-activity, the specific UbcH10 is more potent in promoting the degradation of the APC/CCdh1
in G1-extracts than UbcH5 or E2-25K, as observed over a wide range of E2 concentrations (). These results further suggest that UbcH10, but not UbcH5 or E2-25K, provide the APC/C with specificity for assembling functional K11-linked chains.
To determine the molecular basis underlying the specificity of UbcH10, we compared mutants of UbcH10 and UbcH5 in APC/C-dependent assays. The interaction of E2s with the RING-finger of E3s requires an aromatic side chain in loop 1 of the E2 (Zheng et al., 2000
). As expected, mutation of the respective residue in UbcH10 and UbcH5, UbcH10Y91D
, inactivates both E2s in degradation and ubiquitination assays dependent on APC/CCdh1
(Supp. Fig. 2C–E
). When added to G1-extracts, UbcH10Y91A
, but not UbcH5cF62D
, impair degradation of the APC/C-substrate securin, and thus, behave as dominant negative mutants (Supp. Fig. 2D
). Consistent with this observation in extracts, injection of UbcH10Y91D
into X. tropicalis
embryos delays cell cycle progression (Supp. Fig. 2F
does not interfere with proteasomal degradation, as the SCF-substrate Emi1 is ubiquitinated and degraded in its presence (Supp. Fig. 2G
). Therefore, despite a defective RING-finger-interaction, UbcH10Y91D
can bind the APC/C and compete with endogenous E2s in the extracts, suggesting that UbcH10 contains additional APC/C-binding motifs.
A likely candidate for a second APC/C-binding site in UbcH10 is helix-1 of its UBC-domain, which in other E2s participates in E3-binding (Reverter and Lima, 2005
; Zheng et al., 2000
) and is not conserved between UbcH10 and UbcH5. Indeed, mutations in or close to helix-1 (UbcH10K33D
) significantly reduce the activity of UbcH10 in degradation and ubiquitination assays (Supp. Fig. 2H, I
). In contrast to UbcH10Y91D
do not act as dominant-negatives, indicating their binding to APC/C is disturbed. UbcH10K33D
is also less efficiently charged by E1, which is consistent with findings that E1 and E3-binding sites in E2s overlap (Eletr et al., 2005
). These results imply that residues in or close to helix-1 constitute part of a second APC/C-binding motif in UbcH10. We suggest that the simultaneous engagement of two binding motifs stabilizes UbcH10 binding to APC/C to orient it in the optimal position for assembling K11-linked chains.
Importantly, the assembly of homogenous K11-linked chains by APC/C and UbcH10 allowed us to determine whether these chains function as proteasomal targeting signals. Indeed, APC/C-substrates decorated with K11-linked chains are recognized by the proteasomal substrate receptors Rad23 () and S5a in vitro
(). Consequently, they are efficiently degraded by 26S proteasomes that co-purify with APC/C (; Verma et al., 2000
). APC-substrates modified with K11-linked chains are also rapidly turned over by purified 26S proteasomes from human embryonic kidney cells that were added subsequent to the ubiquitination (). Securin can be modified with K11-linked chains and captured by Rad23 also in 293T cells (). These findings provide strong evidence that K11-linked ubiquitin chains function as efficient proteasomal targeting signals.
K11-linked ubiquitin chains are a proteasomal targeting signal
As described above, APC/C and UbcH10 are able to modify substrates with ubiquitin chains also in the absence of K11, but this occurs with delayed kinetics. In addition, the affinity of APC/C-substrates to Rad23 is reduced, if chains are assembled by UbcH10 using ubi-R11 (), and these chains are less sensitive to proteasome activity in cells (Supp. Fig. 3A
). K11 is not part of the surface of ubiquitin that is recognized by Rad23, as determined by structural analysis (Veradan et al., 2005), and substrates modified with ubi-R11 by E2s other than UbcH10 are efficiently retained by Rad23 (Supp. Fig. 3B
). This indicates that mutation of K11 alters the structure of ubiquitin chains, which are formed by APC/CCdh1
and UbcH10, thereby impeding recognition by Rad23. We conclude that the APC/C and UbcH10 function by preferentially assembling K11-linked chains, which, as shown here, are efficient proteasomal targeting signals.
The TEK-box in ubiquitin is required for assembly of K11-linked chains
In addition to the proper orientation of UbcH10, formation of K11-linked chains by the APC/C requires the alignment of K11 in the acceptor ubiquitin relative to the active site of UbcH10. To identify residues in ubiquitin that help present K11, we mutated surface-exposed amino acids to alanine, and monitored the capacity of these mutants to support APC/C-activity in extracts.
Out of a total of 17 ubiquitin mutants, substituting K6, L8, T9, E34, and I36 with alanine strongly stabilizes securin in extracts (). Accordingly, overexpression of ubi-K6A and ubi-L8A in 293T cells interferes with the degradation of the APC/CCdh1-substrate securinΔD to a similar extent as overexpression of ubi-R11 (). Ubiquitination reactions using purified APC/CCdh1 and UbcH10 revealed that the stabilization of APC/C-substrates is a consequence of impaired chain formation in the presence of these mutants (). Overexpression of ubi-L8A reduced the modification of securin also in cells (). Interestingly, if the positive charge at position 6 is maintained, as in ubi-R6, neither degradation nor ubiquitination of APC/C-substrates is strongly affected (). This suggests that K6 contributes to binding, but is unlikely to be ubiquitinated itself. These experiments identify the ubiquitin residues K6, L8, T9, E34, and I36 to be required for the efficient formation of K11-linked chains by APC/C and UbcH10. Importantly, these residues form a cluster surrounding K11, which we refer to as the TEK-box of ubiquitin ().
The TEK-box in ubiquitin is required for UbcH10-dependent chain formation
In contrast to mutating the TEK-box, altering several other positions of ubiquitin does not affect ubiquitination or degradation of APC/C-substrates. This includes residues shown to support the formation of K29-linkages by a HECT-E3 (E16A/E18A), the formation of K48- and K63-linkages by several E3s (I44A; K48R; Y59A; K63A/E64A), and ubiquitin recognition (I44A, D58A). Moreover, when UbcH5c is used as E2, mutations in the TEK-box inhibit the APC/C-dependent chain formation less severely (Supp. Fig. 4A
). Only ubi-L8A, and to a lesser extent ubi-I36A, are deficient in supporting chain formation by APC/CCdh1
and UbcH5c. None of the TEK-box residues of ubiquitin is important for the monoubiquitination of an unrelated protein (UEV1A) or for the formation of K63-linked ubiquitin dimers by Ube2N/UEV1A (Supp. Fig. 4B
). All ubiquitin mutants are soluble at high concentrations and, with the exception of the slightly impaired E34A-mutant, efficiently loaded onto the active site of UbcH10 (data not shown). These experiments underscore the specific importance of the TEK-box of ubiquitin for UbcH10-dependent chain formation. We conclude that a cluster of residues surrounding K11 of ubiquitin, the TEK-box, is required for the efficient formation of K11-linked chains by APC/C and UbcH10.
The TEK-box is found in APC/C-substrates
Strikingly, we found sequences closely related to the TEK-box of ubiquitin in the APC/C-substrate securin. The two TEK-boxes in securin are located immediately downstream of its D-box, which is an APC/C-binding motif responsible for its processive ubiquitination (; Burton et al., 2005
; Kraft et al., 2005
; Rape et al., 2006
). Especially the second TEK-box of securin is well conserved (Supp. Fig. 5A
). In analogy to the TEK-box in ubiquitin, TEK-boxes in substrates could facilitate the modification of a substrate lysine, thereby nucleating ubiquitin chain formation.
TEK-boxes in securin contribute to APC/C-binding
To test this hypothesis, we first determined whether the TEK-boxes in securin contribute to APC/C-binding. We used a competition assay, in which the ubiquitination and degradation of a radiolabeled APC/C-substrate is competitively inhibited by addition of recombinant securin mutants. As expected, wild-type securin is an efficient competitor of APC/C-dependent degradation in G1-extracts, i.e. it binds well to APC/C (; Supp. Fig. 5B, C
). Even if both the D-box and a redundant motif, the KEN-box, are deleted (securinΔDΔK
), the securin-mutant inhibits APC/C, albeit with reduced efficiency. The same is observed if the D-box, KEN-box and the first TEK-box of securin are removed by deleting the amino-terminal 78 amino acids (securinΔN78
), suggesting that the remaining TEK-box in securinΔN78
is able to mediate APC/C-binding. Indeed, the deletion (securinΔN78ΔTEK
) or mutation (securinΔN78K91A/K92A
) of this TEK-box abolishes competition by securinΔN78
. Moreover, when both TEK-boxes are deleted in a securinΔDΔK
-background, the binding of securin to APC/CCdh1
is strongly impaired (). If more than 78 residues are deleted at the amino-terminus, binding of securin to APC/CCdh1
is also lost, but we cannot exclude that this is caused by misfolding of the truncated proteins. Together, these experiments strongly suggest that just like the D-box, TEK-boxes contribute to the binding of securin to APC/CCdh1
To test whether APC/C recognizes D-boxes and TEK-boxes by using distinct sites, we employed D-box- and TEK-box-peptides in our competition assay. As expected, the addition of a D-box-peptide to G1-extracts stabilizes the labeled APC/C-substrate (). This competition can be overcome by increasing the concentration of UbcH10 in the extracts, which allows the APC/C to ubiquitinate weakly bound substrates (Rape et al., 2006
; ). In a similar manner, competition by the TEK-box peptide securinΔN78
is overcome by addition of UbcH10 (; Supp. Fig. 5D
). In striking contrast, when both the D-box- and TEK-box-binding sites are saturated by the simultaneous addition of the two peptides, even high concentrations of UbcH10 are unable to bypass the competitive inhibition of APC/C. If the labeled substrate itself does not contain TEK-boxes (securinΔTEK1/ΔTEK2
), the D-box-peptide alone inhibits APC/C in the presence of high UbcH10-concentrations (). These results indicate that D-boxes and TEK-boxes are recognized by two non-identical sites on APC/C and/or UbcH10.
Substrate TEK-boxes promote the nucleation of ubiquitin chains
To determine how TEK-box-binding affects the UbcH10-dependent degradation of securin, we monitored securin turnover in extracts of quiescent T24-cells (G0-extracts). These extracts have very low levels of UbcH10, and APC/C-substrates are degraded rapidly only after recombinant UbcH10 has been added. As expected, wild-type securin is degraded in G0-extracts supplemented with UbcH10 (). The deletion of both TEK-boxes (securinΔTEK1/ΔTEK2), but not of each TEK-box alone, strongly stabilizes securin under these conditions. If the first TEK-box is deleted, mutation of K91/K92 in the second TEK-box to alanine (securinΔTEK1/K91A/K92A) is sufficient to stabilize securin in G0-extracts. If K91/K92 are replaced by arginine, securin degradation is not affected, indicating that, reminiscent of K6 of ubiquitin, K91/K92 of securin serve as binding, but not as ubiquitination site. A similar dependency on TEK-boxes is observed in G1-extracts, when we measured the degradation of securinΔD after addition of UbcH10. Again, simultaneous deletion of both TEK-boxes results in stabilization of the substrate in the presence of UbcH10 (). Finally, deletion of both the D-box and the TEK-boxes, but not deletion of either motif alone, strongly stabilizes securin against APC/CCdh1-dependent degradation in intact cells (). The TEK-boxes in securin are therefore important for its APC/C-dependent degradation in extracts and cells.
The TEK-box in securin is required for efficient UbcH10-dependent ubiquitination and degradation
Since the similarity to the TEK-box in ubiquitin implied that the TEK-boxes in securin promote the modification of a securin lysine, we monitored ubiquitination kinetics in the presence of methyl-ubiquitin, which is unable to form chains. As reported previously (Rape et al., 2006
and UbcH10 rapidly modify wild-type securin on several lysine residues (). By contrast, the deletion of both TEK-boxes strongly delays the monoubiquitination of securin and reduces the number of modified lysine residues. A similar reduction in the number of modified lysine residues is observed, when the TEK-box-peptide securinΔN78
is added to block the TEK-box-binding site (Supp. Fig. 6A
). We conclude that the TEK-boxes in securin are required for efficient modification of substrate lysine residues.
As expected from the impaired monoubiquitination of securinΔTEK1/ΔTEK2
, the onset of the UbcH10-dependent multiubiquitination of securinΔTEK1/ΔTEK2
is strongly delayed (). However, following the initial delay, ubiquitin chains approaching full length are rapidly assembled. The same delayed onset of ubiquitin chain formation is observed upon mutation of K91/K92 in securinΔTEK1
to alanine (securinΔTEK1/K91A/K92A
), but not when these residues are replaced by arginine (securinΔTEK1/K91R/K92R
). By contrast, the deletion of the D-box of securin does not delay chain formation, but results in reduced chain length. Consistent with the cooperation between D-box and TEK-boxes, the deletion of both motifs almost completely abrogates securin ubiquitination. The deletion of the TEK-boxes in securin has less severe effects on chain formation by APC/CCdh1
and UbcH5c (Supp. Fig. 6B, C
). These findings all suggest that the TEK-boxes in securin promote the nucleation of ubiquitin chains, especially if UbcH10 is the E2.
If the sole function of TEK-boxes in substrates is to promote ubiquitin chain nucleation, they should be required only for the addition of the first ubiquitin. By contrast, the D-box of substrates should remain important throughout the reaction. To test this hypothesis, we bypassed chain nucleation in D-box- and TEK-box-mutants by fusing ubiquitin to securinΔDΔTEK1/2 (securinΔDΔTEK1/2-UbiΔGG), or by replacing the carboxy-terminus of securinΔD, including both TEK-boxes, with ubiquitin (ΔD/70-Ub). Intriguingly, despite the lack of TEK-boxes, both ubiquitin fusions are degraded in G1-extracts in an APC/C-dependent manner (), and ubiquitinated by purified APC/CCdh1 (). The fused ubiquitin is only functional, if neither its TEK-box nor K11 are mutated (). The degradation of the ubiquitin-fusions is inhibited by the TEK-box peptide securinΔN78, indicating that the TEK-box in the fused ubiquitin recognizes the same site as the TEK-box in securin (). All fusions are degraded only after the extracts are supplemented with UbcH10, which suggests that addition of the first ubiquitin overcomes the lack of TEK-boxes, but not the lack of a D-box in securin. These findings can be reproduced in cells, where the fusion ΔD/70-Ub, but not securinΔDΔTEK1/2, is degraded in an APC/CCdh1-dependent manner (). As in extracts, mutation of the TEK-box in ubiquitin interferes with the APC/C-dependent degradation of the fusion in cells (). Thus, both in extracts and cells, addition of the first ubiquitin eliminates the requirement for TEK-boxes, but not for the D-box in securin. We conclude that the TEK-boxes in securin function primarily in ubiquitin chain nucleation, while the D-box is recognized throughout the ubiquitination reaction.
The TEK-boxes in securin function in ubiquitin chain nucleation
Several APC/C-substrates, including cyclin B1 and geminin, contain TEK-box-like sequences downstream of their D-box (Supp. Fig. 6D
). To test whether TEK-boxes are recognized during the modification of other APC/C-substrates, we monitored their ubiquitination after the TEK-box binding site had been saturated with the TEK-box-peptide securinΔN78
(). With the exception of cyclin A, the monoubiquitination of all APC/C-substrates analyzed in this assay is impaired by securinΔN78
, but not by securinΔN78ΔTEK
. In addition, the multiubiquitination of all APC/C-substrates tested, including cyclin A, is inhibited by securinΔN78
, but not securinΔN78ΔTEK
. Accordingly, addition of securinΔN78
to G1-extracts stabilizes all examined APC/C-substrates, including cyclin A (Supp. Fig. 6E
). Thus, saturation of the TEK-box binding site interferes with the ubiquitination and degradation of several APC/C-substrates. Based on the results presented in this study, we propose that TEK-boxes in substrates facilitate the nucleation of ubiquitin chains, while the TEK-box in ubiquitin promotes the elongation of the K11-linked chains mediating APC/C-dependent reactions.