Molecular Cell
Previews with JQ1 attenuated atherogenesis in both early and late atherosclerosis development. In summary, the present work underscores the importance of SEs as preferential targets of BET-mediated gene regulation in fast transitions and pathological conditions, which could be targeted by BET inhibition therapies. This may underlie the intriguing potency and selectivity of BET inhibitors as reported in different cancers and other diseases (Dawson and Kouzarides, 2012; Shi and Vakoc, 2014; Love´n et al., 2013). It also reinforces the central role of SEs among the repertoire of molecular mechanisms underlying cell identity determination, as well as fast cell state transitions, which are crucial in development, homeostasis, stress response, and disease. It will be
important to determine their idiosyncrasies and interplay with other mechanisms regulating key genes, such as widespread chromatin modifications (Benayoun et al., 2014, Dawson et al., 2012) and stimuli-responsive and cell-specific small and long regulatory RNAs (reviewed in Amaral et al., 2013) (Figure 1). REFERENCES Amaral, P.P., Dinger, M.E., and Mattick, J.S. (2013). Brief Funct Genomics 12, 254–278. Benayoun, B.A., Pollina, E.A., Ucar, D., Mahmoudi, S., Karra, K., Wong, E.D., Devarajan, K., Daugherty, A.C., Kundaje, A.B., Mancini, E., et al. (2014). Cell 158, 673–688. Brown, J.D., Lin, C.Y., Duan, Q., Griffin, G., Federation, A., Paranal, R.M., Bair, S., Newton, G., Lichtman, A., Kung, A., et al. (2014). Mol. Cell 56, this issue, 219–231.
Dawson, M.A., and Kouzarides, T. (2012). Cell 150, 12–27. Dawson, M.A., Foster, S.D., Bannister, A.J., Robson, S.C., Hannah, R., Wang, X., Xhemalce, B., Wood, A.D., Green, A.R., Go¨ttgens, B., and Kouzarides, T. (2012). Cell Rep 2, 470–477. Hnisz, D., Abraham, B.J., Lee, T.I., Lau, A., SaintAndre´, V., Sigova, A.A., Hoke, H.A., and Young, R.A. (2013). Cell 155, 934–947. IIott, N.E., Heward, J.A., Roux, B., Tsitsiou, E., Fenwick, P.S., Lenzi, L., Goodhead, I., Hertz-Fowler, C., Heger, A., Hall, N., et al. (2014). Nat. Commun. 5, 3979. Love´n, J., Hoke, H.A., Lin, C.Y., Lau, A., Orlando, D.A., Vakoc, C.R., Bradner, J.E., Lee, T.I., and Young, R.A. (2013). Cell 153, 320–334. Shi, J., and Vakoc, C.R. (2014). Mol. Cell 54, 728–736. Whyte, W.A., Orlando, D.A., Hnisz, D., Abraham, B.J., Lin, C.Y., Kagey, M.H., Rahl, P.B., Lee, T.I., and Young, R.A. (2013). Cell 153, 307–319.
Ubiquitin Chain Elongation: An Intriguing Strategy Kazuhiro Iwai1 and Keiji Tanaka2,* 1Department
of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2014.10.009 2Laboratory
Using multidisciplinary analyses, in this issue Kelly et al. (2014) and Brown et al. (2014) reveal an unexpected role for the RING finger and substrate recognition adaptor proteins of the anaphase promoting complex/cyclosome (APC/C) in ubiquitin chain elongation. Recently, our knowledge about the ubiquitin conjugation system in controlling protein functions has expanded enormously. In general, the ubiquitin conjugation system attaches polyubiquitin chains to substrate proteins in order to regulate their functions. Because the type of ubiquitin chain determines the mode of protein regulation, the mechanisms underlying selective generation of specific ubiquitin chains are of great interest (Komander and Rape, 2012). Polyubiquitin chains are generated by the repeated actions of three classes of enzymes: ubiquitinactivating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). However, the precise mechanism of polyubiquitin generation by the RING
E3s, the largest family of E3s, has not been convincingly determined. RING ligases recognize both E2s and substrates simultaneously via distinct recognition motifs and then conjugate polyubiquitin chains onto the substrates. A topological constraint seems to arise during chain elongation: the longer the chain, the further the spatial distance between the E3 and the reaction site (Deshaies and Joazeiro, 2009). In two papers in this issue, Kelly et al. (2014) and Brown et al. (2014) used multidisciplinary analyses, including structural, biochemical, and cell biological analyses, to dissect the mechanism of polyubiquitin chain generation by APC/C. This complex is a multisubunit RING finger E3 that mediates
ubiquitin-mediated degradation of regulators of chromosomal segregation, cytokinesis, and mitotic exit, as well as the initiation of DNA replication and other processes, in order to promote cell-cycle progression (Chang et al., 2014). APC/C recognizes substrates via the coordinated functions of APC10 and the adaptor protein CDC20 or CDH1 and conjugates K11- or K48-linked ubiquitin chains to substrates destined for proteasomal degradation. In conjunction with the E2 protein Ube2S, APC/C generates K11 chains by specifically conjugating the C terminus of donor ubiquitin onto the K11 residue of acceptor ubiquitin. The two groups found that CDC20 and CDH1, adaptor proteins involved in
Molecular Cell 56, October 23, 2014 ª2014 Elsevier Inc. 189
Molecular Cell
Previews Ub Ube2S
APC/C
APC10 CDC20 or CDH1
APC/C
APC10
Substrate
Substrate CDC20 Ub or Ub APC CDH1 Ube2S 11
Ub
Ub APC UbcH5/H10 11
APC/C
APC10 trate Subs CDC20 Ub Ub or Ub APC CDH1 Ube2S 11
Figure 1. K11 Chain Formation by Ube2S and APC/C APC/C recognizes substrates via the coordinated functions of APC10 and the adaptor protein CDC20 or CDH1. The complex conjugates the initial ubiquitin together with UbcH5/UbcH10, which binds to APC11, the RING finger subunit of APC/C. CDC20, and possibly CDH1 as well, recruits Ube2S to APC/C by recognizing the C-terminal region of Ube2S and stabilizing the E2 on APC/C. In chain elongation reactions, acceptor ubiquitin in the ubiquitin chain is regarded as the substrate. Ube2S conjugates the C terminus of the donor ubiquitin exclusively to K11 of the acceptor ubiquitin, which binds successively to the substrates. APC/C provides acceptor-ubiquitin binding sites for Ube2S, and the RING of APC11 appears to be involved in acceptor recognition. Thus, APC/C and its adaptor proteins recognize substrates for initial ubiquitylation and ubiquitin bound to the substrates for chain elongation and effectively catalyze polymerization of ubiquitin to form polyubiquitin chains in concert with two E2s, UbcH5/UbcH10 and Ube2S.
substrate recognition that have originally been identified as activators of E3s, enhance diubiquitin synthesis by Ube2S (Visintin et al., 1997). CDC20, and possibly also CDH1, recognizes Ube2S via its unique C-terminal region and transfers it to APC/C. CDC20 appears to stabilize the interaction between APC/C and Ube2S. In the chain elongation reaction, ubiquitin of the existing chain functions as the acceptor, and thus as the substrate. APC/C provides the binding site for the acceptor ubiquitin (Brown et al., 2014; Kelly et al., 2014). Thus, APC/C, together with the adaptor proteins, recognizes both substrates and ubiquitin for the initial ubiquitylation and chain elongation and then conjugates polyubiquitin chains in concert with two ubiquitin-charged E2s, UbcH5/UbcH10 and Ube2S (Figure 1). These results reveal a unique feature of APC/C-mediated polyubiquitylation. The RING E3s bind to E2 proteins, which are bound to ubiquitin via a thioester bond (E2Ub), via their RING finger motif and activate the bound E2Ub for ubiquitin transfer (Plechanovova´ et al., 2012). Indeed, APC/C interacts with UbcH10/ UbcH5 via its RING finger subunit APC11 to activate the E2. However, APC/C binding of Ube2S via its C-terminal region is independent of RING. The results of Brown et al. (2014) strongly suggest that APC11 plays a role in binding to the acceptor ubiquitin and indeed enhances diubiquitin formation by Ube2S.
Two mechanisms underlying ubiquitin chain elongation have been proposed to date. The results of these two papers support a mechanism in which ubiquitins are added one by one to the distal end of a growing ubiquitin chain. In an alternative mechanism, polyubiquitin is generated on ubiquitin bound to E2 via a thioester bond, and the premade chain is then transferred to the substrate en bloc (Li et al., 2007); this strategy would solve the topological constraint mentioned above. In this scenario, the ubiquitin on the active Cys residue of the E2, which functions as the donor ubiquitin, must function simultaneously as the acceptor. However, one ubiquitin cannot function as both acceptor and donor, as the surface of ubiquitin recognized for the acceptor overlaps with that for the donor. Because recognition of acceptor ubiquitin by APC/C solves this steric constraint, and the one-by-one mechanism is favored by kinetic studies (Pierce et al., 2009), it seems likely that polyubiquitin chains are generated by adding ubiquitin to the distal end of the ubiquitin chain, at least in the case of APC/C. APC/C in yeast utilizes a single E2, which is involved in both initial ubiquitylation and chain elongation. However, the APC/C from higher eukaryotes, which was analyzed here, utilizes two E2s, UbcH10/UbcH5 and Ube2S, which are involved in the initial ubiquitylation and the elongation of ubiquitin chains on substrates destined for degradation; this arrangement seems suitable for efficient
190 Molecular Cell 56, October 23, 2014 ª2014 Elsevier Inc.
generation of polyubiquitin chains. The unique characteristics of Ube2S enable it to bind to APC/C noncompetitively with UbcH5/UbcH10, resulting in activation, which also requires binding to the RING domain. Ube2S has a unique C-terminal peptide-like extension that plays a role in interaction with CDC20 and APC/ C. Also, Ube2SUb assumes the active conformation without binding to the RING finger. Although APC11 is independent of Ube2S binding, its RING domain is involved in the binding of acceptor ubiquitin during efficient chain elongation by Ube2S, and the ubiquitin-binding surface of APC11 does not overlap with that of UbcH5/UbcH10. Moreover, the amino acid residues of APC11 critical for acceptor ubiquitin recognition are conserved in organisms that have Ube2S, but not in those that lack this E2. Thus, Ube2S and APC/C appear to have coevolved, possibly to promote efficient ubiquitin-dependent degradation of regulators of chromosomal segregation, cytokinesis, and mitotic exit, as well as the initiation of DNA replication and other processes, in a timely and selective manner. Indeed, Ube2S-mediated K11 chain formation plays an essential role in regulation of the spindle checkpoint. APC/C inactivates the spindle checkpoint in a ubiquitin-dependent manner; however, a ubiquitin-independent function of APC/C in checkpoint inactivation has also been revealed (Mansfeld et al., 2011). Kelly et al. (2014) use gene replacement technology to establish cells that exclusively
Molecular Cell
Previews express a Ube2S mutant that cannot elongate ubiquitin chains. Using this mutant, they elegantly show that ubiquitylation is necessary for inactivation of the spindle checkpoint by APC/C. This finding illustrates how mechanistic analyses can provide a valuable tool for dissecting the physiological function of APC/C. However, several questions persist. Recognition of acceptor ubiquitin by APC11 appears to solve the steric constraint. However, a steric problem still occurs when the chain becomes longer because APC/C could recognize both the substrate and distal end of the ubiquitin chain conjugated to the substrate. Thus, this constraint might limit chain length and facilitate generation of multiple
chains. Despite the fact that Ube2SUb assumes the active conformation, the interaction between the helix D region of Ube2S and APC/C is required for efficient K11 chain formation by an unknown mechanism. Further analyses may elucidate the unexpected mechanism underlying RING E3-mediated formation of ubiquitin chains and reveal whether the mechanism reported here is common to other RING E3s or specific for APC/C.
Deshaies, R.J., and Joazeiro, C.A. (2009). Annu. Rev. Biochem. 78, 399–434.
REFERENCES
Pierce, N.W., Kleiger, G., Shan, S.O., and Deshaies, R.J. (2009). Nature 462, 615–619.
Kelly, A., Wickliffe, K.E., Song, L., Fedrigo, I., and Rape, M. (2014). Mol Cell 56, this issue, 232–245. Komander, D., and Rape, M. (2012). Annu. Rev. Biochem. 81, 203–229. Li, W., Tu, D., Brunger, A.T., and Ye, Y. (2007). Nature 446, 333–337. Mansfeld, J., Collin, P., Collins, M.O., Choudhary, J.S., and Pines, J. (2011). Nat. Cell Biol. 13, 1234–1243.
Brown, N.G., Watson, E.R., Weissmann, F., Jarvis, M.A., Vanderlinden, R., Grace, C.R.R., Frye, J.J., Qiao, R., Dube, P., Petzold, G., et al. (2014). Mol. Cell 56, this issue, 246–260.
Plechanovova´, A., Jaffray, E.G., Tatham, M.H., Naismith, J.H., and Hay, R.T. (2012). Nature 489, 115–120.
Chang, L., Zhang, Z., Yang, J., McLaughlin, S.H., and Barford, D. (2014). Nature 513, 388–393.
Visintin, R., Prinz, S., and Amon, A. (1997). Science 278, 460–463.
Molecular Cell 56, October 23, 2014 ª2014 Elsevier Inc. 191
Previews with JQ1 attenuated atherogenesis in both early and late atherosclerosis development. In summary, the present work underscores the importance of SEs as preferential targets of BET-mediated gene regulation in fast transitions and pathological conditions, which could be targeted by BET inhibition therapies. This may underlie the intriguing potency and selectivity of BET inhibitors as reported in different cancers and other diseases (Dawson and Kouzarides, 2012; Shi and Vakoc, 2014; Love´n et al., 2013). It also reinforces the central role of SEs among the repertoire of molecular mechanisms underlying cell identity determination, as well as fast cell state transitions, which are crucial in development, homeostasis, stress response, and disease. It will be
important to determine their idiosyncrasies and interplay with other mechanisms regulating key genes, such as widespread chromatin modifications (Benayoun et al., 2014, Dawson et al., 2012) and stimuli-responsive and cell-specific small and long regulatory RNAs (reviewed in Amaral et al., 2013) (Figure 1). REFERENCES Amaral, P.P., Dinger, M.E., and Mattick, J.S. (2013). Brief Funct Genomics 12, 254–278. Benayoun, B.A., Pollina, E.A., Ucar, D., Mahmoudi, S., Karra, K., Wong, E.D., Devarajan, K., Daugherty, A.C., Kundaje, A.B., Mancini, E., et al. (2014). Cell 158, 673–688. Brown, J.D., Lin, C.Y., Duan, Q., Griffin, G., Federation, A., Paranal, R.M., Bair, S., Newton, G., Lichtman, A., Kung, A., et al. (2014). Mol. Cell 56, this issue, 219–231.
Dawson, M.A., and Kouzarides, T. (2012). Cell 150, 12–27. Dawson, M.A., Foster, S.D., Bannister, A.J., Robson, S.C., Hannah, R., Wang, X., Xhemalce, B., Wood, A.D., Green, A.R., Go¨ttgens, B., and Kouzarides, T. (2012). Cell Rep 2, 470–477. Hnisz, D., Abraham, B.J., Lee, T.I., Lau, A., SaintAndre´, V., Sigova, A.A., Hoke, H.A., and Young, R.A. (2013). Cell 155, 934–947. IIott, N.E., Heward, J.A., Roux, B., Tsitsiou, E., Fenwick, P.S., Lenzi, L., Goodhead, I., Hertz-Fowler, C., Heger, A., Hall, N., et al. (2014). Nat. Commun. 5, 3979. Love´n, J., Hoke, H.A., Lin, C.Y., Lau, A., Orlando, D.A., Vakoc, C.R., Bradner, J.E., Lee, T.I., and Young, R.A. (2013). Cell 153, 320–334. Shi, J., and Vakoc, C.R. (2014). Mol. Cell 54, 728–736. Whyte, W.A., Orlando, D.A., Hnisz, D., Abraham, B.J., Lin, C.Y., Kagey, M.H., Rahl, P.B., Lee, T.I., and Young, R.A. (2013). Cell 153, 307–319.
Ubiquitin Chain Elongation: An Intriguing Strategy Kazuhiro Iwai1 and Keiji Tanaka2,* 1Department
of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2014.10.009 2Laboratory
Using multidisciplinary analyses, in this issue Kelly et al. (2014) and Brown et al. (2014) reveal an unexpected role for the RING finger and substrate recognition adaptor proteins of the anaphase promoting complex/cyclosome (APC/C) in ubiquitin chain elongation. Recently, our knowledge about the ubiquitin conjugation system in controlling protein functions has expanded enormously. In general, the ubiquitin conjugation system attaches polyubiquitin chains to substrate proteins in order to regulate their functions. Because the type of ubiquitin chain determines the mode of protein regulation, the mechanisms underlying selective generation of specific ubiquitin chains are of great interest (Komander and Rape, 2012). Polyubiquitin chains are generated by the repeated actions of three classes of enzymes: ubiquitinactivating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). However, the precise mechanism of polyubiquitin generation by the RING
E3s, the largest family of E3s, has not been convincingly determined. RING ligases recognize both E2s and substrates simultaneously via distinct recognition motifs and then conjugate polyubiquitin chains onto the substrates. A topological constraint seems to arise during chain elongation: the longer the chain, the further the spatial distance between the E3 and the reaction site (Deshaies and Joazeiro, 2009). In two papers in this issue, Kelly et al. (2014) and Brown et al. (2014) used multidisciplinary analyses, including structural, biochemical, and cell biological analyses, to dissect the mechanism of polyubiquitin chain generation by APC/C. This complex is a multisubunit RING finger E3 that mediates
ubiquitin-mediated degradation of regulators of chromosomal segregation, cytokinesis, and mitotic exit, as well as the initiation of DNA replication and other processes, in order to promote cell-cycle progression (Chang et al., 2014). APC/C recognizes substrates via the coordinated functions of APC10 and the adaptor protein CDC20 or CDH1 and conjugates K11- or K48-linked ubiquitin chains to substrates destined for proteasomal degradation. In conjunction with the E2 protein Ube2S, APC/C generates K11 chains by specifically conjugating the C terminus of donor ubiquitin onto the K11 residue of acceptor ubiquitin. The two groups found that CDC20 and CDH1, adaptor proteins involved in
Molecular Cell 56, October 23, 2014 ª2014 Elsevier Inc. 189
Molecular Cell
Previews Ub Ube2S
APC/C
APC10 CDC20 or CDH1
APC/C
APC10
Substrate
Substrate CDC20 Ub or Ub APC CDH1 Ube2S 11
Ub
Ub APC UbcH5/H10 11
APC/C
APC10 trate Subs CDC20 Ub Ub or Ub APC CDH1 Ube2S 11
Figure 1. K11 Chain Formation by Ube2S and APC/C APC/C recognizes substrates via the coordinated functions of APC10 and the adaptor protein CDC20 or CDH1. The complex conjugates the initial ubiquitin together with UbcH5/UbcH10, which binds to APC11, the RING finger subunit of APC/C. CDC20, and possibly CDH1 as well, recruits Ube2S to APC/C by recognizing the C-terminal region of Ube2S and stabilizing the E2 on APC/C. In chain elongation reactions, acceptor ubiquitin in the ubiquitin chain is regarded as the substrate. Ube2S conjugates the C terminus of the donor ubiquitin exclusively to K11 of the acceptor ubiquitin, which binds successively to the substrates. APC/C provides acceptor-ubiquitin binding sites for Ube2S, and the RING of APC11 appears to be involved in acceptor recognition. Thus, APC/C and its adaptor proteins recognize substrates for initial ubiquitylation and ubiquitin bound to the substrates for chain elongation and effectively catalyze polymerization of ubiquitin to form polyubiquitin chains in concert with two E2s, UbcH5/UbcH10 and Ube2S.
substrate recognition that have originally been identified as activators of E3s, enhance diubiquitin synthesis by Ube2S (Visintin et al., 1997). CDC20, and possibly also CDH1, recognizes Ube2S via its unique C-terminal region and transfers it to APC/C. CDC20 appears to stabilize the interaction between APC/C and Ube2S. In the chain elongation reaction, ubiquitin of the existing chain functions as the acceptor, and thus as the substrate. APC/C provides the binding site for the acceptor ubiquitin (Brown et al., 2014; Kelly et al., 2014). Thus, APC/C, together with the adaptor proteins, recognizes both substrates and ubiquitin for the initial ubiquitylation and chain elongation and then conjugates polyubiquitin chains in concert with two ubiquitin-charged E2s, UbcH5/UbcH10 and Ube2S (Figure 1). These results reveal a unique feature of APC/C-mediated polyubiquitylation. The RING E3s bind to E2 proteins, which are bound to ubiquitin via a thioester bond (E2Ub), via their RING finger motif and activate the bound E2Ub for ubiquitin transfer (Plechanovova´ et al., 2012). Indeed, APC/C interacts with UbcH10/ UbcH5 via its RING finger subunit APC11 to activate the E2. However, APC/C binding of Ube2S via its C-terminal region is independent of RING. The results of Brown et al. (2014) strongly suggest that APC11 plays a role in binding to the acceptor ubiquitin and indeed enhances diubiquitin formation by Ube2S.
Two mechanisms underlying ubiquitin chain elongation have been proposed to date. The results of these two papers support a mechanism in which ubiquitins are added one by one to the distal end of a growing ubiquitin chain. In an alternative mechanism, polyubiquitin is generated on ubiquitin bound to E2 via a thioester bond, and the premade chain is then transferred to the substrate en bloc (Li et al., 2007); this strategy would solve the topological constraint mentioned above. In this scenario, the ubiquitin on the active Cys residue of the E2, which functions as the donor ubiquitin, must function simultaneously as the acceptor. However, one ubiquitin cannot function as both acceptor and donor, as the surface of ubiquitin recognized for the acceptor overlaps with that for the donor. Because recognition of acceptor ubiquitin by APC/C solves this steric constraint, and the one-by-one mechanism is favored by kinetic studies (Pierce et al., 2009), it seems likely that polyubiquitin chains are generated by adding ubiquitin to the distal end of the ubiquitin chain, at least in the case of APC/C. APC/C in yeast utilizes a single E2, which is involved in both initial ubiquitylation and chain elongation. However, the APC/C from higher eukaryotes, which was analyzed here, utilizes two E2s, UbcH10/UbcH5 and Ube2S, which are involved in the initial ubiquitylation and the elongation of ubiquitin chains on substrates destined for degradation; this arrangement seems suitable for efficient
190 Molecular Cell 56, October 23, 2014 ª2014 Elsevier Inc.
generation of polyubiquitin chains. The unique characteristics of Ube2S enable it to bind to APC/C noncompetitively with UbcH5/UbcH10, resulting in activation, which also requires binding to the RING domain. Ube2S has a unique C-terminal peptide-like extension that plays a role in interaction with CDC20 and APC/ C. Also, Ube2SUb assumes the active conformation without binding to the RING finger. Although APC11 is independent of Ube2S binding, its RING domain is involved in the binding of acceptor ubiquitin during efficient chain elongation by Ube2S, and the ubiquitin-binding surface of APC11 does not overlap with that of UbcH5/UbcH10. Moreover, the amino acid residues of APC11 critical for acceptor ubiquitin recognition are conserved in organisms that have Ube2S, but not in those that lack this E2. Thus, Ube2S and APC/C appear to have coevolved, possibly to promote efficient ubiquitin-dependent degradation of regulators of chromosomal segregation, cytokinesis, and mitotic exit, as well as the initiation of DNA replication and other processes, in a timely and selective manner. Indeed, Ube2S-mediated K11 chain formation plays an essential role in regulation of the spindle checkpoint. APC/C inactivates the spindle checkpoint in a ubiquitin-dependent manner; however, a ubiquitin-independent function of APC/C in checkpoint inactivation has also been revealed (Mansfeld et al., 2011). Kelly et al. (2014) use gene replacement technology to establish cells that exclusively
Molecular Cell
Previews express a Ube2S mutant that cannot elongate ubiquitin chains. Using this mutant, they elegantly show that ubiquitylation is necessary for inactivation of the spindle checkpoint by APC/C. This finding illustrates how mechanistic analyses can provide a valuable tool for dissecting the physiological function of APC/C. However, several questions persist. Recognition of acceptor ubiquitin by APC11 appears to solve the steric constraint. However, a steric problem still occurs when the chain becomes longer because APC/C could recognize both the substrate and distal end of the ubiquitin chain conjugated to the substrate. Thus, this constraint might limit chain length and facilitate generation of multiple
chains. Despite the fact that Ube2SUb assumes the active conformation, the interaction between the helix D region of Ube2S and APC/C is required for efficient K11 chain formation by an unknown mechanism. Further analyses may elucidate the unexpected mechanism underlying RING E3-mediated formation of ubiquitin chains and reveal whether the mechanism reported here is common to other RING E3s or specific for APC/C.
Deshaies, R.J., and Joazeiro, C.A. (2009). Annu. Rev. Biochem. 78, 399–434.
REFERENCES
Pierce, N.W., Kleiger, G., Shan, S.O., and Deshaies, R.J. (2009). Nature 462, 615–619.
Kelly, A., Wickliffe, K.E., Song, L., Fedrigo, I., and Rape, M. (2014). Mol Cell 56, this issue, 232–245. Komander, D., and Rape, M. (2012). Annu. Rev. Biochem. 81, 203–229. Li, W., Tu, D., Brunger, A.T., and Ye, Y. (2007). Nature 446, 333–337. Mansfeld, J., Collin, P., Collins, M.O., Choudhary, J.S., and Pines, J. (2011). Nat. Cell Biol. 13, 1234–1243.
Brown, N.G., Watson, E.R., Weissmann, F., Jarvis, M.A., Vanderlinden, R., Grace, C.R.R., Frye, J.J., Qiao, R., Dube, P., Petzold, G., et al. (2014). Mol. Cell 56, this issue, 246–260.
Plechanovova´, A., Jaffray, E.G., Tatham, M.H., Naismith, J.H., and Hay, R.T. (2012). Nature 489, 115–120.
Chang, L., Zhang, Z., Yang, J., McLaughlin, S.H., and Barford, D. (2014). Nature 513, 388–393.
Visintin, R., Prinz, S., and Amon, A. (1997). Science 278, 460–463.
Molecular Cell 56, October 23, 2014 ª2014 Elsevier Inc. 191
