The unique N terminus of the UbcH10 E2 enzyme controls the threshold for APC activation and enhances checkpoint regulation of the APC

In vitro, the anaphase-promoting complex (APC) E3 ligase functions with E2 ubiquitin-conjugating enzymes of the E2-C and Ubc4/5 families to ubiquitinate substrates. However, only the use of the E2-C family, notably UbcH10, is genetically well validated. Here, we biochemically demonstrate preferential use of UbcH10 by the APC, specified by the E2 core domain. Importantly, an additional E2-E3 interaction mediated by the N-terminal extension of UbcH10 regulates APC activity. Mutating the highly conserved N terminus increases substrate ubiquitination and the number of substrate lysines targeted, allows ubiquitination of APC substrates lacking their destruction boxes, increases resistance to the APC inhibitors Emi1 and BubR1 in vitro, and bypasses the spindle checkpoint in vivo. Fusion of the UbcH10 N terminus to UbcH5 restricts ubiquitination activity but does not direct specific interactions with the APC. Thus, UbcH10 combines a specific E2-E3 interface and regulation via its N-terminal extension to limit APC activity for substrate selection and checkpoint control.     

Mutagenic analysis of the destruction signal of mitotic cyclins and structural characterization of ubiquitinated intermediates.

Mitotic cyclins are abruptly degraded at the end of mitosis by a cell-cycle-regulated ubiquitin-dependent proteolytic system. To understand how cyclin is recognized for ubiquitin conjugation, we have performed a mutagenic analysis of the destruction signal of mitotic cyclins. We demonstrate that an N-terminal cyclin B segment as short as 27 residues, containing the 9-amino-acid destruction box, is sufficient to destabilize a heterologous protein in mitotic Xenopus extracts. Each of the three highly conserved residues of the cyclin B destruction box is essential for ubiquitination and subsequent degradation. Although an intact destruction box is essential for the degradation of both A- and B-type cyclins, we find that the Xenopus cyclin A1 destruction box cannot functionally substitute for its B-type counterpart, because it does not contain the highly conserved asparagine necessary for cyclin B proteolysis. Physical analysis of ubiquitinated cyclin B intermediates demonstrates that multiple lysine residues function as ubiquitin acceptor sites, and mutagenic studies indicate that no single lysine residue is essential for cyclin B degradation. This study defines the key residues of the destruction box that target cyclin for ubiquitination and suggests there are important differences in the way in which A- and B-type cyclins are recognized by the cyclin ubiquitination machinery.     

Molecular cloning of cDNA encoding a cyclin-selective ubiquitin carrier protein (E2-C) from Carassius auratus (goldfish) and expression analysis of the cloned gene

Destruction of cyclin B is required for exit from mitosis and meiosis. A cyclin-specific ubiquitinating system, including cyclin-selective ubiquitin carrier protein (E2-C), is thought to be responsible for cyclin B destruction. Here we present the cloning, sequencing and expression analysis of goldfish, Carassius auratus, E2-C which encodes the cyclin-selective ubiquitin carrier protein from goldfish ovary. The cloned cDNA is 677 bp long and encodes 172 amino acids. The deduced amino acid sequence is highly homologous to E2-C from other species. Recombinant goldfish E2-C possesses ubiquitinating activity against cyclin B. The expression of mRNA for E2-C was similar to that of mRNA for cyclin B, occurring at very high level in the ovary. The similarity of the expression pattern of E2-C and cyclin B suggests that E2-C mediates a cyclin-specific ubiquitination.     

APC2 Cullin Protein and APC11 RING Protein Comprise the Minimal Ubiquitin Ligase Module of the Anaphase-promoting Complex

In mitosis, the anaphase-promoting complex (APC) regulates the onset of sister-chromatid separation and exit from mitosis by mediating the ubiquitination and degradation of the securin protein and mitotic cyclins. With the use of a baculoviral expression system, we have reconstituted the ubiquitin ligase activity of human APC. In combination with Ubc4 or UbcH10, a heterodimeric complex of APC2 and APC11 is sufficient to catalyze the ubiquitination of human securin and cyclin B1. However, the minimal APC2/11 ubiquitin ligase module does not possess substrate specificity, because it also ubiquitinates the destruction box deletion mutants of securin and cyclin B1. Both APC11 and UbcH10 bind to the C-terminal cullin homology domain of APC2, whereas Ubc4 interacts with APC11 directly. Zn(2+)-binding and mutagenesis experiments indicate that APC11 binds Zn(2+) at a 1:3 M ratio. Unlike the two Zn(2+) ions of the canonical RING-finger motif, the third Zn(2+) ion of APC11 is not essential for its ligase activity. Surprisingly, with Ubc4 as the E2 enzyme, Zn(2+) ions alone are sufficient to catalyze the ubiquitination of cyclin B1. Therefore, the Zn(2+) ions of the RING finger family of ubiquitin ligases may be directly involved in catalysis.     

The RING-H2-finger protein APC11 as a target of hydrogen peroxide

The anaphase-promoting complex (APC) is a ubiquitin-protein ligase (E3) that targets cell cycle regulators such as cyclin B and securin for degradation. The APC11 subunit functions as the catalytic core of this complex and mediates the transfer of ubiquitin from a ubiquitin-conjugating enzyme (E2) to the substrate. APC11 contains a RING-H2-finger domain, which includes one histidine and seven cysteine residues that coordinate two Zn(2+) ions. We now show that exposure of purified APC11 to H(2)O(2) (0.1 to 1 mM) induced the release of bound zinc as a result of the oxidation of cysteine residues. It also impaired the physical interaction between APC11 and the E2 enzyme Ubc4 as well as inhibited the ubiquitination of cyclin B1 by APC11. The release of HeLa cells from metaphase arrest in the presence of exogenous H(2)O(2) inhibited the ubiquitination of cyclin B1 as well as the degradation of cyclin B1 and securin that were apparent in the absence of H(2)O(2). The presence of H(2)O(2) also blocked the co-immunoprecipitation of Ubc4 with APC11 and delayed the exit of cells from mitosis. Inhibition of APC11 function by H(2)O(2) thus likely contributes to the delay in cell cycle progression through mitosis that is characteristic of cells subjected to oxidative stress.     

The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.

The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has revealed four components required for cyclin-ubiquitin ligation and proteolysis: a nonspecific ubiquitin-activating enzyme E1, a soluble fraction containing a ubiquitin carrier protein activity called E2-C, a crude particulate fraction containing a ubiquitin ligase (E3) activity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel approximately 1500-kDa complex, termed the cyclosome, which contains a cyclin-selective ubiquitin ligase activity, E3-C. E3-C is present but inactive during interphase; it can be activated in vitro by the addition of cdc2, enabling the transfer of ubiquitin from E2-C to cyclin. The kinetics of E3-C activation suggest the existence of one or more intermediates between cdc2 and E3-C. Cyclosome-associated E3-C acts on both cyclin A and B, and requires the presence of wild-type N-terminal destruction box motifs in each cyclin. Ubiquitinated cyclins are then rapidly recognized and degraded by the proteasome. These results identify the cyclosome-associated E3-C as the component of the cyclin destruction machinery whose activity is ultimately regulated by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles.     

The N-terminal Regulatory Domain of Cyclin A Contains Redundant Ubiquitination Targeting Sequences and Acceptor Sites

Cyclin A is destroyed during mitosis by the ubiquitin-proteasome system. Like cyclin B, a destruction box (D-box) motif is required for the destruction of cyclin A. However, Cyclin A degradation is more complicated than cyclin B because cyclin A’s D-box motif is more extensive and proteolysis involves complex signaling in some organisms. In this study, we found that in addition to the D-box, the region between residues 123-157 also contributed to the ubiquitination and degradation of human cyclin A. Indeed, removal of the bulk of the N-terminal regulatory domain was needed to completely stabilize cyclin A and eliminate ubiquitination. A putative second RxxL motif around residue 138 played only a minor role in cyclin A degradation. To distinguish between sequences recognized by the ubiquitination machinery and the ubiquitin acceptor sites per se, we utilized a novel approach involving in vitro cleavage of cyclin A after ubiquitination. We found that several lysine residues proximal to the D-box (Lys37, Lys54, and Lys68) were ubiquitin acceptor sites. Cyclin A lacking the three lysine residues was degraded slower than the wild-type protein. Although these lysines were normally used, ubiquitination could shift to other cryptic sites when the preferred sites were unavailable, suggesting the exact positions of the ubiquitin chains also contributed to degradation. Together, these data revealed that ubiquitination does not occur randomly on cyclin A and open up questions on the precise function of the D-box.     

Dcn1 functions as a scaffold-type E3 ligase for cullin neddylation

Cullin-based E3 ubiquitin ligases are activated through modification of the cullin subunit with the ubiquitin-like protein Nedd8. Dcn1 regulates cullin neddylation and thus ubiquitin ligase activity. Here we describe the 1.9 A X-ray crystal structure of yeast Dcn1 encompassing an N-terminal ubiquitin-binding (UBA) domain and a C-terminal domain of unique architecture, which we termed PONY domain. A conserved surface on Dcn1 is required for direct binding to cullins and for neddylation. The reciprocal binding site for Dcn1 on Cdc53 is located approximately 18 A from the site of neddylation. Dcn1 does not require cysteine residues for catalytic function, and directly interacts with the Nedd8 E2 Ubc12 on a surface that overlaps with the E1-binding site. We show that Dcn1 is necessary and sufficient for cullin neddylation in a purified recombinant system. Taken together, these data demonstrate that Dcn1 is a scaffold-like E3 ligase for cullin neddylation.     

Two ubiquitin-conjugating enzymes,UbcP1/Ubc4 and UbcP4/Ubc11, have distinct functions for ubiquitination of mitotic cyclin

Cell cycle events are regulated by sequential activation and inactivation of Cdk kinases. Mitotic exit is accomplished by the inactivation of mitotic Cdk kinase, which is mainly achieved by degradation of cyclins. The ubiquitin-proteasome system is involved in this process, requiring APC/C (anaphase-promoting complex/cyclosome) as a ubiquitin ligase. In Xenopus and clam oocytes, the ubiquitin-conjugating enzymes that function with APC/C have been identified as two proteins, UBC4 and UBCx/E2-C. Previously we reported that the fission yeast ubiquitin-conjugating enzyme UbcP4/Ubc11, a homologue of UBCx/E2-C, is required for mitotic transition. Here we show that the other fission yeast ubiquitin-conjugating enzyme, UbcP1/Ubc4, which is homologous to UBC4, is also required for mitotic transition in the same manner as UbcP4/Ubc11. Both ubiquitin-conjugating enzymes are essential for cell division and directly required for the degradation of mitotic cyclin Cdc13. They function nonredundantly in the ubiquitination of CDC13 because a defect in ubcP1/ubc4+ cannot be suppressed by high expression of UbcP4/Ubc11 and a defect in ubcP4/ubc11+ cannot be suppressed by high expression of UbcP1/Ubc4. In vivo analysis of the ubiquitinated state of Cdc13 shows that the ubiquitin chains on Cdc13 were short in ubcP1/ubc4 mutant cells while ubiquitinated Cdc13 was totally reduced in ubcP4/ubc11 mutant cells. Taken together, these results indicate that the two ubiquitin-conjugating enzymes play distinct and essential roles in the degradation of mitotic cyclin Cdc13, with the UbcP4/Ubc11-pathway initiating ubiquitination of Cdc13 and the UbcP1/Ubc4-pathway elongating the short ubiquitin chains on Cdc13.     

Structural basis for recruitment of Ubc12 by an E2 binding domain in NEDD8's E1

E2 conjugating enzymes play a central role in ubiquitin and ubiquitin-like protein (ublp) transfer cascades: the E2 accepts the ublp from the E1 enzyme and then the E2 often interacts with an E3 enzyme to promote ublp transfer to the target. We report here the crystal structure of a complex between the C-terminal domain from NEDD8's heterodimeric E1 (APPBP1-UBA3) and the catalytic core domain of NEDD8's E2 (Ubc12). The structure and associated mutational analyses reveal molecular details of Ubc12 recruitment by NEDD8's E1. Interestingly, the E1's Ubc12 binding domain resembles ubiquitin and recruits Ubc12 in a manner mimicking ubiquitin's interactions with ubiquitin binding domains. Structural comparison with E2-E3 complexes indicates that the E1 and E3 binding sites on Ubc12 may overlap and raises the possibility that crosstalk between E1 and E3 interacting with an E2 could influence the specificity and processivity of ublp transfer.     

Implications for the ubiquitination reaction of the anaphase-promoting complex from the crystal structure of the Doc1/Apc10 subunit

The anaphase-promoting complex (APC) is a multi-subunit E3 protein ubiquitin ligase that is responsible for the metaphase to anaphase transition and the exit from mitosis. One of the subunits of the APC that is required for its ubiquitination activity is Doc1/Apc10, a protein composed of a Doc1 homology domain that has been identified in a number of diverse putative E3 ubiquitin ligases. Here, we present the crystal structure of Saccharomyces cerevisiae Doc1/Apc10 at 2.2A resolution. The Doc1 homology domain forms a beta-sandwich structure that is related in architecture to the galactose-binding domain of galactose oxidase, the coagulation factor C2 domain and a domain of XRCC1. Residues that are invariant amongst Doc1/Apc10 sequences, including a temperature-sensitive mitotic arrest mutant, map to a beta-sheet region of the molecule, whose counterpart in galactose oxidase, the coagulation factor C2 domains and XRCC1, mediate bio-molecular interactions. This finding suggests the identification of the functionally important and conserved region of Doc1/Apc10 and, since invariant residues of Doc1/Apc10 colocalise with conserved residues of other Doc1 homology domains, we propose that the Doc1 homology domains perform common ubiquitination functions in the APC and other E3 ubiquitin ligases.     

The SCF ubiquitin ligase: an extended look

The SCF E3 ubiquitin ligases select specific proteins for ubiquitination (and typically destruction) by coupling variable adaptor (F box) proteins that bind protein substrates to a conserved catalytic engine containing a cullin, Cul1, and the Rbx1/Roc1 RING finger protein. A new crystal structure of the SCF(Skp2) ubiquitin ligase shows the molecular organization of this complex and raises important questions as to how substrate ubiquitination is accomplished.     

The N-Terminal Extension of UBE2E Ubiquitin-Conjugating Enzymes Limits Chain Assembly

Protein ubiquitylation depends upon the concerted action of ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). All E2s have a conserved ubiquitin-conjugating (UBC) domain but many have variable extensions N- and C-terminal to the UBC domain. For many E2s, the function of the extension is not well understood. Here, we show that the N-terminal extension of the UBE2E proteins regulates formation of polyubiquitin chains by the processive UBC domain. Target proteins are therefore monoubiquitylated by full-length UBE2E, whereas the UBC domain alone polyubiquitylates proteins. Although the N-terminal extension of UBE2E1 is largely disordered in solution, these residues have a critical role in limiting chain building, and when fused to the highly processive E2, UBE2D2, ubiquitylation is limited. For some E2s, interaction of ubiquitin with the ‘backside’ of the UBC domain promotes polyubiquitylation. However, interaction of ubiquitin with the backside of the UBC domain of UBE2E1 does not appear to be important for processivity. This study underscores the importance of studying full-length E2 proteins and not just the highly conserved core domain.     

Structural and functional analysis of the human mitotic-specific ubiquitin-conjugating enzyme, UbcH10

Cell cycle progression is controlled at several different junctures by the targeted destruction of cell cycle regulatory proteins. These carefully orchestrated events include the destruction of the securin protein to permit entry into anaphase, and the destruction of cyclin B to permit exit from mitosis. These destruction events are mediated by the ubiquitin/proteasome system. The human ubiquitin-conjugating enzyme, UbcH10, is an essential mediator of the mitotic destruction events. We report here the 1.95-A crystal structure of a mutant UbcH10, in which the active site cysteine has been replaced with a serine. Functional analysis indicates that the mutant is active in accepting ubiquitin, although not as efficiently as wild-type. Examination of the crystal structure reveals that the NH2-terminal extension in UbcH10 is disordered and that a conserved 3(10)-helix places a lysine residue near the active site. Analysis of relevant mutants demonstrates that for ubiquitin-adduct formation the presence or absence of the NH2-terminal extension has little effect, whereas the lysine residue near the active site has significant effect. The structure provides additional insight into UbcH10 function including possible sites of interaction with the anaphase promoting complex/cyclosome and the disposition of a putative destruction box motif in the structure.     

Crystal structure of YdaL, a stand-alone small MutS-related protein from Escherichia coli

Sequence homologs of the small MutS-related (Smr) domain, the C-terminal endonuclease domain of MutS2, also exist as stand-alone proteins. In this study, we report the crystal structure of a proteolyzed fragment of YdaL (YdaL??-???), a stand-alone Smr protein from Escherichia coli. In this structure, residues 86-170 assemble into a classical Smr core domain and are embraced by an N-terminal extension (residues 40-85) with an α/β/α fold. Sequence alignment indicates that the N-terminal extension is conserved among a number of stand-alone Smr proteins, suggesting structural diversity among Smr domains. We also discovered that the DNA binding affinity and endonuclease activity of the truncated YdaL??-??? protein were slightly lower than those of full-length YdaL?-???, suggesting that residues 1-38 may be involved in DNA binding.     

The 'destruction box' of cyclin A allows B-type cyclins to be ubiquitinated, but not efficiently destroyed.

The destruction of mitotic cyclins by programmed proteolysis at the end of mitosis is an important element in cell cycle control. This proteolysis depends on a conserved motif of nine residues known as the 'destruction box', which is located 40-50 residues from the N-terminus. The sequences of the A- and B-type destruction boxes are slightly different, which might account for the differences in timing of their destruction. When the cyclin A-type destruction box was substituted for the normal one in cyclin B1 or B2, however, the resulting constructs were unexpectedly stable, although the converse substitution of B-type destruction boxes in cyclin A permitted normal degradation. We compared the ubiquitination of various cyclin constructs, and found that whereas mutation of the highly conserved residues in the destruction box strongly reduced the level of ubiquitinated intermediates, the stable destruction box 'swap' constructs did form such adducts. Thus, while ubiquitination is probably necessary for cyclin destruction, it is not sufficient. We also found that poly-ubiquitinated cyclin derivatives are still bound to p34cdc2, which is not detectably ubiquitinated itself, raising the questions of how cyclin and cdc2 dissociate from one another, and at what stage, in the process of degradation.     

A conserved cysteine is essential for Pex4p-dependent ubiquitination of the peroxisomal import receptor Pex5p

The peroxisomal protein import receptor Pex5p is modified by ubiquitin, both in an Ubc4p-dependent and -independent manner. Here we show that the two types of ubiquitination target different residues in the NH(2)-terminal region of Pex5p and we identify Pex4p (Ubc10p) as the ubiquitin-conjugating enzyme required for Ubc4p-independent ubiquitination. Whereas Ubc4p-dependent ubiquitination occurs on two lysine residues, Pex4p-dependent ubiquitination neither requires lysine residues nor the NH(2)-terminal alpha-NH(2) group. Instead, a conserved cysteine residue appears to be essential for both the Pex4p-dependent ubiquitination and the overall function of Pex5p. In addition, we show that this form of ubiquitinated Pex5p is susceptible to the reducing agent beta-mercaptoethanol, a compound that is unable to break ubiquitin-NH(2) group linkages. Together, our results strongly suggest that Pex4p-dependent ubiquitination of Pex5p occurs on a cysteine residue.     

Structure of LNX1:Ubc13 ~ Ubiquitin Complex Reveals the Role of Additional Motifs for the E3 Ligase Activity of LNX1

LNX1 (ligand of numb protein-X1) is a RING and PDZ domain-containing E3 ubiquitin ligase that ubiquitinates human c-Src kinase. Here, we report the identification and structure of the ubiquitination domain of LNX1, the identification of Ubc13/Ube2V2 as a functional E2 in vitro, and the structural and functional studies of the Ubc13~Ub intermediate in complex with the ubiquitination domain of LNX1. The RING domain of LNX1 is embedded between two zinc-finger motifs (Zn-RING-Zn), both of which are crucial for its ubiquitination activity. In the heterodimeric complex, the ubiquitin of one monomer shares more buried surface area with LNX1 of the other monomer and these interactions are unique and essential for catalysis. This study reveals how the LNX1 RING domain is structurally and mechanistically dependent on other motifs for its E3 ligase activity, and describes how dimeric LNX1 recruits ubiquitin-loaded Ubc13 for Ub transfer via E3 ligase-mediated catalysis.