Mechanism Of Ubiquitin Chain Assembly By The Anaphase Promoting Complex

Background:During protein ubiquitination a lysine residue on a target protein is conjugated to ubiquitin through an enzymatic cascade involving E1 activating enzymes, E2 conjugating enzymes and E3 ligases. Ubiquitin chain formation can proceed in the same manner by repetitively adding ubiquitins to one or more of seven lysines on the conjugated ubiquitin, potentially generating ubiquitin chains of several different topologies. The linkage, topology, and length of the ubiquitin chains determine the fate of the tagged protein. The Anaphase Promoting Complex (APC) is a multi-subunit E3 enzyme that controls cell cycle progression during mitosis and G1 phase by targeting many cell cycle regulators for degradation. Two activator proteins, Cdc20 and Cdh1 collaborate with APC throughout mitosis and G1 phase in substrate binding and targeting. While the function of APC in cell cycle control is well understood, how this enzymatic complex assembles ubiquitin conjugates on its substrates has been difficult to determine. Under this background, we decided to study the mechanism of ubiquitin chain assembly by APC in detail. Methods:1. Degradation assay in cell extracts was used to determine the rates of different APC substrates degradation, and how different components of the pathway affect the activity of APC.2. In vitro ubiquitination reaction combined with autoradiography was used to determine the lengthes and distribution of ubiquitin chains generated on APC substrates.3. Immunoprecipitation and Western blot were used to study the protein interactions between APC and other proteins.Results:1. Securin with a single lysine can support efficient APC dependent degradation To simplify study of the ubiquitination reactions of APC, we made a construct of securin that allows only a single ubiquitin chain to be formed.1.1 We first made a lysine-less version of securin (securin-K0) by mutating all of its 20 lysine residues to arginines. This protein is stable in G1 phase cell extracts and unable to be ubiquitinated in an in vitro ubiquitination reaction using purified APC.1.2 We then added back different lysine residues to the protein and the various securin constructs with single lysines were assayed for their rate of degradation in cell extracts, in comparison to the wildtype protein. Among the mutants, securin-K48 was degraded with kinetics most similar to that of the wildtype securin.1.3 Degradation of securin-K48 was blocked by the APC inhibitor, Emi1, thus excluding non-specific proteolysis in the extracts system used.1.4 In in vitro ubiquitination reactions using purified APC and methylated ubiquitin, only one ubiquitin was added to securin-K48, whereas as least 5 ubiquitins were added to the wildtype protein.1.5 The D-box mutant (DBM) of securin-K48 (securin-K48-DBM) was stable in the cell extracts (Figure 1D), as has been shown for the DBM of wildtype securin. 2. A preference for K-11 linkage in APC catalyzed ubiquitin chain assembly We examined ubiquitination products generated with purified APC, E1, E2s (UbcH10 or UbcH5a), purified securin-K48 and a series of ubiquitin mutants, each containing all but one of its lysine residues converted to arginine.2.1 Ubiquitin K11 generates the longest ubiquitin chain (>6 ubiquitin molecules) on securin-K48 substrate. Wildtype ubiquitin appeared to give somewhat longer chains. Reactions with linkages made through the K6, K27, K29 or K33 of ubiquitin formed short di or tri-ubiquitinated products, with the mono-ubiquitinated species being the predominant product. Reactions with linkages made through K48 and K63 of ubiquitin formed products with slightly longer ubiquitin chains.2.2 When securin-K48-DBM was used, the linkage preferences were almost the same, but the products had comparatively shorter ubiquitin chains.2.3 The linkage preferences in both of the N-terminal fragment of cyclin B1 (N-cyclin B1) mutants were similar to that of securin-K48.2.4 Quantitation of the average lengths of ubiquitin chains in the products obtained with different ubiquitin mutants on securin-K48 and N-cyclin B1-K20 showed very similar pattern.2.5 APC dependent ubiquitination can be mediated by both UbcH10 and UbcH5 E2 enzymes. Regardless of the E2 used, K11 is the preferred linkage on both securin-K48 and cyclin B1-K20.2.6 Adding K48R or K63R ubiquitin mutants has no effect on securin-K48 degradation in G1 extracts, compared with adding the wildtype ubiquitin; by contrast, adding K11R ubiquitin significantly inhibited securin-K48 degradation to the same extent as adding lysine-free ubiquitin (K0 ubiquitin).3. The role of E2-25K in APC dependent reactionsThe E2-25K conjugating enzyme (also know as UbcH1) has recently been shown to mediate the assembly of long ubiquitin chains on APC substrates in conjunction with UbcH10 in yeast. They also extended this model to human cells. We have examined the role of E2-25K in APC catalyzed ubiquitin chain assembly in our experimental system using N-cyclin B1-K20 as a single lysine substrate for ubiquitination.3.1 When high concentrations of UbcH10 and E2-25K were combined in the same reaction, together, a complete conversion of the substrate and formation of products with very long ubiquitin chains were observed (lane 7 and 8), demonstrating a synergism between UbcH10 and E2-25K, as reported.3.2 At high concentrations of E2-25K there was no evidence that the elongation of ubiquitin chains catalyzed by E2-25K was dependent on APC.3.3 When used at these lower concentrations, E2-25K alone was unable to drive any noticeable ubiquitination in the presence or absence of APC; more importantly, it did not act synergistically with UbcH10.4. Ube2S assembles K-11 linked ubiquitin chains on APC substrates4.1 We tested whether Ube2S could support APC-catalyzed ubiquitination reaction in our in vitro system. Purified recombinant Ube2S, unlike UbcH10 and UbcH5a, could not support APC-catalyzed ubiquitination on securin-K48. Interestingly, when added in combination with UbcH10 or UbcH5, Ube2S supported formation of much longer ubiquitin chains on the single lysine substrate.4.2 Securin-K48 was ubiquitinated with APC and UbcH10 first, then APC was removed from the reaction mixture and Ube2S was added. Longer ubiquitin chains were produced when APC was present through the entire course of the reaction. When APC was present only at early stages, the chains were shorter. Ubiquitination reactions with either APC/Cdc20 purified from mitotic extracts or with APC/Cdh1 purified from G1 extracts were indistinguishable.4.3 We found that Ube2S efficiently copurifies with APC during immunoaffinity purification from HeLa cells extracted under normal conditions, using washes with low salt buffers. However, under a high salt wash conditions, very little Ube2S copurifies with APC. While APC purified under the low salt conditions forms long ubiquitin chains (>4 ubiquitins), APC purified under the high salt condition makes mostly mono-ubiquitinated products.4.4 In reactions where Ube2S was present, long ubiquitin chains were formed when wild type ubiquitin or ubiquitin mutants other than the ubiquitin K11R were present. However in the reactions with UbK11R, only short ubiquitin chains were produced and addition of Ube2S did not change their length. Thus the ubiquitin chains formed in an APC/UbcH10/Ube2S catalyzed reaction are linked exclusively through K11. 4.5 Reactions with wildtype Ube2S make long ubiquitin chains with a K11 linkage specificity; however, reactions with the C95S mutant of Ube2S not only produce short ubiquitin chains, but also lose the linkage specificity, as three different K/R ubiquitin mutants generate similar products. Reactions including the Ube2SΔC mutant (Ube2S with the C-terminal domain truncated) generated products similar to those of reaction with UbcH10 alone and the linkage specificity of the ubiquitin chains is maintained. 4.6 Securin-K48 was first ubiquitinated with APC and GST-UbcH5a. APC and GST-UbcH5a were then immuno-depleted from the reaction mixture; the ubiquitinated product of this reaction served as substrate for a second reaction with APC and Ube2S. Under these conditions APC and UbcH5 generated mostly mono-ubiquitinated securin-K48. When both APC and Ube2S were added for the second reaction, the mono-ubiquitinated substrate was converted to products with long ubiquitin chains. However, no additional ubiquitin chains were produced in the presence of APC and Ube2S C95S. Ube2SΔC did convert the substrates to products with longer ubiquitin chains, though not as long as those seen in the presence of wildtype Ube2S.5. The role of Ube2S in substrate degradation5.1 Both securin and geminin remain stable in extracts made from nocodazole-arrested cells. Addition of Ube2S or Ube2S C95S to this extract system had no effect on the degradation of APC substrates, unlike the addition of UbcH10, which drives the system into anaphase and activates APC.5.2 Though the rate of degradation of securin and geminin remain the same on addition of wildtype Ube2S to UbcH10-activated extracts, addition of Ube2S C95S delays degradation.5.3 Longer ubiquitin chains were formed on substrates that were incubated in extracts supplemented with wildtype Ube2S compared to the C95S mutant. The equivalent results were also observed for cyclin B1.5.4 Ube2S also collaborates with UbcH10 during G1. Securin and geminin are quickly degraded in G1 extracts. Addition of Ube2S C95S into the G1 extracts inhibited the degradation of securin and geminin, though addition of additional Ube2S had no measurable effect.Conclusion:1. APC substrates that has only one ubiquitination site can be efficiently ubiquitinated, and this provide a simplified model substrate for studying the formation of a single ubiquitin chain.2. APC ubiquitination reaction preferentially forms K-11 linked ubiquitin chains, and K-11 linked ubiquitin chain can lead to efficient substrate degradation by the proteasome.3. A new ubiquitin conjugating enzyme (E2) Ube2S extends the ubiquitin chain in APC catalyzed ubiquitin chain formation, and it also determines the linkage specificity.4. APC assembles long ubiquitin chain on its substrates in two steps. The first step requires E2 UbcH10 or UbcH5, they monoubiquitinates substrate on multiple lysine residues; the second step requires E2 Ube2S, starting from a ubiquitin that has been coupled to substrate, it forms long ubiquitin chains that are linked only through K-11. This kind of mechanism might be a general mechanism that applies to different ubiquitin ligases (E3), which generates ubiquitin chains of different linkages.