Soc. 126, 3006C3007 (2004). substrates recognized by the OUT screen. table S5. Primers used in this study. References (with a pET vector and that its activity could be enhanced by ammonium sulfate precipitation after eluting the protein from your nickelCnitrilotriacetic acid (Ni-NTA) column. wt fE4B could be efficiently ubiquitinated with wt UB through the wt Uba1-UbcH5b pair, yet it could not be altered by xUB through the xUba1-xUbcH5b pair (Fig. 3A). In contrast, fE4B YS-49 with U-box mutants of KB2 and KB12 (fE4B-KB2 and fE4B-KB12) could be efficiently ubiquitinated with xUB through the xUba1-xUbcH5b pair. We have thus constructed an OUT cascade for xUB transfer to fE4B-KB2 or fE4B-KB12. We also found that xUB could be transferred YAP1 to p53 through xUba1-xUbcH5b relaying with either fE4B-KB2 or fE4B-KB12 and that, with a YS-49 similar efficiency, wt UB could be transferred through wt Uba1-UbcH5b-fE4B to p53 (Fig. 4A). The crossover cascade of xUba1-xUbcH5b-wt fE4B was incapable of transferring xUB to p53, suggesting the orthogonality of the OUT cascade with the native UB transfer cascade. Hence, either fE4B-KB2 or fE4B-KB12 could be used as an xE4B to construct the OUT cascade for profiling E4B substrates. Open in a separate windows Fig. 3 Activity of designed fE4B and CHIP mutants in autoubiquitination with xUB.(A) fE4B-KB2 and fE4B-KB12 are fE4B with mutated U-box domains KB2 and KB12. They could be autoubiquitinated by xUB through the xUba1-xUbcH5b pair. The activity of mutant E4B autoubiquitination was much like wt fE4B autoubiquitination. In contrast, wt fE4B could not be ubiquitinated by xUB through the xUba1-xUbcH5b pair, suggesting the orthogonality of the OUT cascade and the native YS-49 cascade of E4B. (B) wt CHIP displayed on the surface of M13 phage lost activity in autoubiquitination by wt UB and the wt Uba1-UbcH5b pair. (C) CHIP-KB2 and CHIP-KB12 were constructed by replacing the loop1 of the CHIP U-box with corresponding sequences in the KB2 and KB12 mutants of the E4B U-box. This enabled the designed CHIP to be ubiquitinated by xUB through the xUba1-xUbcH5b pair. The efficiency of CHIP-KB2/12 autoubiquitination with xUB was comparable to that of wt CHIP ubiquitination by wt UB through the wt Uba1-UbcH5b pair (fig. S2B). Open in a separate window Fig. 4 xUB transfer through the OUT cascade of E4B and CHIP to p53.(A) fE4B-KB2 and fE4B-KB12 could assemble an OUT cascade with xUba1 and xUbcH5b to mediate xUB transfer to p53. The efficiency of p53 ubiquitination by xUB and the OUT cascade was much like p53 ubiquitination with wt UB and the wt Uba1-UbcH5b-fE4B cascade. In contrast, wt E4B could YS-49 not pair with xUba1-xUbcH5b to transfer xUB to p53, suggesting the orthogonality between the OUT cascade and native E3s. Mutant fE4B KB2 or YS-49 KB12 could not pair with wt Uba1Cwt UbcH5b to transfer wt UB to p53. (B) Much like E4B OUT cascade, CHIP-KB2 and CHIP-KB12 could relay with xUba1-xUbcH5b to transfer xUB to p53. The efficiency of xUB modification of p53 by the CHIP OUT cascades was comparable to that of p53 modification by wt UB going through the wt Uba1-UbcH5b-CHIP cascade. xUB could not be transferred to p53 with the crossover cascade of xUba1-xUbcH5bCwt CHIP. wt UB could not be transferred to p53 with the crossover cascade of wt Uba1Cwt UbcH5bCmutant CHIP (KB2 or KB12). Building an OUT cascade with CHIP We set out to use phage selection to identify U-box mutants of CHIP with restored UB transfer from xUbcH5b. However, even though full-length CHIP including the U-box domain name could be displayed around the phage surface, it was not active in autoubiquitination reactions with wt UB transferred through the wt Uba1-UbcH5b pair (Fig. 3B). CHIP functions as a dimer, so the lack of activity was attributed to the inability of CHIP to form suitable dimers when displayed on phage (fig. S3C) (and set up in vitro ubiquitination reactions with wt fE4B and wt CHIP. Substrates expressed from the might not have the proper posttranslational modification such as phosphorylation to mediate acknowledgement by an E3, or adaptor proteins could be missing to mediate UB transfer. Nevertheless, we observed polyubiquitination.