Figure 1. K11-linked ubiquitin chains mediate APC/C-functions.

A. K11-linked chains are sufficient for degradation of cyclin B1 in mitotic extracts. CP-extracts were supplemented with wt-ubi or single-lysine mutants. APC/C was activated by addition of UbcH10 and p31^comet, and degradation of cyclin B1 was monitored by Western blotting. B. Degradation of APC/C-substrates by K11-linked chains is proteasome-dependent. Degradation of radiolabeled cyclin B1 in CP-extracts was triggered by addition of p31^comet/UbcH10 in the presence wt-ubi or ubi-K11. The proteasome inhibitor MG132 was added when indicated. C. K11 is required for rapid degradation of cyclin B1 in mitotic extracts. CP-extracts were supplemented with ubiquitin mutants and treated as described above. Degradation of cyclin B1 was monitored by Western blotting. D. K11-linkages are required for full activity of APC/C^Cdh1 in G1. The degradation of radiolabeled securin was monitored by autoradiography in G1-extracts in the presence of ubiquitin mutants. E. K11-linked chains target APC/C^Cdh1-substrates for degradation in vivo. The APC/C-dependent degradation of geminin, Plk1, and securin^ΔD was triggered in 293T cells in the presence of indicated ubiquitin mutants (wt-ubi, ubi-R11, ubi-R48) by co-expression of Cdh1. The expression levels were analyzed by Western blotting. F. K11-linkages are required for rapid cell cycle progression in embryos of Xenopus tropicalis. One cell of X. tropicalis embryos at the two-cell stage was injected with recombinant wt-ubi or ubi-R11 and a fluorescent tracer. Injected cells were followed by fluorescence microscopy, and cell division was monitored by phase microscopy. G. K11-linkages are required for X. tropicalis development. Injected embryos were allowed to develop to the tadpole stage. The percentage of embryos without developmental aberrations (“normal”) and that of viable embryos was determined.