Knocking out Ubiquitin Proteasome System Function In Vivo and In Vitro with Genetically Encodable Tandem Ubiquitin

Abstract

At present, the 26S proteasome–specific inhibitor is not available. We constructed polyubiquitin derivatives that contained a tandem repeat of ubiquitins and were insensitive to ubiquitin hydrolases. When these artificial polyubiquitins (tUbs, tandem ubiquitins) were overproduced in the wild‐type yeast strain, growth was strongly inhibited, probably because of inhibition of the 26S proteasome. We also found that several substrates of the ubiquitin‐proteasome pathway were stabilized by expressing tUbs in vivo. tUbs containing four units or more of the ubiquitin monomer were found to form a complex with the 26S proteasome. We showed that tUb bound to the 26S proteasome inhibited the in vitro degradation of polyubiquitinylated Sic1 by the 26S proteasome. When tUB6 (six‐mer) messenger RNA was injected into Xenopus embryos, cell division was inhibited, suggesting that tUb can be used as a versatile inhibitor of the 26S proteasome.