Knocking out ubiquitin proteasome system function in vivo and in vitro with genetically encodable tandem ubiquitin.
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.
Saeki
Y
Y
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.
Isono
E
E
Shimada
M
M
Kawahara
H
H
Yokosawa
H
H
Toh-E
A
A
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D016133:Polymerase Chain Reaction; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
D017931:DNA Primers; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease