ausblenden:
Schlagwörter:
HB-ubiquitin • ubiquitin profiling • SILAC • MG132 • tandem affinity purification
Zusammenfassung:
Ubiquitination regulates a host of cellular processes by labeling proteins for degradation, but also by
functioning as a regulatory, nonproteolytic posttranslational modification. Proteome-wide strategies
to monitor changes in ubiquitination profiles are important to obtain insight into the various cellular
functions of ubiquitination. Here we describe generation of stable cell lines expressing a tandem
hexahistidine-biotin tag (HB-tag) fused to ubiquitin for two-step purification of the ubiquitinated
proteome under fully denaturing conditions. Using this approach we identified 669 ubiquitinated proteins
from HeLa cells, including 44 precise ubiquitin attachment sites on substrates and all seven possible
ubiquitin chain-linkage types. To probe the dynamics of ubiquitination in response to perturbation of
the ubiquitin/proteasome pathway, we combined ubiquitin profiling with quantitative mass spectrometry
using the stable isotope labeling with amino acids in cell culture (SILAC) strategy. We compared
untreated cells and cells treated with the proteasome inhibitor MG132 to identify ubiquitinated proteins
that are targeted to the proteasome for degradation. A number of proteasome substrates were identified.
In addition, the quantitative approach allowed us to compare proteasome targeting by different ubiquitin
chain topologies in vivo. The tools and strategies described here can be applied to detect changes in
ubiquitination dynamics in response to various changes in growth conditions and cellular stress and
will contribute to our understanding of the ubiquitin/proteasome system.
