Global Analysis of Protein Ubiquitination.
Richard
Gardner, Daniel Gottschling
Basic Science, Fred Hutchinson CRC,
1100 Fairview Ave N., Seattle, WA 98109, USA
The cell employs
numerous modifications to alter the behavior of proteins. One
modification, the covalent attachment of the small protein ubiquitin, is
used to alter the stability, localization, and/or activity of proteins
modified with ubiquitin. Although a number of proteins have been shown
to be modified with ubiquitin, the extent of protein ubiquitination
across the panorama of cellular proteins is unknown. To address this, we
have improved a protocol for isolating the pool of ubiquitinated
proteins from yeast. We have analyzed the pool of purified,
ubiquitinated proteins by mass spectrometry and identified a large
number of proteins that are modified with ubiquitin. With this method,
we plan to extend our knowledge beyond characterizing simple identities
to detail the set of substrates targeted for ubiquitination by a single
ubiquitination complex through reproducing the analysis using strains
with known gene knockouts. In particular, we are determining the
ubiquitination substrates and complexes involved in numerous chromatin-related processes, including silencing and DNA repair.
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