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Published June 25, 2013 | Supplemental Material + Published
Journal Article Open

Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking Prokaryotes


In a pulse-chase assay, the in vivo degradation of a protein is measured through a brief labeling of cells with, for example, a radioactive amino acid, followed by cessation of labeling and analysis of cell extracts prepared at different times afterward ("chase"), using immunoprecipitation, electrophoresis and autoradiography of a labeled protein of interest. A conventional pulse-chase assay is fraught with sources of data scatter, as the efficacy of labeling and immunoprecipitation can vary, and sample volumes can vary as well. The ubiquitin reference technique (URT), introduced in 1996, addresses these problems. In eukaryotes, a DNA-encoded linear fusion of ubiquitin to another protein is cleaved by deubiquitylases at the ubiquitin-protein junction. A URT assay uses a fusion in which the ubiquitin moiety is located between a downstream polypeptide (test protein) and an upstream polypeptide (a long-lived reference protein). The cotranslational cleavage of a URT fusion by deubiquitylases after the last residue of ubiquitin produces, at the initially equimolar ratio, a test protein with a desired N-terminal residue and a reference protein containing C-terminal ubiquitin moiety. In addition to being more accurate than pulse-chases without a reference, URT makes it possible to detect and measure the degradation of a test protein during the pulse (before the chase). Because prokaryotes, including Gram-negative bacteria such as, for example, Escherichia coli and Vibrio vulnificus, lack the ubiquitin system, the use of URT in such cells requires ectopic expression of a deubiquitylase. We describe designs and applications of plasmid vectors that coexpress, in bacteria, both a URT-type fusion and Ubp1, a deubiquitylase of the yeast Saccharomyces cerevisiae. This single-plasmid approach extends the accuracy-enhancing URT assay to studies of protein degradation in prokaryotes.

Additional Information

© 2013 Piatkov et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received March 27, 2013; Accepted May 23, 2013; Published June 25, 2013. Funding: This work has been supported by grants to AV from the National Institutes of Health (DK039520 and GM031530). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Editor: Min Wu, University of North Dakota, United States of America. We thank Christopher Brower and Brandon Wadas for helpful comments on the manuscript. We are grateful to the present and former members of the Varshavsky laboratory for their assistance and advice. Author Contributions: Conceived and designed the experiments: KP EG AV. Performed the experiments: KP EG. Analyzed the data: KP EG AV. Contributed reagents/materials/analysis tools: KP EG AV. Wrote the paper: KP AV.

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Published - journal.pone.0067952.pdf

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Supplemental Material - TableS2.docx

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