Cargando…
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, electrop...
Autores principales: | , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2013
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3692480/ https://www.ncbi.nlm.nih.gov/pubmed/23825692 http://dx.doi.org/10.1371/journal.pone.0067952 |
_version_ | 1782274622717165568 |
---|---|
author | Piatkov, Konstantin Graciet, Emmanuelle Varshavsky, Alexander |
author_facet | Piatkov, Konstantin Graciet, Emmanuelle Varshavsky, Alexander |
author_sort | Piatkov, Konstantin |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3692480 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-36924802013-07-02 Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking Prokaryotes Piatkov, Konstantin Graciet, Emmanuelle Varshavsky, Alexander PLoS One Research Article 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. Public Library of Science 2013-06-25 /pmc/articles/PMC3692480/ /pubmed/23825692 http://dx.doi.org/10.1371/journal.pone.0067952 Text en © 2013 Piatkov et al http://creativecommons.org/licenses/by/4.0/ 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 properly credited. |
spellingShingle | Research Article Piatkov, Konstantin Graciet, Emmanuelle Varshavsky, Alexander Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking Prokaryotes |
title | Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking Prokaryotes |
title_full | Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking Prokaryotes |
title_fullStr | Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking Prokaryotes |
title_full_unstemmed | Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking Prokaryotes |
title_short | Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking Prokaryotes |
title_sort | ubiquitin reference technique and its use in ubiquitin-lacking prokaryotes |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3692480/ https://www.ncbi.nlm.nih.gov/pubmed/23825692 http://dx.doi.org/10.1371/journal.pone.0067952 |
work_keys_str_mv | AT piatkovkonstantin ubiquitinreferencetechniqueanditsuseinubiquitinlackingprokaryotes AT gracietemmanuelle ubiquitinreferencetechniqueanditsuseinubiquitinlackingprokaryotes AT varshavskyalexander ubiquitinreferencetechniqueanditsuseinubiquitinlackingprokaryotes |