Using ubiquitin to follow the metabolic fate of a protein
Details
Serval ID
serval:BIB_4E5341F94B86
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Using ubiquitin to follow the metabolic fate of a protein
Journal
Proceedings of the National Academy of Sciences of the United States of America
ISSN
0027-8424 (Print)
Publication state
Published
Issued date
05/1996
Volume
93
Number
10
Pages
4907-12
Notes
Comparative Study
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S. --- Old month value: May 14
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S. --- Old month value: May 14
Abstract
We describe a method that can be used to produce equimolar amounts of two or more specific proteins in a cell. In this approach, termed the ubiquitin/protein/reference (UPR) technique, a reference protein and a protein of interest are synthesized as a polyprotein separated by a ubiquitin moiety. This tripartite fusion is cleaved, cotranslationally or nearly so, by ubiquitin-specific processing proteases after the last residue of ubiquitin, producing equimolar amounts of the protein of interest and the reference protein bearing a C-terminal ubiquitin moiety. In applications such as pulse-chase analysis, the UPR technique can compensate for the scatter of immunoprecipitation yields, sample volumes, and other sources of sample-to-sample variation. In particular, this method allows a direct comparison of proteins' metabolic stabilities from the pulse data alone. We used UPR to examine the N-end rule (a relation between the in vivo half-life of a protein and the identity of its N-terminal residue) in L cells, a mouse cell line. The increased accuracy afforded by the UPR technique underscores insufficiency of the current "half-life" terminology, because in vivo degradation of many proteins deviates from first-order kinetics. We consider this problem and discuss other applications of UPR.
Keywords
Amino Acids/analysis
Animals
Data Interpretation, Statistical
Half-Life
Kinetics
L Cells (Cell Line)
Mice
Protein Processing, Post-Translational
Proteins/genetics/*metabolism
Recombinant Fusion Proteins/genetics/metabolism
Ubiquitins/genetics/*metabolism
beta-Galactosidase/chemistry/genetics/metabolism
Pubmed
Web of science
Open Access
Yes
Create date
28/01/2008 12:17
Last modification date
20/08/2019 15:03