Using ubiquitin to follow the metabolic fate of a protein.
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.
Lévy
F
F
Division of Biology, California Institute of Technology, Pasadena, 91125, USA.
Johnsson
N
N
Rümenapf
T
T
Varshavsky
A
A
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003627:Data Interpretation, Statistical; D006207:Half-Life; D007700:Kinetics; D007739:L Cells; D051379:Mice; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D001616:beta-Galactosidase