Assaying ATE1 Activity in Yeast by β-Gal Degradation.
In 1980s it was found that addition of N-terminal Arg to proteins induces their ubiquitination and degradation by the N-end rule pathway. While this mechanism applies only to the proteins which also have other features of the N-degron (including a closely adjacent Lys that is accessible for ubiquitination), several test substrates have been found to follow this mechanism very efficiently after ATE1-dependent arginylation. Such property enabled researchers to test ATE1 activity in cells indirectly by assaying for the degradation of such arginylation-dependent substrates. The most commonly used substrate for this assay is E. coli beta galactosidase (beta-Gal) because its activity can be easily measured using standardized colorimetric assays. Here we describe this method, which has served as a quick and easy way to characterize ATE1 activity during identification of arginyltransferases in different species.
Department of Animal Biology School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA, 19104, USA, akashina@vet.upenn.edu.
Anna S
AS
Kashina
D019881:Aminoacyltransferases / Q000378:metabolism*; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods; D059748:Proteolysis; D014170:Transformation, Genetic; D015003:Yeasts / Q000201:enzymology / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism*
D019881:Aminoacyltransferases; C019727:arginyltransferase; D001616:beta-Galactosidase