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Assaying ATE1 Activity in Yeast by β-Gal Degradation

  • Anna S. KashinaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1337)

Abstract

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.

Key words

Arginyltransferase Yeast complementation Beta galactosidase N-end rule Ubiquitin Proteasome 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Animal Biology School of Veterinary MedicineUniversity of PennsylvaniaPhiladelphiaUSA

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