Radioactive Assay of in vitro Glutamylation Activity of the Legionella pneumophila Effector Protein SidJ

Abstract

The Legionella effector protein SidJ has recently been identified to perform polyglutamylation on another Legionella effector, SdeA, ablating SdeA’s activity. SidJ is a kinase-like protein that requires the small eukaryotic protein calmodulin to perform glutamylation. Glutamylation is a relatively uncommon type of post-translational modification, where the amino group of a free glutamate amino acid is covalently linked to the γ-carboxyl group of a glutamate sidechain in a substrate protein. This protocol describes the SidJ glutamylation reaction using radioactive [U-14C] glutamate and its substrate SdeA, the separation of proteins by gel electrophoresis, preparation of gels for radioactive exposure, and relative quantification of glutamylation activity. This procedure is useful for the identification of substrates for glutamylation, characterization of substrate and glutamylase activities due to mutations, and identification of proteins with glutamylation activity. Some studies have assayed glutamylation with the use of [3H] glutamate (Regnard et al., 1998) and the use of the GT335 antibody (Wolff et al., 1992). However, the use of [U-14C] glutamate requires a shorter radioactive exposure time with no dependence on antibody specificity.