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.
Highlights
[Background] Legionella pneumophila is an infectious bacteria that causes Legionnaires’ disease (McDade et al, 1977), a potentially fatal form of pneumonia
One process co-opted by Legionella is the ubiquitination system
Studies have demonstrated that the SidE family of proteins can perform phosphoribosyl ubiquitination of substrate proteins independent of E1 and E2 enzymes (Bhogaraju et al, 2016; Qiu et al, 2016; Kotewicz et al, 2017)
Summary
Review the flowchart of the experimental outline for SidJ radioactive glutamylation before beginning procedure (Figure 1). 2. Thaw recombinantly purified SidJ 89-853, SdeA 211-1152, and calmodulin on ice. Thaw radioactive glutamic acid stock at room temperature in a radioactive workspace. 3. Warm water bath to 37 °C and chill centrifuge to 4 °C (if possible). 4. Prepare stock solutions, on ice, listed in Table 1 by diluting in reaction buffer 5. Pipette the volumes of stock solution for a single 12.5 μl reaction listed in Table 2 into a chilled 1.7 ml microcentrifuge tube on ice. Move to radioactive workspace before the addition of [U-14C] glutamic acid. Vortex reaction gently, centrifuge briefly (approximately 10 s max speed), and incubate samples at 37 °C in a water bath for 30 min
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