In vitro Glutamylation Inhibition of Ubiquitin Modification and Phosphoribosyl-Ubiquitin Ligation Mediated by Legionella pneumophila Effectors.

Glutamylation is a posttranslational modification where the amino group of a free glutamate amino acid is conjugated to the carboxyl group of a glutamate side chain within a target protein. SidJ is a Legionella kinase-like protein that has recently been identified to perform protein polyglutamylation of the Legionella SdeA Phosphoribosyl-Ubiquitin (PR-Ub) ligase to inhibit SdeA's activity. The attachment of multiple glutamate amino acids to the catalytic glutamate residue of SdeA by SidJ inhibits SdeA's modification of ubiquitin (Ub) and ligation activity. In this protocol, we will discuss a SidJ non-radioactive, in vitro glutamylation assay using its substrate SdeA. This will also include a second reaction to assay the inhibition of SdeA by using both modification of free Ub and ligation of ADP-ribosylated Ubiquitin (ADPR-Ub) to SdeA's substrate Rab33b. Prior to the identification and publication of SidJ's activity, no SdeA inhibition assays existed. Our group and others have demonstrated various methods to display inhibition of SdeA's activity. The alternatives include measurement of ADP-ribosylation of Ub using radioactive NAD, NAD hydrolysis, and Western blot analysis of HA-Ub ligation by SdeA. This protocol will describe the inhibition of both ubiquitin modification and the PR-Ub ligation by SdeA using inexpensive standard gels and Coomassie staining.