L 14 c glutamate

About 20 uL in vitro glutamylation reactions were performed with 2uM SidJ/CaM and 2uM SdeA, 5 mM ATP, and 50 uM (0.5 nCi) l-[14 C]glutamate (Perkin Elmer) in a buffer consisting of 30 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM β-mercaptoethanol, and incubated at 37 °C for 30 min. Reactions were stopped through the addition of 5 uL of 4x SDS-PAGE sample buffer. The samples were then separated using SDS-PAGE, stained using Coomassie stain, and dried using a Model 583 gel dryer (Bio-Rad). A storage phosphor screen (GE Healthcare) was placed on the gel and exposed for 72 h, and the autoradiography signal was collected using a Typhoon FLA 7000 (GE). Raw autoradiography images of all glutamylation assays are available in the supplementary information file.

EGTA, EDTA, HEPES, Ficoll 400, Sigma-Fluor^® High Performance LSC Cocktail, the analytical grade salts were purchased from Sigma (USA); L-[¹⁴C]glutamate and [³H]GABA (γ-[2,3-3H(N)]-aminobutyric acid) were from Perkin Elmer (Waltham, MA, USA). Rhodamine 6G were obtained from Molecular Probes (USA).