Figure 4. Kinetics of eEF1A glucosylation by Lgt3.

Time courses of glucosylation of purified yeast eEF1A (yEF1A) (A) or mouse eEF1A (mEF1A) (B) by Lgt3 in the absence (triangles) or presence of Phe-tRNA^Phe (filled circles) or uncharged tRNA^Phe (open circles). The glucosylation reactions and analyses were performed under the conditions as described in Figure 1. Values of ¹⁴C-glucose incorporation are shown as the mean (±SD) of at least three independent experiments. The inserts show representative radioimages. (C) eEF1A ternary complex is quantitatively glucosylated by Lgt3. Yeast eEF1A (20 µM) was glucosylated by Lgt3 (5 µM) with Phe-tRNA^Phe (20 µM) and GTP (75 µM) in the absence (lane 1) or presence (lane 2) of cold UDP-glucose (1 mM). Modified and unmodified yEF1A were separated by His-eEF1Bα affinity purification as described in Materials and Methods. Thereafter, the isolated eEF1A (3 µM each) was subjected to second glucosylation reaction with radiolabeled UDP-[¹⁴C]glucose in presence of Phe-tRNA^Phe (1 µM) and GTP (75 µM). The Coomassie gel and the autoradiogram are shown. (D) Glucosylation of yEF1A (2 µM) was performed in the presence of His-tRNA^His or uncharged yeast tRNA with Lgt3 (140 nM).