FIGURE 4.

Toxic effect of Lgt1 in S. cerevisiae variants expressing wild type and mutated TEF1 genes. A, spot-test assay of growth phenotypes of S. cerevisiae containing plasmid-born wild type Tef1 and Tef1-S53A as the only eEF1A present in yeast cells. Yeast variants were transformed with pESC-Ura or pESC-Ura-based plasmids coding for wild type lgt1 and lgt1-D246A/W520A. Strains were analyzed as described under “Experimental Procedures” on SD (left panel, glucose, Glc) and SGal (right panel, galactose, Gal). The transformed variants of lgt1 and types of Tef1 in recipient yeast strains are indicated on the left. B, Western blotting, demonstrating Lgt1 production in S. cerevisiae strains transformed by pESC-Ura-based plasmids coding for wild type lgt1 and lgt1-D246A/W520A. Yeast cultures were grown in SD liquid medium (i.e. with glucose, lanes 1, 3, 5, and 7) or SGal (i.e. with galactose, lanes 2, 4, 6, and 8). Lanes 1 and 2, lgt1-WT in S. cerevisiae expressing wild type TEF1; lanes 3 and 4, lgt1-D246A/W520A in S. cerevisiae expressing wild type TEF1; lanes 5 and 6, lgt1 in S. cerevisiae expressing TEF1-S53A; lanes 7 and 8, lgt1-D246A/W520A in S. cerevisiae expressing Tef1-S53A. Western blotting was probed with anti-Lgt1 serum. The position of the glucosyltransferase is labeled with an asterisk. C, In vitro glucosylation of yeast extract prepared from S. cerevisiae containing Tef1-S53A and transformed with wild type lgt1. After cultivation in SGal, yeast cells were collected, disrupted by glass beads, and tested for Lgt1-dependent glucosylation in the ¹⁴C-glucosylation assay without (lane 1) and with (lane 2) addition of purified His-tagged Tef1. Coomassie-stained purified His-tagged Tef1 (lane 3) and molecular mass markers (lane 4) are shown. Molecular masses of used markers are shown on the right. The position of a protein band representing glucosylated Tef1 is labeled with an asterisk.