Figure 2. FIGURE 2: Overexpression of tRNAs subject to Urm1-dependent U34 thiolation fails to suppress loss of Ahp1 urmylation at 39°C.

(A) Urmylation of Ahp1 is suppressed at 39°C. An urm1∆ strain expressing TAP-URM1 was grown to logarithmic growth phase and split into two cultures prior to cultivation at 30°C or 39°C for three hours (h). Ahp1 urmylation analysis involved anti-TAP-based EMSA and immune blots with anti-Ahp1 to detect non-conjugated (free) Ahp1. Protein loading was controlled with anti-Cdc19 antibodies.

(B) tRNA^Gln, tRNA^Lys and tRNA^Glu (tQKE) overexpression cannot rescue defective Ahp1 urmylation at 39°C. Except for tQKE overexpression from multi-copy vector pQKE (Table S3), cell growth at 30°C or 39°C and urmylation analysis were as described in (A). Arrows (A, B) indicate the positions of non-conjugated (free) forms of TAP-Urm1 and Ahp1, as well as TAP-Urm1 conjugated to Ahp1 and loading control Cdc19.