Analysis of <i>smt3</i> SIM binding mutant alleles.

<p>(A) The identified lethal and conditional <i>smt3</i> mutants found within the SIM binding surface mapped onto the β2-α1 region of Smt3 (PDB: 1EUV). Residues that gave rise to lethal and conditional phenotypes are boxed in red and magenta, respectively. (B) SIM binding mutants are stress sensitive. The indicated strains were cultured overnight and serially diluted and spotted onto plates with or without hydroxyurea (HU). The plates were incubated at 30°C or 39°C, as indicated. (C) The <i>smt3</i> I35A mutant allele accumulates unusual conjugates. Wild type and <i>smt3</i> I35A strains were grown to mid-log phase, diluted 4 fold then shifted to 39°C for 20 hrs. Samples were collected at 0 and 20 hr time points and analyzed by immunoblot analysis. The stacking portion of the gel was left intact so that ultra-high molecular mass conjugates could be visualized. (D) The <i>smt3</i> I35A mutant allele exhibits cell cycle defects. Wild type and <i>smt3</i> I35A strains were grown to mid-log phase at 30°C and then shifted to 39°C for 20 hrs. Cells were collected at 20 hrs, permeabilized and stained with DAPI. Each bar represents the average of 3 independent experiments in which at least 150 cells were counted per experiment. Vertical bars indicate the standard error. Astericks denote a p-value < 0.05. (E) Phosphorylation of T42 and T43 is not critical for <i>smt3</i> function. The <i>smt3Δ</i> shuffle strain was transformed with plasmids coding for the indicated <i>Smt3</i> alleles. The transformants were grown overnight, serially diluted and spotted onto selective media in the presence or absence of 5-FOA at 30°C and 37°C.