Random mutagenesis identifies residues in the unstructured regions of Htz1 that are required for function.

<p><b>[A]</b> Overview of random mutagenesis screen. Yeast strain CMY307 containing <i>P</i>_<i>GAL1</i>-regulated <i>HA-HTZ1</i> and <i>asf1∆</i> was transformed with a library of randomly mutated <i>htz1</i> alleles. Individual clones were picked into 96-well plates and pre-grown in glucose for 72H to repress <i>P</i>_<i>GAL1</i><i>-HA-HTZ1</i> before plating in duplicate onto SG-Ura and SD-Ura to assess growth in the presence and absence of WT Htz1. CMY307 transformed with plasmids carrying WT Htz1 (black box) or empty vector (blue box) were included as controls on each plate. An example of a glucose-sensitive clone is shown boxed in red. <b>[B]</b> Western blot of protein lysates prepared from CMY307 grown in galactose and at various time-points after glucose addition show that WT HA-Htz1 protein is undetectable by 48H. <b>[C]</b> Frequency of mutations at each Htz1 residue recovered from non-functional alleles identified by screening as described in <b>[A]</b>. The coloured bars in the graph represent residues within known structured regions. A schematic of H2A.Z protein structure, where boxes depict α-helices, the black bars indicate unstructured regions and the M6 region is boxed in pink, is shown. The N- and C-terminal tails and the inter-helical loops (LN, L1, L2, LC) are also labelled and residues I109 and S111 are indicated with red arrows. <b>[D]</b> The frequency of mutations at each residue in a sample of clones from the random mutagenesis library that were not screened for Htz1 function in yeast. <b>[E]</b> Summary of mutation frequencies per residue in each structural region of Htz1. The protein sequence was divided into regions including the N- and C-termini, the various alpha helices and the inter-helical loops, as indicated in <b>[C]</b>. The mutation frequency for each region was normalised to the number of residues in that region to allow comparison. S1 (serine 1) was treated separately as most of the N-terminal mutations were at this residue.