10.1371/journal.pgen.1007084.g003 Brenda A. Schilke Brenda A. Schilke Szymon J. Ciesielski Szymon J. Ciesielski Thomas Ziegelhoffer Thomas Ziegelhoffer Erina Kamiya Erina Kamiya Marco Tonelli Marco Tonelli Woonghee Lee Woonghee Lee Gabriel Cornilescu Gabriel Cornilescu Justin K. Hines Justin K. Hines John L. Markley John L. Markley Elizabeth A. Craig Elizabeth A. Craig Substitutions of Y66 in helix IV of the J-domain allow Ydj1<sub>134</sub> to support growth of <i>sis1-Δ</i>. Public Library of Science 2017 Hsp 70-substrate interaction cycle Hsp 70 J-protein specialization SSA 1 suppressor mutations Hsp 70 chaperone systems Ssa 1 function YDJ 1 suppressors Saccharomyces cerevisiae SIS 1 2017-10-30 17:28:46 Figure https://plos.figshare.com/articles/figure/Substitutions_of_Y66_in_helix_IV_of_the_J-domain_allow_Ydj1_sub_134_sub_to_support_growth_of_i_sis1-_i_/5550583 <p>(<b>A</b>) (<i>left</i>) Cells lacking <i>SIS1</i> (<i>sis1-</i>Δ), harboring a plasmid carrying an insert encoding WT Sis1 (<i>SIS1</i>), or Ydj1<sub>134</sub> with no substitutions (WT) or indicated substitution, were plated as 1:10 serial dilutions and grown at 30 or 34°C for 2 days, or at 37°C for 3 days. (<i>right</i>) Lysates prepared from the strains were subjected to immunoblotting using antibodies against the J-domain of Ydj1 or Ssc1 (control). (<b>B</b>) Diagram of Ydj1 J-domain/Gly-rich region with sequence and position of helices in segment encompassing residues 50–85 indicated; suppressor positions Y66 and G70 in red. (<b>C</b>) Averaged NMR structure of Ydj1 J-domain (N-terminal 70 residues) generated from coordinates of 20 lowest-energy conformers presented as ribbon representation and colored by secondary structure (gray for α-helix, green for unstructured regions). (<b>D</b>) (<i>left</i>) Ydj1 J-domain structure with Y66 and G70 (red) and residues with which Y66 interacts (yellow; F7, Y8, I56, L57) in sphere representation. (<i>right</i>) Most affected residues (i.e. those with the largest CSPs (red) and those missing signals in Ydj1<sub>109</sub>G70N (blue) as shown in Fig 3C) mapped on Ydj1 J-domain structure and in the sequence of the Gly-rich region.</p>