Substitutions of Y66 in helix IV of the J-domain allow Ydj1<sub>134</sub> to support growth of <i>sis1-Δ</i>.

<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>