Substitutions of Y66 in helix IV of the J-domain allow Ydj1134 to support growth of sis1-Δ.
(A) (left) Cells lacking SIS1 (sis1-Δ), harboring a plasmid carrying an insert encoding WT Sis1 (SIS1), or Ydj1134 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. (right) Lysates prepared from the strains were subjected to immunoblotting using antibodies against the J-domain of Ydj1 or Ssc1 (control). (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. (C) 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). (D) (left) Ydj1 J-domain structure with Y66 and G70 (red) and residues with which Y66 interacts (yellow; F7, Y8, I56, L57) in sphere representation. (right) Most affected residues (i.e. those with the largest CSPs (red) and those missing signals in Ydj1109G70N (blue) as shown in Fig 3C) mapped on Ydj1 J-domain structure and in the sequence of the Gly-rich region.
