Extended Data Fig. 6: The Hsp70–Hop interface in the GR-loading complex is crucial for cellular functions and client maturation.

a–f, The atomic interactions of Hsp70S_NBD:Hop_TRP2A:Hsp90B_MEEVD in the GR-loading complex. The cryo-EM map from focused classification and refinement is shown in (a and d, left). The atomic model with the corresponding view from (a and d, left) is shown in the right panels of (a, d). Close-up views of the Hsp70S_NTD:Hop_TPR2A interface with the atomic model fit into the density (b). Two key residues (Hop^Y296 and Hop^A328) are buried in the surface with their sidechain density indicated by the arrows (b, top and bottom). Density of the interface residues highlighted in Fig. 2b is shown in (b, bottom). Sequence alignments of Hop with key residues at the Hsp70_NBD:Hop_TPR2A interface highlighted by the red triangles in (c), in which the colour scheme is BLOSUM62. Close-up view of the atomic model of Hop_TPR2A:Hsp90B_MEEVD fit with the cryo-EM map is shown in (e). Close-up view of the atomic interactions of the MEEVD fragment from Hsp90B (light blue) and Hop_TPR2A (pink) from e is shown in f, in which polar interactions are depicted with dashed lines. g–j, In vivo validation of the Hsp70S_NBD:Hop_TPR2A interface in the loading complex. The two buried residues in Hsp70S_NBD-IIA:Hop_TPR2A interface, which were chosen for the mutational studies, are shown in g. Components of the GR-loading complex (g, top left) are coloured as in other figures and as labelled. Hsp70S_NBD is shown in surface-charge representation (blue: positive; red: negative) calculated using PyMOL. Note that the corresponding residue numbering of the two positions in yeast Hop (Sti1) are shown in parentheses of the labels of the bottom panels in g. In h, Sti1^Y332A-T364H accumulates at levels similar to WT Sti1 (see also Supplementary Fig. 10 for the uncropped gels/blots); data in h are representative of two independent experiments. Extracts from WT cells (JJ762), sti1 cells (JJ623), or sti1 cells transformed with a plasmid that expresses WT Sti1 or Sti1^Y332A-T364H were analysed by SDS–PAGE and immunoblotted with a polyclonal antisera specific for Sti1. Loading control is antibody against mitochondrial protein Tim44. In i, sti1-Y332A-T364H is inviable in hsc82hsp82 cells expressing hsc82-G309S. hsc82hsp82 (JJ117) or sti1hsc82hsp82 (JJ1443) strains harbouring YEp24-HSP82 were transformed with plasmids expressing WT HSC82 or hsc82-G309S. Strains that lacked STI1 were also transformed with an empty plasmid or a plasmid expressing WT STI1 or sti1-Y332A-T364H. Transformants were grown in the presence of 5-FOA for 3 days to counter-select for the YEp24-HSP82 plasmid. STI1 is essential under these conditions and the growth of cells expressing sti1-Y332A-T364H was indistinguishable from those expressing the empty plasmid. In j, WT cells, sti1 cells or sti1 cells transformed with a plasmid that expresses WT Sti1 or Sti1-Y332A-T364H were transformed with an empty plasmid or a plasmid that expresses GAL-v-src. v-src induction in the presence of galactose sharply reduces the growth of WT cells, but not cells lacking STI1. The growth of cells expressing sti1-Y332A-T364H was very similar to those expressing the empty plasmid, indicating that sti1-Y332A-T364H is unable to support v-src function. The growth of cells in the presence of glucose was indistinguishable. 10-fold serial dilutions of cultures were grown for 3 days in the presence of galactose or glucose.