Ydj1 interaction at nucleotide-binding-domain of yeast Ssa1 impacts Hsp90 collaboration andclient maturation
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Date
2022-11
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Abstract
Hsp90constitutes one of the major chaperone machinery in the cell. The Hsp70 assists
Hsp90inits client maturation though the underlying basis of the Hsp70 role remains to be
explored. In the present study, using S. cerevisiae strain expressing Ssa1 as sole Ssa
Hsp70, weidentified novel mutations in the nucleotide-binding domain of yeast Ssa1 Hsp70
(Ssa1-T175N andSsa1-D158N)that adversely affect the maturation of Hsp90 clients v-Src
andSte11. The identified Ssa1 amino acids critical for Hsp90 function were also found to be
conserved across species such as in E.coli DnaK and the constitutive Hsp70 isoform
(HspA8) in humans. These mutations are distal to the C-terminus of Hsp70, that primarily
mediates Hsp90 interaction through the bridge protein Sti1, and proximal to Ydj1 (Hsp40 co
chaperone of Hsp70 family) binding region. Intriguingly, we found that the bridge protein
Sti1 is critical for cellular viability in cells expressing Ssa1-T175N (A1-T175N) or Ssa1
D158N(A1-D158N)assoleSsaHsp70.Thegrowthdefectwasspecific forsti1Δ,as deletion
of none of the other Hsp90 co-chaperones showed lethality in A1-T175N or A1-D158N.
Mass-spectrometry based whole proteome analysis of A1-T175N cells lacking Sti1 showed
an altered abundance of various kinases and transcription factors suggesting compromised
Hsp90activity. Further proteomic analysis showed that pathways involved in signaling, sig
nal transduction, and protein phosphorylation are markedly downregulated in the A1-T175N
upon repressing Sti1 expression using doxycycline regulatable promoter. In contrast to
Ssa1, the homologous mutations in Ssa4 (Ssa4-T175N/D158N), the stress inducible Hsp70
isoform, supported cell growth even in the absence of Sti1. Overall, our data suggest that
Ydj1 competes with Hsp90 for binding to Hsp70, and thus regulates Hsp90 interaction with
the nucleotide-binding domain of Hsp70. The study thus provides new insight into the
Hsp70-mediated regulation of Hsp90 and broadens our understanding of the intricate com
plexities of the Hsp70-Hsp90 network.