The C-terminal domain of Hsp70 is responsible for paralog-specific regulation of ribonucleotide reductase

Abstract

The Hsp70 family of molecular chaperones is well-conserved and expressed in all organisms. In budding yeast, cells express four highly similar cytosolic Hsp70s Ssa1, 2, 3 and 4 which arose from gene duplication. Ssa1 and 2 are constitutively expressed while Ssa3 and 4 are induced upon heat shock. Recent evidence suggests that despite their amino acid similarity, these Ssas have unique roles in the cell. Here we examine the relative importance of Ssa1-4 in the regulation of the enzyme ribonucleotide reductase (RNR). We demonstrate that cells expressing either Ssa3 or Ssa4 as their sole Ssa are compromised for their resistance to DNA damaging agents and activation of DNA damage response (DDR)-regulated transcription. In addition, we show that the steady state levels and stability of RNR small subunits Rnr2 and Rnr4 are reduced in Ssa3 or Ssa4-expressing cells, a result of decreased Ssa-RNR interaction. Interaction between the Hsp70 co-chaperone Ydj1 and RNR is correspondingly decreased in cells only expressing Ssa3 and 4. Through studies of Ssa2/4 domain swap chimeras, we determined that the C-terminal domain of Ssas are the source of this functional specificity. Taking together, our work suggests a distinct role for Ssa paralogs in regulating DNA replication mediated by C-terminus sequence variation.

The APE2 Exonuclease Is a Client of the Hsp70–Hsp90 Axis in Yeast and Mammalian Cells

“…Several studies have suggested that these chaperone paralogs have overlapping but unique interactomes [ 34 ]. Recently, work using the model substrate ribonucleotide reductase (RNR) showed a clear preference for this client in binding Ssa1 and Ssa2 [ 35 ]. Although Apn2 binds cytosolic Hsp70 and Hsp90 paralogs equally, cells expressing Ssa1 as their sole cytosolic Ssa1 are unable to support WT levels of Ape2 as depicted by compromised stability in Figure 1 B,C.…”