Hsp70 nucleotide exchange factor Fes1 is essential for ubiquitin-dependent degradation of misfolded cytosolic proteins

Abstract

Protein quality control systems protect cells against the accumulation of toxic misfolded proteins by promoting their selective degradation. Malfunctions of quality control systems are linked to aging and neurodegenerative disease. Folding of polypeptides is facilitated by the association of 70 kDa Heat shock protein (Hsp70) molecular chaperones. If folding cannot be achieved, Hsp70 interacts with ubiquitylation enzymes that promote the proteasomal degradation of the misfolded protein. However, the factors that direct Hsp70 substrates toward the degradation machinery have remained unknown. Here, we identify Fes1, an Hsp70 nucleotide exchange factor of hitherto unclear physiological function, as a cytosolic triaging factor that promotes proteasomal degradation of misfolded proteins. Fes1 selectively interacts with misfolded proteins bound by Hsp70 and triggers their release from the chaperone. In the absence of Fes1, misfolded proteins fail to undergo polyubiquitylation, aggregate, and induce a strong heat shock response. Our findings reveal that Hsp70 direct proteins toward either folding or degradation by using distinct nucleotide exchange factors.

Fig. 1.

Inactivation of FES1 results in hypersensitivity to induced protein misfolding and strong activation of the heat shock response. (A) fes1Δ strains are hypersensitive to the amino acid analogs azetidine-2-carboxylic acid (AzC, 0.1 μM) and canavanine (Can, 3.6 mM). (B) Total protein extracts from WT and fes1Δ cells grown in YPD at 25 °C separated by SDS/PAGE and stained with Coomassie Brilliant Blue. The arrows mark the positions of induced Hsp70, Hsp90, and Hsp104 (Results). (C) Western analysis of HA-epitope tagged Hsp70s Ssa1, Ssa2, Ssa3, and Ssa4 expressed from their endogenous loci in WT and fes1Δ cells. Protein extract gel loading was adjusted as indicated (%). (D) Hsf1-Myc hypo- and hyperphosphorylation status in WT and fes1Δ cells was monitored using a gel shift assay based on Phos-Tag. Heat shocked (HS) cells were transferred from 25 °C to 37 °C for 30 min. Standard SDS/PAGE (Right). (E) Heat shock induction in Hsp70 NEF single- and double-mutant strains analyzed as in B and growth phenotypes at different temperatures.

Fig. 2.

Fes1 is required for UPS degradation of misfolded DHFR mutants. (A, B) Two folding-deficient variants of DHFR (DHFR^MutC-Ura3, DHFR^MutD-Ura3) fused to Ura3 were expressed from the P_CUP1 promoter (Fig. S3) in isogenic WT, fes1∆, sse1∆, and snl1∆ strains. (C) A third substrate (Ub-V76-Ura3) is a noncleavable ubiquitin fusion protein that is targeted by the UFD pathway. Following cycloheximide (CH) addition, extracts were prepared by glass bead lysis of cells collected at the indicated time points and analyzed by quantitative Western blot. The data represent at least three experiments with independent transformants (error bars indicate SD).

Fig. 3.

Fes1 interacts with and is required for UPS degradation of Hsp70-associated misfolded protein Rpo41^T920-L1217. (A) Growth on medium selective for interaction of a yeast two-hybrid reporter strain expressing Fes1 or the Hsp70-binding mutant Fes1^{A79R, R195A} as baits and Hsp70 (SSA4), Hsp40 (SIS1), or a fragment of RPO41 as preys Vector control (VC). The isolated RPO41 expresses a truncated polypeptide (T920-L1217) that corresponds to an internal region of the C-terminal domain (CTD) of mitochondrial RNA polymerase (marked in black in the crystal structure of human homolog). The N-terminal domain (NTD) is indicated. (B) Western analysis of total lysate (T) and supernatant (S) and pellet (P) fractions following ultracentrifugation (541,000 × g). Pgk1 functions as a soluble control protein. (C) Degradation of Rpo41^T920-L1217 in WT and fes1Δ cells after translation arrest by cycloheximide (CHX). (D) Degradation at a restrictive temperature (37 °C) as in C in strains carrying mutations in Hsp70 (ssa1-45 ssa2Δ ssa3Δ ssa4Δ), the proteasome (cim3-1) or the ubiquitin E3 ligase Ubr1 (ubr1Δ). (E) Degradation as in C in strains carrying Hsp70 binding (A79R, R195A) mutations in the chromosomal FES1 locus. (F) UPS degradation (CHX) of transcription factor Stp1-HA (Stp1). Induced Hsp70-dependent ubiquitin-proteasome degradation of fructose-1,6-bisphosphatase (Fbp1-HA) followed after addition of 2% glucose to Yeast extract peptone-ethanol medium.

Fig. 4.

Fes1 is required for the release of misfolded proteins from Hsp70 and their targeting to the UPS. (A) Western blot (WB) analysis of amounts of Hsp70 Ssa2-HA copurifying with DHFR^MutC-FLAG immunoprecipitated (IP) from WT and fes1Δ protein extracts. (B) Western analysis of total polyubiquitin levels (Ubi_(n)) in WT, fes1Δ, and sse1Δ cells grown at 25 °C or heat-shocked (HS) at 42 °C for 30 min. Loading was normalized to amount of cells used for denaturing protein extraction. (C) WB analysis of amounts of Ubr1-HA copurifying with DHFR^MutD-FLAG IP from WT and fes1Δ protein extracts. Quantification of the amount of Ubr1-HA copurifying with DHFR^MutD-FLAG normalized to WT (100%) (error bars represent SD of experiments performed with five independent transformants). (D) Growth of cells (fes1Δ) transformed with vector control (vc) or plasmids expressing Fes1, Snl1^cat, or Sse1. Heat shock response of each transformant was assessed by immunoblotting (Hsp104). (E) UPS degradation after cycloheximide (CHX) of Rpo41^T920-L1217 followed in fes1Δ cells expressing Fes1, Snl1^cat or Sse1.

Fig. 5.

Model for Hsp70 substrate triage regulated by NEFs (Discussion).