HSP70-binding motifs function as protein quality control degrons

Abstract

Protein quality control (PQC) degrons are short protein segments that target misfolded proteins for degradation through the ubiquitin-proteasome system (UPS). To uncover how PQC degrons function, we performed a screen in Saccharomyces cerevisiae by fusing a library of flexible tetrapeptides to the C-terminus of the Ura3-HA-GFP reporter. The identified degrons exhibited high sequence variation but with marked hydrophobicity. Notably, the best scoring degrons constitute predicted Hsp70-binding motifs. When directly tested, a canonical Hsp70 binding motif (RLLL) functioned as a dose-dependent PQC degron that was targeted by Hsp70, Hsp110, Fes1, several Hsp40 J-domain co-chaperones and the PQC E3 ligase Ubr1. Our results suggest that multiple PQC degrons overlap with chaperone-binding sites and that PQC-linked degradation achieves specificity via chaperone binding. Thus, the PQC system has evolved to exploit the intrinsic capacity of chaperones to recognize misfolded proteins, thereby placing them at the nexus of protein folding and degradation.

Prediction of quality-control degradation signals in yeast proteins

“…Most notably, we find that the presence of the negatively charged side chains in Asp and Glu counteract degradation whereas the positively charged side chains in Lys and Arg have a more neutral effect. These results complement our recent observation that Hsp70 binding motifs, which are often hydrophobic and positively charged, can act as degrons 17, 27 . The effects of hydrophobic, positively and negatively charged residues are also validated by experiments that show that replacing hydrophobic residues with Glu in a degron can prevent degradation, whereas replacing the same amino acids with Arg has a substantially smaller effect 18 .…”

“…First, in the library we have screened we only vary 17-residue long peptides, and so our model only predicts degrons that fit within this length. More importantly, an unfolded or misfolded protein may have multiple degrons and we have previously shown that an increased number of exposed degrons lead to progressively lower abundance (Abildgaard et al, 2021). Thus, future work should quantify the relationship between the length and number of degrons and protein abundance.…”

“…These experiments revealed a large breadth in the sequences that cause degradation without any obvious individual motifs. More recently, we used the same reporter system to show that Hsp70-binding motifs may function as PQC degrons (Abildgaard et al, 2021). Specifically, we found that inserting known Hsp70 binding motifs into the reporter system led to Hsp70-dependent degradation.…”