Blocking protein quality control degradation leads to structural stabilization of <scp>DHFR</scp> indel variants

Gene variants leading to insertions or deletions of amino acid residues (indels) often have detrimental consequences for the folding of the encoded protein. Yet, at some positions, indels are tolerated or result merely in partial unfolding and hypomorphic (reduced function) phenotypes. Typically, unfolded proteins are targeted for protein quality control (PQC) degradation via the ubiquitin‐proteasome system, which in yeast is mediated by specific E3 ubiquitin‐protein ligases, including Ubr1 and San1. Here, we systematically probed the folding of a library of indel variants in the DHFR protein using a sensitive yeast‐based protein folding reporter. We show that deletion of Ubr1 or San1 leads to a greater fraction of folded DHFR indel variants, primarily positioned toward the N‐ and C‐terminal regions in DHFR. Intriguingly, most of the DHFR indels that are structurally stabilized in the E3 knockout strains are also stabilized at lowered temperatures and upon binding the DHFR inhibitor methotrexate. This suggests that blocking PQC degradation can restore sufficient folding and increase function of hypomorph variants, thus providing a potential therapeutic avenue for protein misfolding diseases.