UC San Diego

As a part of protein quality control (PQC), the ubiquitin-proteosome system (UPS) ubiquitinates and degrades aberrant proteins. In this role, the UPS must identify damaged, orphaned, and misfolded species amidst a proteome-worth of normal, folded proteins. This exquisite specificity is achieved by PQC E3 ligases, each of which recognizes a broad range of PQC substrates. Alongside efforts to understand how PQC ligases effect selectivity, there has been sustained interest in PQC substrates and their distinguishing structural characteristics. Perhaps the best studied substrates are disease causative mutants, such as the most common cause of cystic fibrosis, CFTRΔF508. Numerous high-throughput screens have also aimed to identify “degrons,” discrete amino-acid sequences that elicit degradation. Despite these efforts, isolating and characterizing PQC substrates remains an exigent mode of inquiry in the field. Here, we present insights gained from PQC substrates both new and old. An initial study underlines the benefits of integrating transgenes onto yeast chromosomes: Stable integration of GFP-tagged proteins allows rapid and reliable quantitation of protein steady-state levels, a crucial indicator of substrate (in)stability. A second study demonstrates the value of one model, misfolded substrate, Sec61-2-GFP. Sec61-2-GFP allowed us to monitor each step of inner-nuclear-membrane-associated degradation (INMAD), from ubiquitination to retrotranslocation of the full-length protein. Sec61-2-GFP was also lethal in the combined absence of ERAD and INMAD, further evincing the elucidative power of appropriate model substrates. These studies and others justify the “Structure-Misfunction” screen, a platform to isolate minimally misfolded versions of cytosolic proteins. Tellingly, the screen seems to have uncovered a novel PQC pathway in the Saccharomyces cerevisiae cytosol. Structure-Misfunction analysis also provided insight into misfolding itself. Our studies show that different destabilizing point mutations within one domain can lead to entirely distinct PQC outcomes. These data suggest that minimal misfolding can cause “local” as opposed to “global” unfolding in vivo. The screen also identified mutants of chorismate mutase that can be stabilized by the allosteric effector tryptophan, a striking example of chemical chaperoning. Thus, the screen is a simple genetic approach to uncovering novel features of cell and structural biology.