| Protein quality control (PQC) systems selectively target misfolded, or otherwise aberrant proteins for ubiquitin/proteasome-mediated degradation. How cells discern abnormal from normal proteins remains largely unknown. Specific recognition is thought to occur between PQC machinery and the degradation signals (degrons) within PQC substrates, allowing cells to selectively target degron-presenting proteins for degradation, while sparing others lacking such signals. The unifying goal of my thesis work has been to understand the specificity in ubiquitin/proteasome-mediated degradation of cytosolic protein quality control (CytoQC) substrates. To investigate both the machinery and the signals that target aberrant proteins for degradation, I significantly expanded the number of model degrons available for analysis. Using a URA3-based classical genetic screen, I isolated and characterized a comprehensive library of 109 novel degrons that target cytosolic Ura3p for proteasome-dependent degradation. To probe known PQC machinery for roles in recognizing these novel degrons, I established and validated a high-throughput system to rapidly and methodically interrogate candidate genes, such as those encoding E3 ubiquitin ligases. Using this system in conjunction with traditional techniques for monitoring protein degradation, I identified degrons for the known PQC E3 ligases Doa10p, San1p, Ubr1p, and Rkr1p. Notably, loss of Doa10p, an ER membrane-bound E3, stabilizes about half of my Ura3p-degron proteins. This finding, combined with our previously published work, suggests that the ER membrane serves as a platform for the ubiquitination of certain CytoQC substrates, but clearly not for all. Also notable, I found that many of my novel degrons target Ura3p for Rkr1p-dependent degradation. Rkr1p ubiquitinates nonstop proteins, whose translation causes ribosome stalling during synthesis of polylysine from the poly(A) tail. My constructs, however, contain stop codons which I show are correctly recognized by the ribosome. Importantly, I demonstrate that these sequences, which are neither polylysine nor any other polymeric sequence, do not induce ribosome stalling. My results indicate that Rkr1p can mediate the degradation of proteins other than those containing ribosome stall-inducing sequences, revealing additional roles for Rkr1p in PQC. Overall, my results suggest that the degron library I generated and the analytical approach I developed will be a valuable resource for the PQC field. |
