This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The ubiquitin proteasome system (UPS) governs most of the regulated proteolysis in eukaryotes. Substrates destined for proteasomal degradation are often modified with ubiquitin. The ubiquitin is attached to these proteins by a series of enzymes called E1, E2, and E3. A degron is a primary degradation signal of UPS substrates that is recognized by E3 enzymes or chaperones. We have designed a high-resolution strategy to map the sequence-function relationship of known degrons and to discover degrons in a systematic and high throughput manner. We will combine simple genetic tools with high-throughput sequencing to characterize the degradation signal. Our system is based on the fact that yeast cells that express Ura3 die in the presence of 5-FOA because Ura3 converts 5-FOA into a toxic product. We can alter the stability of Ura3 by fusing it to degrons, which can affect the sensitivity of yeast to 5-FOA. We are currently optimizing this system using the well-characterized degradation signal Deg1 from Matα2 fused to Ura3. This fusion protein is under the control of galactose-regulatable promoter. After the expression of the Ura3-degron fusion is shut off by addition of glucose to the media, the stability of the Ura3-degron fusion in the cells is determined by testing how sensitive the cells are to 5-FOA. We plan to generate a library of mutant Deg1 degrons fused to Ura3 to determine the effect of mutations in the degron for their ability to destabilize Ura3. Cells that harbor functional Ura3-degron fusions will degrade the Ura3 and grow in media containing 5-FOA, and cells that express non-functional degrons will be lost. By comparing the sequences of degrons in the selected yeast to the input culture we will gain insight into how mutations affect the stability of Ura3.
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