Regulated protein degradation in eukaryotes is performed predominantly by the ubiquitin-proteasome pathway. Prior to their degradation by the 26S proteasome, protein substrates become covalently modified with ubiquitin chains. Such ubiquitination enables the 19S regulatory particle of the proteasome to recognize the doomed protein substrate. My thesis research focuses on defining how the 19S regulatory particle of the proteasome recognizes ubiquitinated substrates. When I began my thesis research, S5a/Rpn10 was the only known proteasomal ubiquitin receptor; yet, it is not essential for degradation of ubiquitinated substrates by the proteasome. My thesis research helped establish Adrm1/ARM1/Rpn13 as the missing proteasomal ubiquitin receptor. I used NMR spectroscopy to provide mechanistic insights into how human Rpn13 binds ubiquitinated substrates. I adapted a protocol developed by the late Cecile Pickart to fine tune polyubiquitin synthesis for use in NMR structural studies. By using this approach, I selectively labeled individual subunits within polyubiquitin to determine Rpn13's selective binding to the proximal subunit of K48-linked diubiquitin (diUb). These results, along with additional results from my lab and our collaborators' labs, are described in Chapters 2 and 3.I continued to explore Rpn13's role as a ubiquitin receptor by testing whether it is able to work in cooperation with S5a/Rpn10. Along with other members of my lab, we solved the structure the S5a/K48-linked diUb complex by utilizing the method I optimized for synthesizing selectively labeled polyubiquitin. I then helped expand this work to reveal that Rpn13 and S5a are able to bind a common ubiquitin chain and thereby work cooperatively to capture ubiquitinated substrates. These results are described in Chapter 4.In Chapter 5, I describe a novel interaction of Rpn13's Pru domain to a ubiquitin processing enzyme, namely E2 ubiquitin conjugating enzyme Cdc34. NMR experiments reveal that an Rpn13 surface that neighbors its ubiquitin-binding loops binds to Cdc34's unique C-terminal tail and that this interaction does not restrict Rpn13 binding to ubiquitin. Immunoprecipitation experiments performed on HeLa cells with endogenous protein levels demonstrate Rpn13 and Cdc34 to be in complex in the cellular environment. The Rpn13:Cdc34 interaction suggests that Rpn13 may play a role in SCF-mediated ubiquitination. Proteasome dysfunction is implicated in many diseases, such as cancer, cystic fibrosis, heart disease, and neurodegenerative diseases and the ubiquitin-proteasome pathway has therefore become a major pharmaceutical target. My thesis research in trying to understand how the proteasome recognizes ubiquitinated substrates provides structural and mechanistic information that could be used to help develop new drugs and possible treatments for these diseases.