Regulation of the Rsp5 HECT Domain Ubiquitin Ligase

Michael French

doi:https://doi.org/10.21985/N2943T

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Regulation of the Rsp5 HECT Domain Ubiquitin Ligase

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Rsp5 is a ubiquitin ligase that controls a broad range of cellular processes in budding yeast and is part of a large family of proteins that controls analogous processes in mammalian cells. Although Rsp5 targets a number of different substrates for ubiquitination, the mechanisms that regulate Rsp5 catalytic activity are poorly characterized. This dissertation examines several previously unknown molecular factors that are likely to play a role in the regulation of Rsp5 activity. The first is the presence of a noncovalent ubiquitin-binding site located in the catalytic HECT domain of Rsp5. Protein interaction studies and mutagenesis were used to demonstrate that the N-terminal lobe of the HECT domain mediates binding to ubiquitin, and the results of in vivo growth assays and in vitro ubiquitination assays indicated that the Rsp5 ubiquitin-binding site regulates the ability of the Rsp5 HECT domain to assemble polyubiquitin chains. The second factor likely to regulate Rsp5 activity is an intramolecular interaction between the WW and catalytic HECT domains of the ligase. Finally, phosphorylation of Rsp5 is examined as a potential mechanism of regulation, and a kinase responsible for the phosphorylation known as Cbk1 is identified. The results of these studies help contribute to our understanding of the mechanisms that regulate the activity of Rsp5 and related ubiquitin ligases. Mammalian homologues of Rsp5 such as Nedd4 and Itch are critical regulators of many important biological processes, and the identification of regulatory factors that control the catalytic activity of these ubiquitin ligases is critical to understanding how these enzymes carry out their functions. The results described in this thesis suggest that the activity of Rsp5 is regulated by at least three different factors, and recent work from other labs indicates that additional regulatory factors exist. Finally, recent data indicates that mammalian homologues of Rsp5 are regulated by mechanisms that are similar to those described in this dissertation, suggesting that the results of these studies will be generally applicable to understanding how this family of ubiquitin ligases functions in higher eukaryotes.

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