IN VITRO UBIQUITINATION OF AN ARRESTIN-RELATED TRAFFICKING ADAPTOR BY THE SCHIZOSACCHAROMYCES POMBE E3 LIGASE PUB1
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The ubiquitination of proteins at the plasma membrane (PM) serves as an endocytosis signal in the downregulation of PM protein levels and cellular responses to their environment. In Saccharomyces cerevisiae, the E3 ubiquitin ligase Rsp5 ubiquitinates its PM targets by forming complexes with arrestin-related trafficking (ART) adaptor proteins that enable Rsp5 to recognize specific sets of PM proteins. The range of specificity for some of the ARTs has been uncovered in yeast models, but the molecular mechanisms by which the ART proteins present substrates to the Rsp5 catalytic domain for ubiquitination remains poorly understood. The ubiquitination of Art1 on lysine residue K486 by Rsp5 has been shown to play a critical role in the Art1-Rsp5 complex’s function, which suggests that this adaptor ubiquitination likely plays a role in stabilization of the Art1-Rsp5 complex or in forming the correct complex structure to enable recognition of substrates. To better understand the mechanism by which Art1 presents PM substrates to Rsp5, and to uncover the functional significance of Art1 ubiquitination, our lab seeks to structurally characterize the Art1-Rsp5 complex. Here I report the purification and biochemical characterization of Pub1 and Any1, the homologous E3-adaptor pair from S. pombe, and an approach to generate the ubiquitinated form of Any1 in vitro for the purposes of structural studies and further biochemical investigation.
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2019-08-30
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Ubiquitin; adapter; Art1; NEDD4; Rsp5; E3 Ligase; Biochemistry
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Mao, Yuxin
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Sondermann, Holger
Emr, Scott David
Emr, Scott David
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Biochemistry, Molecular and Cell Biology
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M.S., Biochemistry, Molecular and Cell Biology
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Master of Science
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dissertation or thesis