Description
TitleCharacterization of Rad23, as a shuttle factor
Date Created2019
Other Date2019-10 (degree)
Extent1 online resource (xiii, 189 pages) : illustrations
DescriptionThe ubiquitin proteasome pathway (UPP) is the primary proteolytic system for the spatial and temporal elimination of intracellular proteins, and is conserved from yeast to humans. Proteins that are targeted for degradation become covalently linked to a small protein called ubiquitin and are subsequently degraded by the 26S proteasome. Key enzymes of this pathway and their function are well characterized, but the regulation of its activities is not well understood. For instance, it is widely believed that nuclear proteins are degraded inside the nucleus despite the evidence that some nuclear proteins are degraded following their export. There were a number of discoveries regarding the site of protein turnover. First, the Madura group and others reported that the degradation of some nuclear proteins required export from the nucleus. Second, substrates were stabilized inside the nucleus when nuclear export was blocked, strongly suggesting that proteasomes do not operate inside the nucleus. Third, it was reported that catalytically active proteasomes exist predominantly in the cytosol, since purified nuclei lacked proteasome peptidase activity. Fourth, it was determined that Sts1 plays a central role in proteasome localization. Through its interaction with Srp1 (an importin-α protein), and Rpn11 (a 19S proteasome subunit), Sts1 appears to localize proteasomes to the nuclear surface. Fifth, Srp1 was shown to harbor two distinct functions; it can act as nuclear import factor, and can tether the proteasome to the nuclear surface. These findings provide a basis for a mechanism for targeting proteasomes to the nucleus. Sixth, Rad23 functions as a shuttle factor that can translocate ubiquitylated proteins to the proteasome, and is known to control the stability of the nuclear protein Rad4. The shuttle factor Rad23 is present in both the nucleus and cytosol. This suggests that Rad23 might deliver nuclear substrates to cytosolic proteasomes. Using two genetic mutants, I tested this hypothesis and found that Rad23 bound high levels of polyUb substrates when it was trapped inside the nucleus. In contrast, when it was trapped in the cytosol it interacted with low levels of polyUb substrates. Whereas previous studies examined artificial substrates, I confirmed this binding pattern using the physiological substrate Ho endonuclease, which functions in mating type switching in yeast. Therefore I propose that the function of Rad23 is to deliver nuclear substrate to proteasomes in the cytosol. The mode of nucleocytoplasmic trafficking of Rad23 remains to be discovered.
NotePh.D.
NoteIncludes bibliographical references
Genretheses, ETD doctoral
LanguageEnglish
CollectionSchool of Graduate Studies Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.