Ubiquitin proteasome pathway is an important cellular pathway that affects the fate of almost all intracellular proteins. Misregulation of this pathway has been found to be associated with a broad range of human diseases, such as cancer, neurodegenerative diseases, as well as viral infections. Ubiquitin recognition by the proteasome is of central importance to this pathway. So far, two proteasome subunits, Rpn10 and Rpn13, have been identified as ubiquitin receptors. An alternative pathway is mediated by shuttling factors. In yeast, three shuttling factors, known as UBL-UBA proteins, have been found. A UBL receptor activity of the proteasome has been attributed to Rpn1. However, yeast cell mutated all five proteasomal ubiquitin receptors is still viable.
To identify the additional proteasomal ubiquitin receptor in cells, I first obtained and characterized a new Rpn13 mutant allele. This Rpn13 mutant completely abolished its ubiquitin binding activity, and functionally resembles a null allele. Rpn13 substrate pool has also been sought in this mutant cells.
In the second part of this dissertation, I reported a novel ubiquitin binding site on proteasomal subunit Rpn1. With the help of NMR analysis, Rpn1's ubiquitin and UBL binding surfaces were resolved at high resolution and found to substantially overlap. A specific Rpn1 mutation that disrupts both ubiquitin and UBL binding while not compromising the folding of Rpn1 was obtained. This mutant allele shows a pleiotropic proteasomal defect in vivo. Moreover, I found that the dual ubiquitin/UBL binding activity is not unique in Rpn1, but a common feature in all three proteasomal ubiquitin receptors. In summary, the proteasome adopts a multilayer ubiquitin/UBL binding surface to ensure flexible substrate recognition.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/13070024 |
| Date | January 2014 |
| Creators | Shi, Yuan |
| Contributors | Finley, Daniel J. |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | closed access |
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