Ubiquitination is a post-translational modification tht mediates sorting of integral membrane proteins to lysosomes for their degradation. ESCRTs (Endosomal Sorting Complex Required For Transport) bind and sequester ubiquitinated membrane proteins and direct them into multivesicular bodies (MVBs). ESCRTs themselves become covalently ubiquitinated, simply by virtue of non-covalently binding Ub. However, it is unclear whether this regulates a critical aspect of ESCRT function. In yeast, many MVB cargo proteins are ubiquitinated by the HECT-type Ub-ligase Rsp5, sometimes via the action of Rsp5 adaptor proteins. While many Rsp5 targets are modified by polyubiquitination, it remains unclear whether polyubiquitination is a necessary signal for their incorporation into MVBs. Despite years of research, these and related questions have been difficult to resolve because it is technically quite challenging to control the level of a given protein's ubiquitination. The aim of this research was to develop a novel technique, which can render proteins resistant to ubiquitination. The technique involved the fusion of the Ub-peptidase to a protein of interest via a flexible linker, essentially creating a "DUb module". The intent of this module would be to cleave any Ub form the target protein, essentially immunizing it from the effects of ubiquitination. This novel method was used in combination with several conventional methods to examine the role of ubiquitination within the endocytic pathway and in particular focus on the questions of what type of ubiquitin signal was sufficient for sorting into MVB vesicles and whether ubiquitination of ESCRTs was required for their sorting activity. We found that a single Ub was sufficient for membrane protein entry into MVBs in the absence of ESCRT ubiquitination.
| Identifer | oai:union.ndltd.org:uiowa.edu/oai:ir.uiowa.edu:etd-2674 |
| Date | 01 December 2010 |
| Creators | Stringer, Daniel Kenneth |
| Contributors | Piper, Robert C. |
| Publisher | University of Iowa |
| Source Sets | University of Iowa |
| Language | English |
| Detected Language | English |
| Type | dissertation |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | Copyright © 2010 Daniel Kenneth Stringer |
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