Document Type


Date of Degree

Fall 2010

Degree Name

PhD (Doctor of Philosophy)

Degree In

Molecular Physiology and Biophysics

First Advisor

Piper, Robert C

First Committee Member

Stamnes, Mark

Second Committee Member

Snyder, Peter

Third Committee Member

Yeaman, Charles

Fourth Committee Member

Campbell, Kevin

Fifth Committee Member

Henry, Michael


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.


Endosome, ESCRT, Lysosome, Membrane Protein, MVB, Ubiquitin


2, xi, 219 pages


Includes bibliographical references (pages 203-219).


This thesis has been optimized for improved web viewing. If you require the original version, contact the University Archives at the University of Iowa:


Copyright © 2010 Daniel Kenneth Stringer

Included in

Biophysics Commons