Genetic Inhibition of the Ubiquitin-Proteasome Pathway: Insights Into Proteasomal Targeting

Citation

Ghaboosi, Nazli (2007) Genetic Inhibition of the Ubiquitin-Proteasome Pathway: Insights Into Proteasomal Targeting. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/PNPS-JR40. https://resolver.caltech.edu/CaltechETD:etd-05242007-234932

Abstract

Regulated proteolysis plays a major role in diverse cellular processes, including cell-cycle progression, endocytosis, apoptosis, transcription, and signal transduction. Proteins destined for proteolysis undergo two key steps in the ubiquitin-proteasome pathway. First, a complex enzymatic cascade controls conjugation of a multiubiquitin chain onto a protein. Once ubiquitinated, protein substrates must be recognized and targeted to the proteasome complex, where they are unfolded and degraded. Receptor proteins, such as Rad23, recognize ubiquitinated proteins and deliver them to the proteasome.

While the list of enzymes involved in ubiquitination is steadily growing, the first enzymatic reaction required for all ubiquitin-dependent processes is catalyzed by one protein, the ubiquitin-activating enzyme, E1. In this work, we describe a genetic screen that targets the Saccharomyces cerevisiae E1 gene, UBA1. We report the isolation of uba1-204, a temperature-sensitive allele UBA1 that exhibits dramatic inhibition of the ubiquitin-proteasome pathway. Shifting mutant cells to the restrictive temperature results in the depletion of cellular ubiquitin conjugates within minutes, accompanied by stabilization of multiple protein substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play the recognition and delivery of substrates to the proteasome.

It is possible to purify intact and active proteasomes complexes from uba1-204 cells. In the absence of ubiquitin activation, these proteasome complexes are depleted of ubiquitin conjugates and the ubiquitin-binding receptor proteins Rad23 and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo. As an additional example of the value of this novel genetic mutant in the study of the ubiquitin-proteasome system, we present preliminary results from a quantitative mass spectrometric analysis of the proteins associated with proteasome complexes isolated from uba1-204 cells.

In summary, we have created a genetic method of rapidly inhibiting the ubiquitin-proteasome system. This will enable future exploration of the ubiquitin-proteasome system and potentially many other ubiquitin-dependent cellular processes.

Thesis Files