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The Rao Laboratory
 
The University of Texas Health Science Center, San Antonio, TX, USA
BioTechniques, Vol. 42, No. 2, February 2007, p. 125
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The Research

Our long-term goal is to understand the underlying mechanism and functions of the ubiquitin/proteasome system, which regulates the majority of regulated proteolysis in eukaryotes. Ubiquitin (Ub) is a small protein that can be covalently attached onto other cellular proteins. Many proteins marked by Ub are subsequently degraded by the proteasome. Specifically, we are interested in studying how the ubiquitylated substrates are delivered to the proteasome. It is now widely accepted that Ub-binding proteins (e.g., Rad23, Rpn10, and Cdc48) play important roles in this process. We use various genetic and biochemical approaches to characterize the function of Ub-binding proteins, such as Rad23 and Dsk2, in proteasome-mediated degradation. In addition, our laboratory is trying to understand the significance of proteolysis in neurodegenerative disorders, including prion diseases. Prion protein PrP is the cause of several devastating diseases, including mad cow disease and Creutzfeltd-Jacob disease. Mammalian PrP is known to be regulated by the ubiquitin/proteasome system. However, the specific pathway responsible for PrP degradation is not known. Our laboratory is using yeast as the model system to identify the degradation pathway for PrP, and we hope to understand the role of proteolysis in PrP-related disorders.

molecularmedicine.uthscsa.edu/FAC_Profile.asp?facID=81



The Technique

We study the ubiquitin/proteasome system using yeast as our primary model system. In the past a mutant strain was needed in order to observe the effects of small chemicals that induce proteasome inhibition. The need for a more user-friendly technique arose from complications endured with the mutant strain. Slow growth and inconsistent results with the erg6 mutant lead to our search for an alternative strategy; therefore we adapted a simple approach used in studying protein trafficking. With this method, small chemicals can be used effectively with wild-type yeast by simply modifying the growth media to contain an alternate nitrogen source and by adding a small amount of sodium dodecyl sulfate. Because of the simplicity of the technique, our data output has accelerated. Furthermore, monitoring the effects of proteasome inhibition in the presence of another knockout no longer requires two genes to be altered or knocked out, saving time and money. We have also used this technique to enrich proteins that are rapidly degraded for detecting their bindings to other proteins. Detecting ubiquitin conjugates sometimes requires enrichment with the use of proteasome inhibitors, and this technique allows for this without the use of a mutant. The ease and simplicity of our technique will greatly benefit any laboratory studying proteasome-mediated proteolysis in yeast. This method also allows simple chemical inhibition of the proteasome in wild-type yeast cells.

Proteasome inhibition in wild-type yeast Saccharomyces cerevisiae cells, p. 158.