Exploring the ubiquitin-proteasome protein degradation pathway in yeast

✍ Scribed by Tamara J. Will; Melissa K. McWatters; Kristi L. McQuade

• Publisher: The American Society for Biochemistry and Molecular Biology
• Year: 2006
• Tongue: English
• Weight: 74 KB
• Volume: 34
• Category: Article
• ISSN: 1470-8175
• DOI: 10.1002/bmb.2006.494034062683

✦ Synopsis

Abstract

This article describes an undergraduate biochemistry laboratory investigating the ubiquitin‐proteasome pathway in yeast. In this exercise, the enzyme β‐galactosidase (β‐gal) is expressed in yeast under the control of a stress response promoter. Following exposure to heat stress to induce β‐gal expression, cycloheximide is added to halt translation, and β‐gal degradation is monitored by measuring enzyme activity as a function of time. Students observe that an N‐Ile‐β‐gal variant with an N‐terminal isoleucine has a significantly lower stability than wild‐type β‐gal, whose N‐terminal residue is methionine. This strong dependence of protein stability on the N‐terminal residue is known as the “N‐end rule.” To corroborate the enzyme activity assay results, students perform denaturing protein electrophoresis and immunoblotting of lysates, observing that the time‐dependent loss of enzyme activity is coincident with the disappearance of the β‐gal protein.