title: Exploring the ubiquitin-proteasome protein degradation pathway in yeast.
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.
authors.affiliation: Department of Chemistry and Biochemistry, Bradley University, Peoria, Illinois 61625.
authors.forename: Tamara J
authors.initials: TJ
authors.lastname: Will
authors.forename: Melissa K
authors.initials: MK
authors.lastname: McWatters
authors.forename: Kristi L
authors.initials: KL
authors.lastname: McQuade