Exploring the ubiquitin-proteasome protein degradation pathway in yeast

This article describes an undergraduate biochemistry laboratory investigating the ubiquitin-proteasome pathway in yeast. In this exercise, the enzyme beta-galactosidase (beta-gal) is expressed in yeast under the control of a stress response promoter. Following exposure to heat stress to induce beta-gal expression, cycloheximide is added to halt translation, and beta-gal degradation is monitored by measuring enzyme activity as a function of time. Students observe that an N-Ile-beta-gal variant with an N-terminal isoleucine has a significantly lower stability than wild-type beta-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 beta-gal protein.