Our studies of the yeast ubiquitin-proteasome pathway have uncovered a number of general principles that govern substrate selectivity and proteolysis in this complex system. Much of the work has focused on the destruction of a yeast transcription factor, MATα2. The α2 protein is polyubiquitinated and rapidly degraded in α–haploid cells. One pathway of proteolytic targeting, which depends on two distinct endoplasmic reticulum–localized ubiquitin–conjugating enzymes, recognizes the hydrophobic face of an amphipathic helix in α2. Interestingly, degradation of α2 is blocked in a/α–diploid cells by heterodimer formation between the α2 and a1 homeodomain proteins. The data suggest that degradation signals may overlap protein–protein interaction surfaces, allowing a straightforward steric mechanism for regulated degradation. Analysis of α2 degradation led to the identification of both 20S and 26S proteasome subunits, and several key features of proteasome assembly and active–site formation were subsequently uncovered. Finally, it has become clear that protein (poly)ubiquitination is highly dynamic in vivo, and our studies of yeast de–ubiquitinating enzymes illustrate how such enzymes can facilitate the proteolysis of diverse substrates.

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