I think there are three reasons for this. First, our paper provided direct biochemical evidence for the idea that the proteasome subunit Rpn10 and proteins bearing ubiquitin-like (UBL) and ubiquitin-binding (UBA) domains function as receptors that link ubiquitinated substrates to the proteasome so that they can be degraded. Although this idea was floating about based on genetic evidence pointing to a positive role for these proteins in proteolysis, in fact there were alternative proposals that these proteins promote proteolysis indirectly by shielding ubiquitin chains from being cleaved from proteasome substrates. So, there was some controversy. Our results cut through the uncertainty surrounding the biochemical functions of these proteins, which will now allow the study of these proteins to move forward on firm footing. Second, our analysis of the turnover of about half a dozen different physiological substrates of the ubiquitin system in yeast yielded the surprising observation that different substrates are guided to the proteasome by different receptor pathways, which points at yet more complexity and potential regulation in what is already an extraordinarily complex system. Third, our paper came along at the right time. Many labs have entered the ubiquitin ligase field during the past 5-10 years, and now it is starting to dawn on many of us that ubiquitin ligases do not account for all of the specificity and regulation in the ubiquitin system. Proteins that bind ubiquitin and different types of ubiquitin chains have become a hot topic.

Both. We report the first biochemical assay that monitors the ability of Rpn10 and Rad23 proteins to sustain turnover of ubiquitinated proteins by the proteasome. We also report the discovery that different physiological substrates of the proteasome rely on different receptor proteins (or combinations of receptor proteins) for their delivery to the proteasome. This finding reveals an unexpected layer of specificity in the ubiquitin system downstream of the ubiquitin ligases.

Cells are constantly making new proteins and eliminating existing proteins. By analogy, consider your local supermarket. New goods are constantly being added to the shelves as existing ones are removed by customers who purchase them. Thus, although the supermarket may look pretty much the same from day-to-day, there is a constant flux of goods into and out of the store. The same is true of cells. Proteins are removed from cells when they become old and damaged—i.e., past their "expiration date," much like meat or milk poised on the brink of spoilage. Other times, proteins are eliminated from cells because they are blocking something from happening or the cell is in the process of reconfiguring itself. Similarly, perfectly serviceable products can be removed from a supermarket as part of a renovation that truncates shelf space or upon the acquisition of a new product line. Even though this analogy obviously has its limits, it projects the essential notion that even though cells may look stable, they are filled with thousands of different proteins that are constantly being removed and replenished.

The process of removing proteins is quite complicated. First, they are tagged with a signal (known as ubiquitin) that indicates that they are to be removed. Next, they are taken to a shredder (known as the proteasome), where they are broken down so that their individual components can be recycled. Our paper describes how a protein that is tagged for removal with ubiquitin is taken to the proteasome "shredder." Although one might naively think that the cell employs a single agent that goes around looking for ubiquitin-tagged proteins and then brings them to the proteasome, in fact there are multiple agents—on the order of a dozen or so. Even more surprising, these agents are specialists—they are dedicated to the removal of specific proteins. Back to the earlier example, it is as if the supermarket hired a group of people to go around putting brightly colored stickers on all food items that should be removed because they are past their expiration date, and then hired a second group of people to go around picking up these tagged items to bring them to the trash compactor. One individual might specialize in collecting the past-due meat, whereas another one might handle milk and cheese, and yet a third focuses on the produce section. On its face this system seems inefficient, so presumably the cell gains something in terms of the accuracy with which ubiquitin-tagged proteins are disposed, or the additional complexity improves the cell’s ability to control the surveillance process—e.g., the supermarket might want the person who handles the meat section to do more frequent rounds and have a more discerning eye than the one who monitors the potato chip aisle.

We became involved quite by accident. The trigger was that we made proteasomes from a mutant lacking Rpn10 protein and observed that these purified proteasomes were unable to degrade ubiquitinated Sic1 (Sic1 is a Cdk inhibitor that we use as a model physiological substrate of the ubiquitin system). This was a major surprise to us, because published data indicated that Rpn10 is completely dispensable for proteasome function in vivo. The paper emerged from our efforts to chase down the basis for this surprising result. In short, purified proteasomes are highly dependent upon Rpn10 for degradation of Sic1 because Sic1 degradation in vivo requires either Rpn10 or Rad23, and in the process of purifying the proteasomes other receptor proteins that could cover for Rpn10’s absence—including Rad23 —are left behind. Naturally there were many successes and failures—this is experimental science, after all!

The major social implication is that our work identifies a potential approach to inhibit the turnover of subsets of proteins by the proteasome. The reason why this might be useful is that inhibition of the proteasome has emerged as a therapeutic strategy in cancer. Perhaps an inhibitor that blocks a specific receptor pathway would be efficacious in killing cancer cells, but would have fewer side effects compared to inhibitors that block the degradation of all proteasome substrates.