Research Topics

The lab’s main interest is the ubiquitin-proteasome pathway. We focus mainly on the proteasome, the most intricate enzyme of the pathway and a key regulator of cellular function. Although it has over 35 components, the proteasome is very amenable to biochemistry—it is abundant, stable, and easy to purify and assay. Using yeast genetics we can manipulate its structure with relative freedom. Some of the questions we are interested in are, how does the proteasome recognize its substrates, how does it coordinate deubiquitination with degradation, how does it assemble, and how does unfold and translocate the substrate in preparation for degradation.

Several years ago, we found that the proteasome as it had been studied in different labs for decades is missing several key factors, since routine purifications strip them off. One is a deubiquitinating enzyme, Ubp6. This turns out to be a powerful inhibitor of the proteasome. Ubp6 functions noncatalytically to delay the degradation of ubiquitinated substrate proteins. While it inhibits degradation, Ubp6 gradually deubiquitinates the target protein. Thus, delay of degradation by Ubp6 appears to provide a time window allowing progressive deubiquitination of the substrate by Ubp6. With time, deubiquitination proceeds towards completion, the substrate loses its chain, and degradation is irreversibly inhibited. 

Proteasomes also contain another deubiquitinating enzyme, Rpn11, which, in contrast to Ubp6, promotes protein breakdown through degradation-coupled deubiquitination. Rpn11 catalyzes substrate-proximal en bloc chain cleavage, and Ubp6 interferes with degradation at or upstream of this step, so that degradation delay by Ubp6 is accompanied by a switch in the mode of ubiquitin chain processing.

Another proteasome-associated factor is Hul5, a ubiquitin ligase. Our studies suggest that Hul5 functions as an E4 enzyme, that is, a factor dedicated to the elongation of multiubiquitin chains. The binding of Hul5 to the proteasome is promoted by Ubp6. Moreover, progressive deubiquitination of the substrate by Ubp6 is antagonized by Hul5. Thus, ubiquitin chains are in a highly dynamic state on the proteasome, which is governed especially by the functional interactions among Ubp6, Hul5, and Rpn11. These chain dynamics regulate substrate selection by the proteasome. 


A related problem is how ubiquitin-conjugates and ubiquitin-like proteins are recognized by the proteasome. We find that ubiquitin conjugates are recognized both by specific integral subunits of the proteasome and by other proteins that associate reversibly with the proteasome via ubiquitin-like domains, such as Rad23, Dsk2, and Ddi1. Rad23-like proteins, which mediate the indirect pathway of recognition, are not only ubiquitin-like proteins also ubiquitin chain binding proteins, thus when they dock onto proteasomes they deliver ubiquitin conjugates. Proteasome subunits that mediate ubiquitin chain recognition include Rpn10 and Rpn13. We don’t understand why so many pathways of conjugate recognition are used or the basis for their selectivity. Two neighboring proteasome subunits serve as receptors for the reversibly-bound factors that assist in proteasome function. Together they form a scaffold that arranges Rad23, Ubp6, and Hul5 in proximity to one another on the proteasome. Interestingly, each of these associated proteins is active on multiubiquitin chains, serving either to bind, disassemble, or extend chains.

Finally, we have begun to study how the proteasome assembles. Our interest is in assembly of the 19-subunit regulatory particle (RP) of the proteasome. The key step in RP assembly seems to be the formation of a hexameric ATPase ring within the RP. Four dedicated chaperones guide the assembly of this ring, each binding to a different ATPase. Among other things these chaperones regulate the association of the ATPase ring of the RP with the hexameric “alpha ring” of the 28-subunit CP complex.