The proteasome regulatory particle (RP) initiates the degradation of ubiquitin-protein conjugates by unfolding the substrate and translocating it into the proteasome core particle (CP) to be degraded1. Here we show that in yeast three proteins are found associated with RP but not RP-CP holoenzyme: Nas6, Rpn14, and Hsm3. Mutations in these genes confer proteasome loss of function phenotypes, despite their virtual absence from holoenzyme. These effects result from deficient RP assembly. Thus, Nas6, Rpn14, and Hsm3 are RP chaperones. The RP contains six ATPases–the Rpt proteins–and each RP chaperone binds to the C-terminal domain of a specific Rpt. We show in an accompanying study2 that RP assembly is templated through the Rpt C-termini, apparently by their insertion into binding pockets in the CP. Thus, RP chaperones may regulate proteasome assembly by directly restricting the accessibility of Rpt C-termini to the CP. In addition, competition between the CP and RP chaperones for Rpt engagement may explain the release of RP chaperones as proteasomes mature.

The Anaphase-Promoting Complex (APC) is an E3 ubiquitin ligase that regulates mitosis and G1 by sequentially targeting cell-cycle regulators for ubiquitination and proteasomal degradation. The mechanism of ubiquitin chain formation by APC and the resultant chain topology remains controversial. By using a single-lysine APC substrate to dissect the topology of ubiquitinated substrates, we find that APC-catalyzed ubiquitination has an intrinsic preference for the K11 linkage of ubiquitin that is essential for substrate degradation. K11 specificity is determined by an E2 enzyme, UBE2S/E2-EPF, that elongates ubiquitin chains after the substrates are pre-ubiquitinated by UbcH10 or UbcH5. UBE2S copurifies with APC; dominant-negative Ube2S slows down APC substrate degradation in functional cell-cycle extracts. We propose that Ube2S is a critical, unique component of the APC ubiquitination pathway.uring protein ubiquitination, a lysine residue on a target protein is conjugated to ubiquitin through an enzymatic cascade involving E1 activating enzymes, E2 conjugating enzymes, and E3 ligases (1). Chain formation can proceed in the same manner by repetitively adding ubiquitins to one or more of seven lysines on conjugated ubiquitin, potentially generating ubiquitin chains of several different topologies (2, 3). The linkage, topology, and length of the ubiquitin chains are thought to determine the fate of the tagged protein: K48-linked ubiquitin for degradation and K63 for signaling DNA damage and inflammation (4).We have studied the linkage and topology of ubiquitin chains generated by the APC, a multisubunit E3 enzyme that controls cell-cycle progression (5). Recently, two interesting observations have been made about chain topology: First, different E2s might be required in concert to form long ubiquitin conjugates on APC substrates (6). Second, the density of ubiquitin, rather than the length of the chains, may qualify substrates for degradation (7).Like most E3 ligases, APC modifies multiple lysine residues on each substrate, generating a diverse mixture of products. Poor resolution obtained in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) hinders analysis. Even when distinct bands are observed, they may not correspond to single products. A band of a discrete molecular weight may reflect ubiquitination on multiple lysine residues or, alternatively, the formation of ubiquitin chains on single-lysine sites.Dissecting the topology of the ubiquitin chains requires a careful simplification of the ubiquitination reaction under meaningful conditions. To achieve that, following earlier work of Petroski and Deshaies (8), we have modified the well-characterized APC substrate, securin, and generated a model substrate that has only a single-lysine residue available for ubiquitination, thereby limiting chain formation to a single chain. By using this model substrate, we discovered that the ubiquitin conjugates formed on APC substrates are preferentially linked through ubiquitin lysine 11. Similar results have r...