The proteasome plays a pivotal role in the cellular response to oxidative stress. Here, we used biochemical and mass spectrometric methods to investigate structural changes in the 26S proteasomes from yeast and mammalian cells exposed to hydrogen peroxide (H 2 O 2 ). Oxidative stress induced the dissociation of the 20S core particle from the 19S regulatory particle of the 26S proteasome, which resulted in loss of the activities of the 26S proteasome and accumulation of ubiquitinated proteins. H 2 O 2 triggered the increased association of the proteasome-interacting protein Ecm29 with the purified 19S particle. Deletion of ECM29 in yeast cells prevented the disassembly of the 26S proteasome in response to oxidative stress, and ecm29 mutants were more sensitive to H 2 O 2 than were wild-type cells, suggesting that separation of the 19S and 20S particles is important for cellular recovery from oxidative stress. The increased amount of free 20S core particles was required to degrade oxidized proteins. The Ecm29-dependent dissociation of the proteasome was independent of Yap1, a transcription factor that is critical for the oxidative stress response in yeast, and thus functions as a parallel defense pathway against H 2 O 2 -induced stress.

Covalent attachment of ubiquitin to proteins regulates a host of cellular events by proteolysis dependent and independent mechanisms. A variety of protein domains that bind non-covalently to ubiquitin have been described and functionally linked to diverse cellular processes. Overall, however, the understanding and knowledge of the mechanisms by which ubiquitin-binding domains (UBDs) regulate these processes is limited. Here, we describe identification of a UBD in the yeast transcription factor Met4. Met4 activity, but not its stability, is regulated by polyubiquitination. We found that the UBD restricts the length of the polyubiquitin chain that is assembled on Met4, and prevents proteasomal recognition and degradation of polyubiquitinated Met4. Inactivation of the UBD allowed synthesis of longer ubiquitin chains on Met4 and transformed the normally stable polyubiquitinated Met4 into a short-lived protein. Our results demonstrate a function for UBDs in ubiquitin-chain synthesis and regulation of protein degradation.

Cells have developed a variety of mechanisms to respond to heavy metal exposure. Here, we show that the yeast ubiquitin ligase SCF Met30 plays a central role in the response to two of the most toxic environmental heavy metal contaminants, namely, cadmium and arsenic. SCF Met30 inactivates the transcription factor Met4 by proteolysis-independent polyubiquitination. Exposure of yeast cells to heavy metals led to activation of Met4 as indicated by a complete loss of ubiquitinated Met4 species. The association of Met30 with Skp1 but not with its substrate Met4 was inhibited in cells treated with cadmium. Cadmium-activated Met4 induced glutathione biosynthesis as well as genes involved in sulfuramino acid synthesis. Met4 activation was important for the cellular response to cadmium because mutations in various components of the Met4-transcription complex were hypersensitive to cadmium. In addition, cell cycle analyses revealed that cadmium induced a delay in the transition from G 1 to S phase of the cell cycle and slow progression through S phase. Both cadmium and arsenic induced phosphorylation of the cell cycle checkpoint protein Rad53. Genetic analyses demonstrated a complex effect of cadmium on cell cycle regulation that might be important to safeguard cellular and genetic integrity when cells are exposed to heavy metals. INTRODUCTIONHeavy metals are a major environmental hazard and present a danger to human health. The cause of the cytotoxic effects of heavy metals is not completely understood, but it has been suggested that at least part of their toxicity is due to the formation of hydroxyl radicals, which lead to lipid, protein, and DNA damage (Stohs and Bagchi, 1995;Brennan and Schiestl, 1996;Halliwell and Gutteridge, 1984).As with any cytotoxic and genotoxic insults, all organisms have developed strategies to respond to heavy metal exposure to maintain cellular and genetic integrity. These strategies include detoxification, repair, or removal of damaged molecules, and delay of cell division to prevent propagation of damaged cellular components (Jamieson, 1998).The biological effects of cadmium are perhaps better studied than that of other heavy metals. High affinity for sulfhydryl groups, competition with Zn(II) in proteins, nonspecific interaction with DNA, generation of reactive oxygen species, and depletion of glutathione have been shown to contribute to the toxicity of cadmium (Stohs and Bagchi, 1995;Zalups and Ahmad, 2003;McMurray and Tainer, 2003). Recently, it has been demonstrated in yeast that the genotoxic effects of cadmium are indirect (Jin et al., 2003;McMurray and Tainer, 2003). Rather than by direct DNA damage, cadmium leads to genome instability by inhibition of the DNA mismatch repair system (Jin et al., 2003). Although the mechanism of how cadmium inhibits DNA repair is not clear, it has been suggested that damage of sulfhydryl groups containing components of the mismatch repair system might be responsible (Jin et al., 2003).The damaging effect of cadmium on sulfhydryl groups containing pro...

Summary A large group of E3 ubiquitin ligases is formed by the multisubunit SCF complex, whose core complex (Rbx1/Cul1-Cdc53/Skp1) binds one of many substrate recruiting F-box proteins to form an array of SCF ligases with diverse substrate specificities. It has long been thought that ubiquitylation by SCF ligases is regulated at the level of substrate binding. Here we describe an alternative mechanism of SCF regulation by active dissociation of the F-box subunit. We show that cadmium stress induces selective recruitment of the AAA+ ATPase Cdc48/p97 to catalyze dissociation of the F-box subunit from the yeast SCFMet30 ligase to block substrate ubiquitylation and trigger downstream events. Our results not only provide an additional layer of ubiquitin ligase regulation but also suggest that targeted, signal-dependent dissociation of multisubunit enzyme complexes is an important mechanism in control of enzyme function.

Ubiquitination regulates a host of cellular processes and is well known for its role in progression through the cell division cycle. In budding yeast, cadmium and arsenic stress, the availability of sulfur containing amino acids, and the intracellular concentration of S-adenosylmethionine are linked to cell cycle regulation through the ubiquitin ligase SCF Met30 . Regulation is achieved by ubiquitination of the transcription factor Met4. Met4 activity is controlled by a regulatory K48-linked ubiquitin chain that is synthesized by Cdc34/SCF Met30 . A ubiquitin-interacting-motif (UIM) present in Met4 prevents degradation of ubiquitinated Met4 allowing the ubiquitin chain to function as a reversible switch of Met4 activity. Here we discuss mechanisms of Met4 and SCF Met30 regulation in response to intracellular and environmental conditions, and describe the integration of these signals with cell cycle control.