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...

A 33-kDa alkaline serine protease secreted by Penicillium citrinum strain 52-5 is shown to be an allergenic agent in this fungus. The protein, designated Pen c 1, was purified by sequential DEAE-Sepharose and carboxymethyl (CM)-Sepharose chromatographies. Pen c 1 has a molecular mass of 33 kDa and a pI of 7.1. The caseinolytic enzyme activity of this protein was studied. The protein binds to serum IgE from patients allergic to Penicillium citrinum. The cDNA encoding Pen c 1 is 1420 bp in length and contains an open reading frame for a 397-amino-acid polypeptide. Pen c 1 codes for a larger precursor containing a signal peptide, a propeptide and the 33-kDa mature protein. Sequence comparison revealed that Pen c 1 possesses several features in common with the alkaline serine proteases of the subtilisin family. The essential Asp, His, and Ser residues that make up the catalytic triad of serine proteases are well conserved. Northern blots demonstrated that mRNAs transcribed from this gene are present at early stages of culture. The allergen encoded by Pen c 1 gene was expressed in Escherichia coli as a fusion protein bearing an N-terminal histidine-affinity tag. The protein, purified by affinity chromatography with a yield of 130 mg of pure protein per liter of culture, was able to bind to both a monoclonal anti-Pen c 1 antibody and IgE from the serum of patients allergic to Penicillium. Recombinant Pen c 1 can therefore be expressed in E. coli in large quantities and should prove useful as a standardized specific allergen for immuno-diagnosis of atopic disorders. In addition, full caseinolytic enzyme activity could be generated in the purified recombinant protein by sulfonation and renaturation, followed by removal of the affinity tag, indicating that the refolded protein can assume the same conformation as the native protein.

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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.

Sall4 is important for the activation of ATM-dependent cellular responses to DNA double-stranded breaks (DSBs) in mouse embryonic stem cells and confers resistance to DSB-induced cytotoxicity.

Met30 is the substrate recognition subunit of the essential ubiquitin ligase SCF Met30 . The essential function of Met30 is the inactivation of the Saccharomyces cerevisiae transcription factor Met4, because fully activated Met4 induces a cell cycle arrest. Met4 regulates expression of genes involved in the sulfur assimilation pathway and coordinates the transcriptional program and cell cycle progression in response to cadmium and arsenic stress. Met4 lacks DNA binding activity and requires either Cbf1 or one of the two homologous proteins Met31 and Met32 for promoter association. Accordingly, met4 mutants, cbf1 mutants, and met31 met32 double mutants are methionine auxotroph. We isolated a truncated version of Met32 (Met32 ⌬145-192 ) as a dominant suppressor of the cell cycle defect of met30 mutants. Expression of Met32 ⌬145-192 significantly reduced induction of Met4-regulated genes. Interestingly, both Cbf1-and Met31/32-dependent genes were affected by Met32 ⌬145-192 . Mechanistically, Met32 ⌬145-192 prevented recruitment of Met4 to both Cbf1 and Met31/32-dependent promoters. We further demonstrated that Met32 is part of the Cbf1-Met4 complex bound to Cbf1-recruiting promoter elements and that Met31/32 are required for formation of a stable Met4-Cbf1 transcription complex. These results suggest a regulatory role of Met32 as part of the Cbf1-Met4 complex and provide molecular insight into coordination of cell cycle response and modulation of gene expression programs.

The mould genus, Penicillium, is known to be a significant source of environmental aero-allergens. One important allergen from Penicillium citrinum, Pen c 2, has been identified by means of two-dimensional immunoblotting using IgE-containing patients' sera. This novel allergen was cloned, sequenced and expressed in Escherichia coli. The cloned cDNA encodes a large 457-amino acid protein precursor containing a 16-amino acid signal peptide, a 120-amino acid propeptide and the 321-amino acid mature protein. Comparison of the Pen c 2 sequence with known protein sequences revealed shared high sequence similarities with two vacuolar serine proteases from Aspergillus niger and Saccharomyces cerevisiae. Asp-46, His-78 and Ser-244 were found to constitute the catalytic triad of the 39-kDa Pen c 2. The DNA coding for Pen c 2 was cloned into vector PQE-30 and expressed in E. coli as a His-tag fusion protein that bound serum IgE from Penicillium-allergic patients on immunoblots. Recombinant Pen c 2 could therefore be used effectively for diagnosis and also potentially for the treatment of mould-derived allergic disorders.

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