The Yeast Ubiquitin Ligase SCF^Met30Regulates Heavy Metal Response

Abstract

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...

Combined Proteome and Metabolite-profiling Analyses Reveal Surprising Insights into Yeast Sulfur Metabolism

“…This putative pathway would activate the transcriptional activator Met4 since all the genes of the sulfur amino acid pathway are induced by cadmium in a Met4-dependent way (7). Consistent with our data, recent work showed that Met4, which is normally inactivated following methionine supplementation (through the increased pool of cysteine), remains active when methionine-supplemented cells are treated with cadmium (28,29). Under cadmium conditions, Met4 is stabilized as a result of the dissociation of the Skp1-Met-30 interaction in the SCF Met30 ubiquitin ligase complex (28) that targets the ubiquitylation and the degradation of Met-4 upon methionine addition (30).…”

Survival of starving yeast is correlated with oxidative stress response and nonrespiratory mitochondrial function

“…This implied connection is strengthened by a study of glucose repletion in yeast, which shows that methionine biosynthetic genes are highly induced upon relief from glucose starvation (8). The methionine-regulated transcription factors are also well known to regulate the response to various toxins, including heavy metals and oxidizing agents (9,10).…”

Overexpression of <i>RPN8, SKP1, MIA40</i> or<i> MES1</i> increases resistance to cadmium in <i>Saccharomyces cerevisiae </i>

“…Since the literature suggests that cadmium promotes disassembly of the SCF complex (Yen et al, 2012(Yen et al, , 2005, overexpression of SKP1 may reduce the degree of SCF complex disassembly following cadmium exposure, thereby leading to attenuation of cadmium toxicity. RPN8 is a gene encoding a non-ATPase regulatory subunit of the 26S proteasome (Finley et al, 1998), MES1 encodes a methionyl-tRNA synthetase (Chatton et al, 1987) and MIA40 encodes a mitochondrial oxidoreductase (Chacinska et al, 2004).…”