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A ubiquitin-proteasome system is responsible for the protection of yeast and human cells against methylmercury¹

Laboratory of Molecular and Biochemical Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan

²Correspondence: Laboratory of Molecular and Biochemical Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan. E-mail: naganuma{at}mail.pharm.tohoku.ac.jp

SPECIFIC AIMS

The aim of our study was to elucidate the mechanism responsible for the toxic effects of methylmercury (MeHg). In this report, a ubiquitin-proteasome system is shown for the first time to be involved in the protection against MeHg toxicity.

PRINCIPAL FINDINGS

1. Effect of overexpression of Cdc34 on toxicity of MeHg
To characterize the mechanisms of toxicity and the defense against MeHg, we searched for factors that determine the sensitivity of yeast cells to MeHg. We screened yeast cells that had been transformed with a yeast genomic DNA library for resistance to MeHg and isolated clones that grew in the presence of a normally toxic concentration of MeHg. Analysis of the clone with the highest resistance to MeHg revealed that the CDC34 gene conferred resistance to MeHg. As shown in Fig. 1 A, yeast cells that overexpressed the product of CDC34 exhibited significant resistance to MeHg. CDC34 encodes a ubiquitin-conjugating enzyme, Cdc34, that is involved in ubiquitin-dependent proteolysis.

View larger version (40K): [in this window] [in a new window]   Figure 1. Effects of the overexpression of Cdc34 on the sensitivity of yeast cells to MeHg. A) Yeast strains carrying pRS425 (control) or pRS425-CDC34 were grown on a plate of agar-solidified synthetic dextrose (SD) medium (-leu) for 3 days with or without MeHgCl. B) Structural domains of Cdc34 and construction of mutant proteins. C) Yeast strains carrying pRS425 (control), pRS425-CDC34, pRS425-cdc34C95A, pRS425-cdc34E109, D111, E113A, or pRS425-cdc341–170aa were grown in SD (-leu) medium in the presence of various concentrations of MeHgCl. D) Lysates of each strain of yeast cells cultured in control medium were subjected to immunoblotting analysis with multiubiquitin-specific antibody. Staining with Coomassie blue (lower panel) indicates the amount of total protein loaded. E) Yeast cells carrying pRS425 (control) or pRS425-CDC34 were cultured for 3 h in the presence or absence of MeHgCl (100 nM). Lysates of these cells were subjected to immunoblotting analysis with multiubiquitin-specific antibody.

The ubiquitin-dependent pathway to protein degradation involves the covalent attachment of ubiquitin to substrate proteins to yield ubiquitin-protein conjugates. Ubiquitin is activated initially by ubiquitin-activating enzyme (E1) via formation of a thiolester bond with this enzyme. The activated ubiquitin is then transferred to one of many distinct ubiquitin-conjugating enzymes (E2s) by transthiolation. The E2 enzymes catalyze the ubiquitination of substrate proteins either directly or in conjunction with a distinct ubiquitin ligase (E3s) composed of multiple proteins. The ubiquitination of a substrate protein is followed by degradation of the protein by the proteasome.

2. Role of activity of Cdc34 to form a complex with ubiquitin
To investigate the way Cdc34 confers resistance to MeHg, we examined the ability of Cdc34 to form a complex with ubiquitin. Cdc34 has only one cysteine moiety, located at position 95, known to be the binding site for ubiquitin (Fig. 1B ). As shown in Fig. 1C , the yeast cells that overexpressed Cdc34^C95A, in which cysteine 95 was replaced by alanine, failed to exhibit resistance to MeHg. This suggested that the activity of Cdc34 to form a complex with ubiquitin might be required for such resistance. Alternatively, MeHg might bind strongly to cysteine 95 of Cdc34 overexpressed in MeHg-resistant yeast cells, since MeHg has high affinity for the thiol groups on cysteine residues in proteins. Such strong binding might reduce the intracellular concentration of free MeHg and suppress the toxicity of MeHg in yeast. Therefore, we next examined the effects of overexpression of mutant Cdc34 proteins (Cdc34^{E109, D111, E113A} and Cdc34^1–170aa) on the sensitivity of yeast cells to MeHg.

The mutant Cdc34^{E109, D111, E113A} protein in which glutamate 109, aspartate 111, and glutamate 113 are replaced by alanine is unable to perform the function of Cdc34 essential for cell growth. The altered amino acids are located in a 12-residue segment (Fig. 1B ) of Cdc34 not found in most other E2s and might be involved in the unique activity of Cdc34. The second mutant, Cdc34^1–170aa, has no E3 binding domain, which is also essential for the ubiquitination of substrate proteins by Cdc34. The sensitivity of yeast cells that overexpressed Cdc34^{E109, D111, E113A} or Cdc34^1–170aa to MeHg was not significantly different from that of control yeast cells (Fig. 1C ). These results indicated that the binding of MeHg to cysteine 95 of Cdc34 is not involved in the resistance to MeHg since the mutant proteins both include cysteine 95. Binding of Cdc34 not only to ubiquitin, but also to E3, might be required for protection against the toxicity of MeHg. Our results suggest that the function of Cdc34 as a ubiquitin-conjugating enzyme is essential for resistance to MeHg. The level of total ubiquitinated proteins in yeast cells that overexpressed Cdc34 was higher than that in control and yeast cells that expressed Cdc34^{E109, D111, E113A} or Cdc34^1–170aa (Fig. 1D ). Although multiple enzymes (E1, E2, and E3) are involved in the ubiquitination of target proteins, this result indicates that overexpression of Cdc34 alone is sufficient to increase the ubiquitination of proteins.

3. Effect of overexpression of ubiquitination-related enzymes other than Cdc34
SCF (Skp1, Cdc53/cullin, F-box protein) is one of several E3 complexes and is involved in the Cdc34-mediated ubiquitination of proteins. Four subunits of SCF—Cdc53, Hrt1, Skp1, and F-box protein—have been identified. Overexpression of individual subunits of SCF (particularly Cdc53, Hrt1, and Skp1) did not significantly affect the sensitivity of yeast cells to MeHg or the level of total ubiquitin-protein conjugates. By contrast, overexpression of E1(Uba1) conferred limited resistance to MeHg but the level of total ubiquitinated proteins did not change significantly. Increased levels of total ubiquitinated proteins were observed only in yeast cells that overexpressed Cdc34. Thus, Cdc34 seems to be a rate-limiting enzyme in the protein-ubiquitination system that requires Cdc34 as E2. The basal cellular concentration of Cdc34 might be lower than that of the other components of the ubiquitin system.

4. Effect of MeHg on ubiquitin-conjugating activity of Cdc34
Treatment with MeHg did not decrease levels of total ubiquitinated proteins in control yeast cells nor reduce the elevated levels of ubiquitinated proteins that were induced by overexpression of Cdc34(Fig. 1E ). Slight increases in the levels of ubiquitinated proteins were observed in control cells and Cdc34-overexpressing yeast cells after treatment with MeHg. These data suggest that MeHg has no inhibitory effects on the ubiquitin-conjugating activities of Cdc34 and most other E2s.

5. Involvement of proteasome activity
The conjugation of ubiquitin to target proteins serves as a signal for degradation of these proteins in proteasomes. We postulated that MeHg might induce the accumulation of some protein(s) with an undesirable effect on cell growth and an inherent signal for ubiquitination. Degradation of the protein(s) by proteasomes after ubiquitination might act as a cellular defense against MeHg toxicity. To examine this possibility, we investigated the effects of a proteasome inhibitor, carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132), on the resistance to MeHg conferred by overexpression of Cdc34 (Fig. 2 A). We constructed yeast strain erg6 (ise1), which is verypermeable to MG132, because proteasome inhibitors such as MG132 are unable to penetrate wild-type yeast cells. The erg6 mutant cells that overexpressed Cdc34 (erg6/CDC34) were significantly more resistant to MeHg than erg6 cells that harbored the control vector (erg6/pRS425). Treatment with MG132 almost completely eliminated the protective effect of the overexpression of Cdc34 against MeHg toxicity, which suggested that proteasome activity is essential for the Cdc34-mediated resistance to MeHg. We also observed the enhancement of MeHg toxicity by MG132 (Fig. 2A ). MG132 alone had only a slightly inhibitory effect on the growth of erg6 mutant cells under our experimental conditions. To confirm the role of proteasome activity in the protection against the toxicity of MeHg, we performed an experiment with the proteasome-defective strain WCG4–11a. In WCG4–11a cells, the mutant PRE1 gene encodes a missense version of the 22.6 kDa subunit of the yeast proteasome and the cells cannot degrade proteins that undergo ubiquitin-dependent proteolysis in wild-type yeast cells. As shown in Fig. 2B , proteasome-defective WCG4–11a cells were hypersensitive to MeHg compared with wild-type cells (WCG4a). These results clearly indicated that proteasomes play a protective role in the defense against MeHg toxicity.

View larger version (30K): [in this window] [in a new window]   Figure 2. Involvement of proteasome activity in resistance to MeHg. A) Yeast erg6 cells that harbored pRS425 (control) or pRS425-CDC34 were grown in SD (-leu) medium with or without the proteasome inhibitor MG132 in the presence of various concentrations of MeHgCl. B) Yeast WCG4a cells (control) and WCG4–11a cells (proteasome defective cells) were grown on a plate of agar-solidified YPD (yeast extract-peptone-dextrose) medium for 3 days with or without MeHgCl.

CONCLUSIONS AND SIGNIFICANCE

The results presented here suggest a novel hypothetical mechanism for MeHg toxicity and the defense against such toxicity. MeHg might accelerate the synthesis or modification of a certain protein(s). The protein (designated X-protein) modified by MeHg suppresses cell growth and includes a signal for ubiquitination by Cdc34 and related enzymes. Ubiquitination of X-protein and its subsequent degradation by the proteasome protect the cell against MeHg toxicity. In normal cells, MeHg might be cytotoxic when the cellular concentration of X-protein exceeds the cell’s capacity for ubiquitination. X-Protein might still have growth-suppressive activity after ubiquitination since the sensitivity to MeHg observed in the presence of MG132 was almost identical in erg6 cells that overexpressed Cdc34 (erg6/CDC34) and in cells that harbored the empty vector (erg6/pRS425) (Fig. 2A ) even though levels of total ubiquitinated proteins after treatment with MG132 were significantly higher in erg6/CDC34 cells than in erg6/pRS425 cells (data not shown). Thus, not only the ubiquitination of X-protein, but also proteasome activity for degradation of ubiquitinated X-protein, seem to be crucial for protection against the cytotoxicity of MeHg. The identification and characterization of X-protein or the protein converted to X-protein by MeHg-initiated modification should help us to explain the mechanism of toxicity of MeHg.

The ubiquitin-proteasome system is strongly conserved from yeast to human cells. To determine the effects of overexpression of human Cdc34 (hCdc34) in human cells (HEK293 cells), we established three lines of transfectants that stably expressed hCdc34. These clones all exhibited significant resistance to MeHg compared with two control clones that had been transfected with the empty vector (data not shown). This result indicates that the ubiquitin-proteasome system plays an important role in the protection against MeHg toxicity not only in yeast cells, but also in human cells.

View larger version (16K): [in this window] [in a new window]   Figure 3.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0899fje; to cite this article, use FASEB J. (March 12, 2002) 10.1096/fj.01-0899fje

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