A set of mutant coding sequences for Chinese hamster metallothionein (MT) 2 in which codons for individual cysteines were replaced by serine codons was cloned into a yeast expression system. MT gene expression was placed under control of a constitutive promoter on a multicopy Escherichia coli-yeast shuttle vector. MTs were expressed in a metal-sensitive host that lacks the endogenous MT gene. The expressed MTs conferred increased metal resistance to the yeast host. A sensitive assay for cadmium resistance was developed in which population doubling times were monitored in rich liquid medium supplemented with a sublethal dose of CdCl2. Measurements on mutants with single cysteine replacements at 12 positions revealed two mutant classes. One class (Cys----Ser at position 5, 13, 19, or 33) did not affect the detoxification capacity of MT. A second class (Cys----Ser at position 7, 15, 26, 29, 44, 48, 50, or 60) conferred to the host markedly less resistance to cadmium. Bridging cysteines were more critical to cadmium resistance. All five bridging cysteine mutants studied (at positions 7, 15, 44, 50, and 60) conferred lower cadmium resistance. In contrast, mutation of four out of seven terminal cysteines (at position 5, 13, 19, or 33) was shown to be inconsequential. Mutations tend to be more detrimental in the alpha domain than in the beta domain in conveying cadmium resistance, suggesting that the contribution of individual cysteine to the detoxification function of MT is site specific.

• PMID: 2014225
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Journal Article | Research Support, U.S. Gov't, P.H.S.

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Chernaik ML, Huang PC

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