Differential effect of cysteine-to-serine substitutions in metallothionein on cadmium resistance.

Abstract

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 metalsensitive 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. AM 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 a domain than in the (I domain in conveying cadmium resistance, suggesting that the contribution of individual cysteine to the detoxification function of MT is site specific.Metallothioneins (MTs) are metal binding proteins present in all eukaryotic species examined to date (for reviews, see refs. 1 and 2). Mammalian MTs share extensive primary sequence homology, including the invariant positions of 20 cysteine residues out of 61 amino acids. Eight lysine residues, as well as serine or threonine residues, as part of the sequence Cys-(Ser,Thr)-Cys, are also highly conserved (3). The significance of the conservation of these amino acids to the structure and function of MT is a subject of considerable interest.The role of cysteine in MT is to bind metal atoms through coordinate covalent bonds. The sulfhydryl group of cysteine is well suited for this role. Free cysteine has a high affinity for a variety'of transition metal atoms (4, 5). All MT cysteine residues participate in metal binding; none occurs as intramolecular or intermolecular disulfides under native conditions.A prominent structural feature of MT is the arrangement of bound metal atoms through thiolate-metal coordination into two distinct clusters, termed the a and if domains (6). The ( domain consists of amino acids 1-30 and contains 9 cysteine residues that bind to three atoms of zinc or cadmium. The a domain consists of amino acids 31-61 and contains 11 cysteine' residues that bind four atoms of zinc or cadmium (7). Two lysine residues at positions 30 and 31 mark the interdomain boundary.The function ofMT probably depends on the metal it binds. Thus, although the probable function of MT in normal metal metabolism is the storage and mobilization...

Functional homologs of fungal metallothionein genes from Arabidopsis.

“…The significance of two distinct MT genes in Arabidopsis is not yet understood. Whereas the cysteine-rich domains are responsible for metal binding, as demonstrated in animal MTs (Otvos and Armitage, 1980;Chernaik and Huang, 1991), the central domain has no known function. Nevertheless, conservation of the central domain in plant MTs implies that it is functionally important.…”

“…The cup7 A mutant is highly sensitive to heavy metals because of the lack of endogenous MT, and this mutant has been widely used to test the function of MT genes from several organisms (Ecker et al, 1986;Thiele et al, 1986;Chernaik and Huang, 1991 Figure 4A, and introduced into yeast strain ABDE1, a cup7…”

“…Saccharomyces cerevisiae ABDE-1 (arg4-8,leu2-112, his7-2, trpl-289, ade5, and cupIA) (Ecker et al, 1986) and pYCHEMT (Chernaik and Huang, 1991), which contains a Chinese hamster metallothionein (MT) gene, were kindly provided by F?-C. Huang (Johns Hopkins University, Baltimore). pYCHEMT ( Figure 4A) carries the N-(5'-phosphoribosyl)anthranilate isomerase (TRPI) gene and a 2 p replication origin conferring high copy number in yeast cells.…”

“…The strains were streaked on plates as shown. The yeast strains carrying pYCHE (vector alone), pYAMTI (Arabidopsis MT1), pYAMT2 (Arabidopsis MT2), or pYCHEMT (hamster MT) were grown on synthetic agar medium (Chernaik and Huang, 1991) containing various amounts of CuSO 4 or CdSO 4 as indicated below the plates and photographed after incubation at 30°C for 4 days.…”

“…Yeast (ABDE1) cells were transformed with different constructs using a lithium acetate procedure (Stearns et al, 1991), and transformants were selected by growth on tryptophan-deficient agar medium (1.7 glL yeast nitrogen base, 5 glL ammonium sulfate, 5 glL casamino acids [autoclaved for 30 minto remove tryptophan], 20 glL glucose, 30 mglL adenine, 20 mglL histidine, 30 mglL arginine, 40 mglL leucine, 40 mglL lysine, and 2% bactoagar) (Chernaik and Huang, 1991). Transformed yeast strains were streaked on tryptophan-deficient plates containing various amounts of CdS04 or CuS04 and photographed after incubation for 4 days at 3OOC.…”