The plant CDF family member TgMTP1 from the Ni/Zn hyperaccumulator Thlaspi goesingense acts to enhance efflux of Zn at the plasma membrane when expressed in Saccharomyces cerevisiae

Abstract

SummaryTo avoid metal toxicity, organisms have evolved mechanisms including ef¯ux of metal ions from cells and sequestration into internal cellular compartments. Members of the ubiquitous cation diffusion facilitator (CDF) family are known to play an important role in these processes. Overexpression of the plant CDF family member metal tolerance protein 1 (MTP1) from the Ni/Zn hyperaccumulator Thlaspi goesingense (TgMTP1), in the Saccharomyces cerevisiae D zinc resistance conferring (zrc)1D cobalt transporter (cot)1 double mutant, suppressed the Zn sensitivity of this strain. T. goesingense was found to contain several allelic variants of TgMTP1, all of which confer similar resistance to Zn in Dzrc1Dcot1. Similarly, MTP1 from various hyperaccumulator and non-accumulator species also confer similar resistance to Zn. Dzrc1Dcot1 lacks the ability to accumulate Zn in the vacuole and has lower accumulation of Zn after either long-or short-term Zn exposure. Expression of TgMTP1 in Dzrc1Dcot1 leads to further lowering of Zn accumulation and an increase in Zn ef¯ux from the cells. Expression of TgMTP1 in a V-type ATPase-de®cient S. cerevisiae strain also confers increased Zn resistance. In vivo and in vitro immunological staining of hemagglutinin (HA)-tagged TgMTP1::HA reveals the protein to be localized in both the S. cerevisiae vacuolar and plasma membranes. Taken together, these data are consistent with MTP1 functioning to enhance plasma membrane Zn ef¯ux, acting to confer Zn resistance independent of the vacuole in S. cerevisiae. Transient expression in Arabidopsis thaliana protoplasts also reveals that TgMTP1::green¯uorescent protein (GFP) is localized at the plasma membrane, suggesting that TgMTP1 may also enhance Zn ef¯ux in plants.

Examining the Specific Contributions of Individual Arabidopsis Metallothioneins to Copper Distribution and Metal Tolerance

“…Growth of the Dzrc1 Dcot1 mutant was inhibited by 0.1 mM ZnSO 4 . Expression of MTP1, a plasma-membrane Zn efflux transporter from the nickel/Zn hyperaccumulator Thlaspi goesingense (Kim et al, 2004), restored Zn tolerance almost to that of the wild type ( Fig. 2A).…”

“…Cells of the Dzrc1 Dcot1 mutant had a lower level of Zn because of their inability to accumulate Zn in the vacuole (MacDiarmid et al, 2000). Expression of the T. goesingense efflux transporter MTP1 in Dzrc1 Dcot1 further decreased Zn accumulation in yeast cells (Kim et al, 2004;Fig. 2B).…”

“…A construct containing the yeast MT gene (CUP1) was also transformed into Dcup1 to provide a positive control for Cu tolerance experiments. The yeast Dzrc1 Dcot1 mutant expressing MTP1, a Zn transporter from the nickel/Zn hyperaccumulator Thlaspi goesingense (Kim et al, 2004), was a gift from Dr. David Salt.…”

“…Three replicates were inoculated for each strain. The cultures were grown at 30°C for 24 h. For both Cu and Zn content analyses, cells from 4 mL of cultures were collected by filtration (0.45 mm nitrocellulose filter; Millipore) and washed with citrate buffer (20 mM sodium citrate, 1 mM EDTA, pH 4.2) as described previously (Kim et al, 2004). The filters with cells were dried at 92°C for 24 h and digested in 2 mL of concentrated nitric acid at 115°C for 6 h. Digested samples were diluted to 4 mL with water and analyzed by atomic absorption spectrometry (Perkin Analyst 400; Perkin Elmer).…”

A Vacuolar Arsenite Transporter Necessary for Arsenic Tolerance in the Arsenic Hyperaccumulating Fern Pteris vittata Is Missing in Flowering Plants

“…Membrane proteins from yeast were isolated according to Kim et al (2004). To isolate P. vittata membrane proteins, 5 g of 4-week-old gametophytes grown in the presence of 2 mM arsenate were washed twice with deionized water, filtered, and homogenized at 48C in a prechilled glass homogenizer with grinding buffer (25 mM HEPES-KOH, pH 8.5, 0.29 M sucrose, 3 mM DTT, 4 mM EDTA, 0.5% polyvinylpolypyrrolidone, 0.1 mM phenylmethylsulfonyl fluoride, and Plant Protease Inhibitor Cocktail [Sigma-Aldrich P9599]).…”