Drosophila ABC Transporter, DmHMT-1, Confers Tolerance to Cadmium

Abstract

Half-molecule ATP-binding cassette transporters of the HMT-1 (heavy metal tolerance factor 1) subfamily are required for Cd 2؉ tolerance in Schizosaccharomyces pombe, Caenorhabditis elegans, and Chlamydomonas reinhardtii. Based on studies of S. pombe, it has been proposed that SpHMT-1 transports heavy metal⅐phytochelatin (PC) complexes into the vacuolysosomal compartment. PCs are glutathione derivatives synthesized by PC synthases (PCS) in plants, fungi, and C. elegans in response to heavy metals. Our previous studies in C. elegans, however, suggested that HMT-1 and PCS-1 do not necessarily act in concert in metal detoxification. To further explore this inconsistency, we have gone on to test whether DmHMT-1, an HMT-1 from a new source, Drosophila, whose genome lacks PCS homologs, functions in heavy metal detoxification. In so doing, we show that heterologously expressed DmHMT-1 suppresses the Cd 2؉ hypersensitivity of S. pombe hmt-1 mutants and localizes to the vacuolar membrane but does not transport Cd⅐PC complexes. Crucially, similar analyses of S. pombe hmt-1 mutants extend this finding to show that SpHMT-1 itself either does not transport Cd⅐PC complexes or is not the principal Cd⅐PC/apoPC transporter. Consistent with this discovery and with our previous suggestion that HMT-1 and PCS-1 do not operate in a simple linear metal detoxification pathway, we demonstrate that, unlike PCS-deficient cells, which are hypersensitive to several heavy metals, SpHMT-1-deficient cells are hypersensitive to Cd 2؉ , but not to Hg 2؉ or As 3؉ . These findings significantly change our current understanding of the function of HMT-1 proteins and invoke a PC-independent role for these transporters in Cd 2؉ detoxification.The adverse health effects of heavy metals such as cadmium (Cd 2ϩ ), mercury (Hg 2ϩ ), and lead (Pb 2ϩ ) from food and air are well established (1-4). Despite this knowledge, exposure to heavy metals continues, and has even increased in some areas, due to their sustained production and emission into the environment. At the cellular level, the toxicity of heavy metals results from the displacement of endogenous cofactors from their cellular binding sites, the oxidation of essential enzymes and other proteins, and promotion of the formation of reactive oxygen species (3, 4). The variety of ways by which heavy metals exert their effects places demands on a wide range of distinct cellular detoxification mechanisms in which ATP-binding cassette (ABC) 3 transporters are clearly implicated (5-9). The ABC transporter family is one of the largest families of membrane proteins. Although 60 ABC transporter family members are known in Caenorhabditis elegans, 49 in humans, 57 in Drosophila, 103 in Arabidopsis, 30 in Saccharomyces cerevisiae, and 11 in Schizosaccharomyces pombe (10 -13), the exact role played by the many that are implicated in heavy metal detoxification remains to be determined. What is known is that ABC transporters mediate the Mg⅐ATP-energized transmembrane transport of a wide range of substrates, reside on ...

Fission Yeast HMT1 Lowers Seed Cadmium through Phytochelatin-Dependent Vacuolar Sequestration in Arabidopsis

“…However, previous studies suggested that GSH instead of PCs is required for SpHMT1 function in yeast (Prévéral et al, 2009;Sooksa-Nguan et al, 2009), in contrast to the current result in Arabidopsis. The inconsistency possibly resulted from the unusual conditions used in yeast, where high abundance of SpHMT1 protein might recognize GSH that is devoid of competition from PCs.…”

“…Subsequent studies from overexpression of SpHMT1 also implicated GS2-Cd as a likely substrate when PCs were absent (Prévéral et al, 2009;Sooksa-Nguan et al, 2009). The production of both PCs and GSH can be abolished by BSO that inhibits a key enzyme in GSH biosynthesis (Howden et al, 1995a).…”

“…In any case, the substrates recognized by SpHMT1 might be the reason that SpHMT1 enhanced As tolerance in plants but not in yeast, as PCs are known to be more efficient than GSH in metal(loid)s detoxification (Howden et al, 1995a;Gong et al, 2003;Song et al, 2010). Further support for this model came from the result that SpHMT1 did effect As sensitivity in a dose-dependent manner in yeast (Prévéral et al, 2009;Sooksa-Nguan et al, 2009). …”

“…Despite the subsequent identification of CeHMT1, a C. elegans PC transporter, and more recently a Drosophila melanogaster HMT1 homolog that likely transports GSH conjugates, a HMT1 ortholog has not been identified in higher plants (Vatamaniuk et al, 2005;Sooksa-Nguan et al, 2009). The likelihood that higher plants have evolved a different PC transport system has received recent support from the discovery of two ABCC subfamily members of ABC transporters in Arabidopsis (Arabidopsis thaliana), for which vacuolar PC-As(III) transport was shown .…”

Tonoplast-localized Abc2 Transporter Mediates Phytochelatin Accumulation in Vacuoles and Confers Cadmium Tolerance

“…This was particularly intriguing after the completion of the Arabidopsis genome project, where it was clear that this reference plant lacks an Hmt1 ortholog, but retains the capacity to sequester PCs and Cd in vacuoles to form HMWCs (30,32). Hmt1-like proteins are not abundant in nature, being restricted to a limited number of organisms, and their function is not completely understood (33). The closest Hmt1 homologues in Arabidopsis are the ATM transporters localized in the mitochondrion (30,31,33,34).…”