Heavy-metal detoxification in Caenorhabditis elegans and other animals has almost exclusively been attributed to three classes of heavy-metal-binding molecules: the thiol tripeptide glutathione (GSH); a family of small (6-7 kDa) thiol-rich peptides, metallothioneins (MTs); and an assortment of structurally unrelated proteins. Here we present the results from our recent studies demonstrating that another class of heavy-metal-binding peptides, phytochelatins (PCs), formerly considered to be restricted to plants and some fungal species, play a critical, if not predominant, role in heavy-metal tolerance in C. elegans. PCs, ((gamma-Glu-Cys)n Gly polymers, where n = 2-11) derived from GSH by the action of PC synthases (gamma-glutamylcysteine dipeptidyl transferases), chelate heavy-metals with high affinity by thiol coordination and, in the case of plants and fungi, promote their sequestration in the vacuole, a lysosome-like compartment. In one of the better characterized systems, the fission yeast, Schizosaccharomyces pombe, the vacuolar sequestration of Cd.PCs is mediated by the half-molecule ATP-binding cassette (ABC) transporter, SpHMT1 (alias heavy metal tolerance factor 1). Previous investigations by our group and two others have resulted in the cloning of a novel cDNA (AtPCS1) from the model plant Arabidopsis thaliana encoding a 55 kDa soluble protein that is sufficient for metal-activated PC synthesis in vitro. Here we describe how we have determined that a homologous gene (ce-
pcs-1) from C. elegans also encodes a functional PC synthase. Heterologous expression of ce-
pcs-1 in Saccharomyces cerevisiae promotes cadmium-elicited PC accumulation and yields cell-free extracts with PC synthetic activities comparable to those obtained from cells expressing AtPCS1. Crucially, RNA-mediated interference (RNAi) experiments demonstrate that the endogenous ce-
pcs-1 gene is needed for metal detoxification in the intact organism. The progeny of worms injected with dsce-
pcs-1 RNA exhibit increased sensitivity to cadmium, manifest as irreversible larval arrest and extensive necrosis, at the concentrations tolerated by wild-type worms. Given this functional equivalence between AtPCS1 and ce-
pcs-1, we have gone on to explore the processes downstream of PC fabrication in C. elegans. In so doing we have isolated a cDNA encoding a 90.7 kDa half-molecule ABC transporter (CeHMT1), the only one of 58 ORFs in this organism that encode half-molecule ABC transporters that is topologically equivalent to and bears greater than 50% similarity to SpHMT1. Phenotypically, CeHMT1 satisfies all the requirements of a heavy-metal tolerance factor involved in the sequestration and/or elimination of heavy-metal-PC complexes. Heterologous expression of ce-
hmt-1 in S. pombe alleviates the Cd2+ -hypersensitivity of
hmt1- mutants and when worms are injected with dsce-
hmt-1 their progeny acquire a cadmium-hypersensitive phenotype even more extreme than that exhibited by ce-
pcs-1-deficient worms. Evidently, efficient heavy-metal detoxification in C. elegans is not only contingent on a functional
pcs-1 gene but also on a functional
hmt-1 gene. These results demonstrate for the first time that PC-dependent, HMT1-mediated heavy metal detoxification pathways are not restricted to plants and fungi but are also operative in some animals. This is a possibility that had not even been speculated previously. Given that C. elegans is only one of at least 100,000 nematode species, many of which are pathogenic, discovery of this pathway in C. elegans will likely prove to be of wide toxicological significance. The conditional lethality of the ce-
pcs-1 and ce-
hmt-1 mutations hand in hand with the identification of PC synthase homologs in the EST databases of other nematodes as well as a number of pathogenic trematodes and round worms reinforces this contention. This work was funded by NSF grant MCB-0077838 awarded to P.A.R.