Phytochelatins (PCs), a family of small thiol-rich peptides play a central role in heavy metal, primarily Cd 2+ , detoxification in plants, some fungi such as Schizosaccharomyces pombe , and some invertebrates as exemplified by C. elegans . PCs are derived from the tripeptide glutathione by the action of a constitutively expressed dipeptidyl transpeptidase, phytochelatin synthase (PCS). PCs thiol-coordinate heavy metals and promote their sequestration into the vacuolysosomal compartment however, the transport processes associated with PC-dependent Cd 2+ detoxification remain to be defined precisely. Perhaps the best understood system is that of S. pombe . In this organism, a half-molecule vacuolar ATP-binding cassette (ABC) transporter, SpHMT1, participates in the detoxification of Cd 2+ by catalyzing the MgATP-energized transport of apo-PC and CdPC complexes into the vacuole (Ortiz et al 1992, 1995). Our recent discovery of the PC-dependent pathway for Cd 2+ detoxification in C. elegans has provided us with a unique opportunity to examine the role of HMT1-like proteins in organisms distinct from S. pombe . We identified one out of a total of 31 ORFs encoding half-molecule ABC transporters with an equivalent topology and bearing greater than 51% sequence similarity to SpHMT1. Designated ce-
hmt-1 , the 2.4 kb cDNA encodes a 90.7 kDa polypeptide which satisfies the requirements of a heavy metal tolerance factor involved in the PC-dependent heavy metal tolerance. When heterologously expressed in S. pombe , CeHMT1 localizes to the vacuolar membrane and alleviates the Cd 2+ -hypersensitivity of hmt 1- mutants. Crucially, CeHMT1 is required for Cd 2+ tolerance in the intact organism. The progeny of the wild type control worms develop into normal-sized, gravid adults even at high concentrations of Cd 2+ (50 and 100 microM) in the growth media, whereas the progeny of worms injected with ds ce-
hmt-1 RNA are acutely sensitive to Cd 2+ . They are developmentally arrested in the early L1-L2 stages and eventually die, never reaching adulthood even at the lowest Cd 2+ concentrations (5, 10 microM). Furthermore, the Cd 2+ -hypersensitivity of ce-
hmt-1 RNAi worms is considerably greater than that of PCS-deficient worms (Vatamaniuk et al 2001). In addition, cellular morphological phenotypes of these two classes of RNAi mutant are readily distinguishable. Whereas the intestinal epithelial cells of ce-
pcs-1 RNAi worms become necrotic upon exposure to Cd 2+ , the corresponding cells of ce-
hmt-1 RNAi worms do not necrose per se but instead accumulate punctuate, apoptotic inclusions. These results and those from our previous investigations of the requirement for PCS for heavy metal tolerance in C. elegans demonstrate that PCS-dependent, HMT-1-mediated heavy metal detoxification pathways exist not only in S. pombe but also in some invertebrates, a possibility that had not even been speculated previously. Future studies of the tissue-specificity of ce-
hmt-1 RNA expression and the subcellular localization of its translation product will provide insights into which cell types, tissues, and subcellular compartments are responsible for the sequestration and/or elimination of Cd 2+ and other heavy metals not only in C. elegans but also in other animals that might deploy an equivalent mechanism for metal detoxification. This work was funded by NSF Grant No. MCB-0077838 awarded to P.A.R.