N-Terminal Extension and C-Terminal Domains Are Required for ABCB6/HMT-1 Protein Interactions, Function in Cadmium Detoxification, and Localization to the Endosomal-Recycling System in Caenorhabditis elegans

Abstract

The chronic exposure of humans to toxic metals such as cadmium from food and air causes dysfunction of vital organs, neurodegenerative conditions, and cancer. In this regard, members of the ABCB sub-family of the ATP-binding cassette (ABC) transporter superfamily, ABCB6/HMT-1, are acutely required for the detoxification of heavy metals and are present in genomes of many organisms including the nematode worm, Caenorhabditis elegans and humans. We showed previously that C. elegans ABCB6/HMT-1 detoxifies cadmium, copper, and arsenic, and is expressed in liver-like cells, the coelomocytes, head neurons and intestinal cells, which are the cell types that are affected by heavy metal poisoning in humans. The subcellular localization of ABCB6/HMT-1 proteins is unclear. ABCB6/HMT-1 proteins have a distinguishing topology: in addition to one transmembrane domain and one nucleotide-binding domain, they possess a hydrophobic N-terminal extension (NTE) domain encompassing five to six transmembrane spans. The role of the NTE domain in the function of ABCB6/HMT-1 in the native organism remains to be investigated. We used a versatile, multicellular model system, C. elegans, to establish the subcellular localization of ABCB6/HMT-1 and refine its structure-function studies in the native organism. We show that ABCB6/HMT-1 localizes mainly to the apical recycling endosomes and, in part, to early and late endosomes of intestinal cells. We also show that ABCB6/HMT-1 lacking the NTE domain is mistargeted to the plasma membrane and is unable to confer cadmium resistance. Although the NTE domain is essential for ABCB6/HMT-1 interaction with itself, the absence of NTE does not fully prevent this interaction. As a result, ABCB6/HMT-1 lacking the NTE domain, and expressed in wild-type worms or co-expressed with the full-length polypeptide, inactivates and mistargets the full-length ABCB6/HMT-1. We also show that the 43 amino acid residue stretch at the COOH-terminus is required for the ABCB6/HMT-1 interaction with itself and cadmium detoxification function. These results suggest that both NTE and COOH-terminus must be present to allow the protein to interact with itself and confer cadmium resistance. Considering that ABCB6/HMT-1 proteins are highly conserved, this study advances our understanding of how these proteins function in cadmium resistance in different species. Furthermore, these studies uncover the role of the endosomal-recycling system in cadmium detoxification.

The human ABCB6 protein is the functional homologue of HMT-1 proteins mediating cadmium detoxification

“…In C. elegans , ABCB6 was expressed under the control of the endogenous CeHMT-1 promoter, offering an opportunity to study ABCB6 localization in an intact organism without the burden of artifacts associated with overexpression. In complete agreement with a recent report [41], we find that CeHMT-1 is localized to the endosomal compartment in the intestinal cells of the nematode (Fig. 3).…”

“…3). Our results confirm recent studies establishing the relevance of the N-terminal domain in the localization of ABCB6 and CeHMT-1 [13, 41]. Determining the subcellular localization of a protein is a key step toward understanding the cellular function of a protein.…”

Cryo‐electron microscopy structure of human ABCB6 transporter

“…ATP‐driven translocation of porphyrin into mitochondria is severely impaired in the absence of ABCB6, 5 highlighting the role of ABCB6 in heme precursor translocation. Several studies have suggested that ABCB6 is also presented in other membrane systems like intracellular endo/lysosomal compartment 6–9 or the plasma membrane, 10 which is consistent with its ability to confer tolerance to toxic heavy metals 1,3 . ABCB6 has also been shown to protect cells from oxidative stress 11 and drug toxicity 12,13 .…”

“…In the ATPase hydrolysis assay, we observed stimulated ATPase activity in the presence of both Cu 2+ and GSH. Previous studies have shown that ABCB6 is involved in cell resistance to arsenic and cadmium, 3,9 most likely through GSH conjugation. Although our data did not show direct binding or stimulated ATPase activity of GSH for ABCB6, it cannot be ruled out that GSH may bind to ABCB6 with low affinity.…”

An inventory of lysosomal ABC transporters

“…Taken together, these results indicate that TMD0 is dispensable for the folding, dimerization, membrane insertion and ATP binding/hydrolysis of the core–ABCB6 complex, but has a crucial role in the lysosomal targeting of ABCB6 [108]. Almost identical results were obtained with CeHMT‐1 ( C. elegans Heavy Metal Tolerance factor 1), which shares significant sequence and topological similarity with ABCB6 [116,117]. The evolutionarily conserved role of the HMT‐1 proteins is to confer tolerance to heavy metals through the intracellular sequestration of metal complexes.…”