We are just beginning to understand the metabolism of heavy metals and to use their metabolic functions in biotechnology, although heavy metals comprise the major part of the elements in the periodic table. Because they can form complex compounds, some heavy metal ions are essential trace elements, but, essential or not, most heavy metals are toxic at higher concentrations. This review describes the workings of known metal-resistance systems in microorganisms. After an account of the basic principles of homoeostasis for all heavy-metal ions, the transport of the 17 most important (heavy metal) elements is compared.

Microbial heavy-metal resistance

Trace elements in human physiology and pathology. Copper.

The cellular uptake and intracellular distribution of the essential but highly toxic nutrient, copper, is a precisely orchestrated process. Copper homeostasis is coordinated by several proteins to ensure that it is delivered to specific subcellular compartments and copper-requiring proteins without releasing free copper ions that will cause damage to cellular components. Genetic studies in prokaryotic organisms and yeast have identified membrane-associated proteins that mediate the uptake or export of copper from cells. Within cells, small cytosolic proteins, called copper chaperones, have been identified that bind copper ions and deliver them to specific compartments and copper-requiring proteins. The identification of mammalian homologues of these proteins reveal a remarkable structural and functional conservation of copper metabolism between bacteria, yeast and humans. Furthermore, studies on the function and localization of the products of the Menkes and Wilson's disease genes, which are defective in patients afflicted with these diseases, have provided valuable insight into the mechanisms of copper balance and their role in maintaining appropriate copper distribution in mammals.

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A Delicate Balance: Homeostatic Control of Copper Uptake and Distribution

Copper-transporting ATPases (Cu-ATPases) ATP7A and ATP7B are evolutionarily conserved polytopic membrane proteins with essential roles in human physiology. The Cu-ATPases are expressed in most tissues, and their transport activity is crucial for central nervous system development, liver function, connective tissue formation, and many other physiological processes. The loss of ATP7A or ATP7B function is associated with severe metabolic disorders, Menkes disease, and Wilson disease. In cells, the Cu-ATPases maintain intracellular copper concentration by transporting copper from the cytosol across cellular membranes. They also contribute to protein biosynthesis by delivering copper into the lumen of the secretory pathway where metal ion is incorporated into copper-dependent enzymes. The biosynthetic and homeostatic functions of Cu-ATPases are performed in different cell compartments; targeting to these compartments and the functional activity of Cu-ATPase are both regulated by copper. In recent years, significant progress has been made in understanding the structure, function, and regulation of these essential transporters. These studies raised many new questions related to specific physiological roles of Cu-ATPases in various tissues and complex mechanisms that control the Cu-ATPase function. This review summarizes current data on the structural organization and functional properties of ATP7A and ATP7B as well as their localization and functions in various tissues, and discusses the current models of regulated trafficking of human Cu-ATPases.

Function and Regulation of Human Copper-Transporting ATPases

Molecular mechanisms of copper uptake and distribution

Menkes disease arises from a genetic impairment in copper transport. The gene responsible for the phenotype has been identified as a copper transporting ATPase ( ATP7A ). Recently, the protein encoded by the ATP7A gene has been localized to the Golgi complex. In order to investigate the role of the Menkes disease protein in copper transport, recombinant constructs containing both the full-length open reading frame and an alternatively spliced form have been successfully expressed and localized in mammalian cells. Other studies of a patient with occipital horn syndrome, an allelic variant of Menkes disease, have demonstrated that only this alternatively spliced isoform and not the full-length form is expressed in this patient. The milder form of this patient's phenotype suggests that the alternatively spliced isoform has some functional role in copper transport. In the present study the full-length recombinant Menkes protein was shown by immunofluorescence to localize to the Golgi apparatus and the alternatively spliced form, lacking sequences for transmembrane domains 3 and 4 encoded by exon 10, was shown to localize to the endoplasmic reticulum. Using sequences from exon 10 fused to a non-Golgi reporter molecule, a 38 amino acid sequence containing transmembrane domain 3 of the Menkes protein was found to be sufficient for localization to the Golgi complex. Therefore, the protein sequence encoded by exon 10 may be responsible for this differential localization and both isoforms may be required for comprehensive transport of copper within the cell.

A Golgi Localization Signal Identified in the Menkes Recombinant Protein

The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.

Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane.

Menkes disease is a fatal X-linked disorder of copper metabolism. The gene defective in Menkes disease (ATP7A) encodes a copper transporting P-type ATPase (MNK or ATP7A) with six copper-binding domains at its N-terminus. MNK is normally localized to the trans -Golgi network in cultured cells, but relocates to the plasma membrane in the presence of elevated extracellular copper. In this study, the role of the six copper-binding domains on copper-induced redistribution is investigated. In a recombinant clone, when all the wild-type copper-binding motifs are mutated from GMXCXXC to GMXSXXS and the cells grown in medium containing elevated copper, relocalization of the recombinant protein to the plasma membrane was not observed. Using the same assay with any one of the six copper-binding domains intact, MNK moves to the plasma membrane in a way indistinguishable from the wild-type protein. Therefore, the copper-binding domains are vital for MNK trafficking and only a single domain is sufficient for this redistribution to occur.

Emma E. Jones

Papers

A Golgi Localization Signal Identified in the Menkes Recombinant Protein

Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane.

Characterization of the Menkes Protein Copper-Binding Domains and Their Role in Copper-Induced Protein Relocalization