Contents Summary  799 Introduction  800 The origins of Cu homeostasis  800 Copper homeostasis in unicellular photosynthetic model organisms  801 Functions of Cu in plants  802 Typical levels of Cu in plants, deficiency and toxicity  802 Copper abundance in soils and appropriate Cu concentrations in media  804 Uptake in the root and distribution to aerial tissues  804 Uptake in the shoot symplast, redistribution of Cu during flowering, seed set and senescence  806 Cu delivery inside the cell  806 Regulation of Cu homeostasis  809 Conclusions and outlook  811 Acknowledgements  811 References  811 Summary Copper (Cu) is a cofactor in proteins that are involved in electron transfer reactions and is an essential micronutrient for plants. Copper delivery is accomplished by the concerted action of a set of evolutionarily conserved transporters and metallochaperones. As a result of regulation of transporters in the root and the rarity of natural soils with high Cu levels, very few plants in nature will experience Cu in toxic excess in their tissues. However, low Cu bioavailability can limit plant productivity and plants have an interesting response to impending Cu deficiency, which is regulated by an evolutionarily conserved master switch. When Cu supply is insufficient, systems to increase uptake are activated and the available Cu is utilized with economy. A number of Cu‐regulated small RNA molecules, the Cu‐microRNAs, are used to downregulate Cu proteins that are seemingly not essential. On low Cu, the Cu‐microRNAs are upregulated by the master Cu‐responsive transcription factor SPL7, which also activates expression of genes involved in Cu assimilation. This regulation allows the most important proteins, which are required for photo‐autotrophic growth, to remain active over a wide range of Cu concentrations and this should broaden the range where plants can thrive.

Wilson disease is a severe genetic disorder associated with intracellular copper overload. The affected gene, ATP7B, has been identified, but the molecular events leading to Wilson disease remain poorly understood. Here, we demonstrate that genetically engineered Atp7b-/- mice represent a valuable model for dissecting the disease mechanisms. These mice, like Wilson disease patients, have intracellular copper accumulation, low-serum oxidase activity, and increased copper excretion in urine. Their liver pathology developed in stages and was determined by the time of exposure to elevated copper rather than copper concentration per se. The disease progressed from mild necrosis and inflammation to extreme hepatocellular injury, nodular regeneration, and bile duct proliferation. Remarkably, all animals older than 9 months showed regeneration of large portions of the liver accompanied by the localized occurrence of cholangiocarcinoma arising from the proliferating bile ducts. The biochemical characterization of Atp7b-/- livers revealed copper accumulation in several cell compartments, particularly in the cytosol and nuclei. The increase in nuclear copper is accompanied by marked enlargement of the nuclei and enhanced DNA synthesis, with these changes occurring before pathology development. Our results suggest that the early effects of copper on cell genetic material contribute significantly to pathology associated with Atp7b inactivation.

Copper is essential for human physiology, but in excess it causes the severe metabolic disorder Wilson disease. Elevated copper is thought to induce pathological changes in tissues by stimulating the production of reactive oxygen species that damage multiple cell targets. To better understand the molecular basis of this disease, we performed genome-wide mRNA profiling as well as protein and metabolite analysis for Atp7b ؊/؊ mice, an animal model of Wilson disease. We found that at the presymptomatic stages of the disease, copper-induced changes are inconsistent with widespread radical-mediated damage, which is likely due to the sequestration of cytosolic copper by metallothioneins that are markedly up-regulated in Atp7b ؊/؊ livers. Instead, copper selectively up-regulates molecular machinery associated with the cell cycle and chromatin structure and down-regulates lipid metabolism, particularly cholesterol biosynthesis. Specific changes in the transcriptome are accompanied by distinct metabolic changes. Biochemical and mass spectroscopy measurements revealed a 3.6-fold decrease of very low density lipoprotein cholesterol in serum and a 33% decrease of liver cholesterol, indicative of a marked decrease in cholesterol biosynthesis. Consistent with low cholesterol levels, the amount of activated sterol regulatory-binding protein 2 (SREBP-2) is increased in Atp7b ؊/؊ nuclei. However, the SREBP-2 target genes are dysregulated suggesting that elevated copper alters SREBP-2 function rather than its processing or re-localization. Thus, in Atp7b ؊/؊ mice elevated copper affects specific cellular targets at the transcription and/or translation levels and has distinct effects on liver metabolic function, prior to appearance of histopathological changes. The identification of the network of specific copper-responsive targets facilitates further mechanistic analysis of human disorders of copper misbalance.

The human copper-transporting ATPases (Cu-ATPases) are essential for dietary copper uptake, normal development and function of the CNS, and regulation of copper homeostasis in the body. In a cell, Cu-ATPases maintain the intracellular concentration of copper by transporting copper into intracellular exocytic vesicles. In addition, these P-type ATPases mediate delivery of copper to copper-dependent enzymes in the secretory pathway and in specialized cell compartments such as secretory granules or melanosomes. The multiple functions of human Cu-ATPase necessitate complex regulation of these transporters that is mediated through the presence of regulatory domains in their structure, posttranslational modification and intracellular trafficking, as well as interactions with the copper chaperone Atox1 and other regulatory molecules. In this review, we summarize the current information on the function and regulatory mechanisms acting on human Cu-ATPases ATP7A and ATP7B. Brief comparison with the Cu-ATPase orthologs from other species is included.

NifS-like proteins catalyze the formation of elemental sulfur (S) and alanine from cysteine (Cys) or of elemental selenium (Se) and alanine from seleno-Cys. Cys desulfurase activity is required to produce the S of iron (Fe)-S clusters, whereas seleno-Cys lyase activity is needed for the incorporation of Se in selenoproteins. In plants, the chloroplast is the location of (seleno) Cys formation and a location of Fe-S cluster formation. The goal of these studies was to identify and characterize chloroplast NifS-like proteins. Using seleno-Cys as a substrate, it was found that 25% to 30% of the NifS activity in green tissue in Arabidopsis is present in chloroplasts. A cDNA encoding a putative chloroplast NifS-like protein, AtCpNifS, was cloned, and its chloroplast localization was confirmed using immunoblot analysis and in vitro import. AtCpNIFS is expressed in all major tissue types. The protein was expressed in Escherichia coli and purified. The enzyme contains a pyridoxal 5Ј phosphate cofactor and is a dimer. It is a type II NifS-like protein, more similar to bacterial seleno-Cys lyases than to Cys desulfurases. The enzyme is active on both seleno-Cys and Cys but has a much higher activity toward the Se substrate. The possible role of AtCpNifS in plastidic Fe-S cluster formation or in Se metabolism is discussed.NifS-like proteins are pyridoxal 5Ј phosphate (PLP)-dependent enzymes with sequence similarity to the Cys desulfurase encoded by nifS of Azotobacter vinelandii (Zheng et al., 1993). These proteins have been found in most organisms tested, where they play a role in S or Se metabolism (Mihara et al., 1997). NifS-like proteins catalyze the breakdown of Cys to form Ala and elemental S, or they may act on related substrates such as seleno-Cys to form Ala and elemental Se (Mihara et al., 1997). The nifS of A. vinelandii is required under nitrogen fixation conditions for the formation of Fe-S clusters in nitrogenase (Zheng et al., 1993). A. vinelandii NIFS is present in a gene cluster with several other genes (nifU, nifA, and cysE) all thought to be involved in Fe-S cluster formation. A second NifS-like protein of A. vinelandii, IscS, has a housekeeping function in the formation of other cellular Fe-S proteins (Zheng et al., 1993). Interestingly, iscS is present in a gene cluster that contains paralogs of the nif genes (iscU and iscA), thus, the nif and isc clusters share a similar organization (Zheng et al., 1998). Homologs of the nif/isc genes, all thought to play a role in cellular Fe-S cluster formation have been discovered in several other bacteria including in Escherichia coli (Zheng et al., 1998). In the eukaryotes, Fe-S clusters are essential cofactors for mitochondrial respiration, as well as for many cytosolic proteins. Recent work has suggested that in yeast and in mammals, all Fe-S clusters are made in the mitochondria (for review, see Lill and Kispal, 2000). Fe-S cluster formation in the mitochondria of eukaryotes involves homologs of the genes encoded by the nif/isc clusters of bacteria (Kispal et a...