Delivering Copper Inside Yeast and Human Cells

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Science  31 Oct 1997:
Vol. 278, Issue 5339, pp. 817-818
DOI: 10.1126/science.278.5339.817

Copper is absolutely required for aerobic life [HN2], [HN3], [HN4] and yet, paradoxically, is highly toxic. [HN5] Within the living cell, it coexists with high concentrations of electron-rich molecules such as thiols or ascorbate that are essential to life. But, in the laboratory, it is a superb catalyst for the oxidative destruction of those same molecules. This apparent contradiction has been rationalized by assuming that Cu, like other redox-active metals, is sequestered in nonreactive forms as it is transported into cells and moves through cellular compartments. However, the agents of such trafficking and the mechanisms of delivery of Cu to its final destinations have, until recently, remained largely unknown. Our knowledge of this area has increased substantially during the past 2 years with the identification of two proteins involved in Cu trafficking in yeast: Atx1 (1) and Cox17 (2). [HN6] Two reports now add even more to our knowledge in this area. The first, by Pufahl et al. on page 853 of this issue (3), concerns the mechanism of in vivo Cu transfer by Atx1; and the second, by Culotta et al. (4), reports the identification of a similar Cu-trafficking function for Lys7 in catalyzing Cu incorporation into apo-copper-zinc superoxide dismutase (CuZnSOD) in yeast.

The uptake of Cu in yeast begins with reduction by one of several plasma membrane reductases. The reduced Cu is then transported across the membrane by the high-affinity Cu transporter Ctr1 (5) (see the figure). [HN7] Three different proteins have been identified that transport Cu from Ctr1 to three different cellular locations: Cox17 (2) guides Cu to the mitochondria for insertion into cytochrome c oxidase (CCO), the terminal oxidase of the respiratory chain; [HN8], [HN9], [HN10], [HN11] Lys7 (4) targets Cu to CuZnSOD, a primary antioxidant enzyme in the cytosol; and Atx1 (1) directs Cu to a post-Golgi compartment, by way of Ccc2 (6), a P-type adenosine triphosphatase (ATPase) transmembrane Cu transporter, for final insertion into Fet3, a multicopper oxidase essential for high-affinity iron uptake.

Copper trafficking in yeast.

Cu in the form of Cu(I) is transported across the plasma membrane by Ctr1 and then transferred to small, soluble cytoplasmic Cu transporters, or Cu chaperones; the three currently known Cu transporters—Atx1, Lys7, and Cox17—are shown in orange. Each feeds Cu to a specific protein—Fet3, Sod1 (CuZnSOD), and CCO (cytochrome c oxidase)—in a different cellular compartment. A physical interaction between the components of these pathways (yellow highlighting) has been directly demonstrated only for Atx1 and Ccc2; the precise mechanism of the transfer to the other proteins is unknown and could involve unidentified intervening proteins. In mitochondria, more components are likely (dashed brown boxes); Cu must cross at least one membrane and a second aqueous compartment (the intermembrane space) to reach its final destination. One of these components may be Sco1, an inner membrane protein involved in CCO assembly. Sco1 overexpression can rescue yeast lacking Cox17 (12). The copper pathways are indicated by red arrows. Green dashed arrows, the diffusion of the Cu chaperones in the cytoplasm; membrane transporters, brown; final target proteins for Cu, pink.

Illustration: K. Sutliff

For each of these three proteins, it is assumed that molecular recognition and binding events are essential parts of the individual Cu-trafficking pathways because of the apparent specificity each protein shows for its final site of Cu delivery. Nevertheless, inhibiting Cu delivery by genetically eliminating one of these proteins does not in itself prove that a particular protein interacts directly either with Ctr1 or with the next (genetically defined) acceptor of Cu delivery along that pathway. Pufahl et al. (3) now present the first direct evidence for an interaction and recognition event in Cu transfer from a Cu-trafficking protein to the acceptor protein with their finding, based on a yeast two-hybrid experiment, that Atx1 interacts directly with the metal-binding domain of Ccc2 in vivo (see the figure). In their physical studies of the Cu-binding site of Atx1, Pufahl et al. also provide the first characterization of the metal-binding motif, Met-X-Cys-X-X-Cys (X, any residue), that is found in these proteins involved in the transport and transfer of metal ions. They demonstrate that both a three-coordinate and a two-coordinate geometry are possible for such a site, suggesting the very reasonable possibility that interconversion between the two geometries plays a role in the mechanism of metal ion exchange from one protein to another. The Cu chemistry of Atx1 is likely to be similar to that of the Atx1-like domains in several other Cu proteins including the Menkes and Wilson disease ATPases. [HN12]

This information about Cu metabolism in yeast gains even more significance because the three soluble cytoplasmic Cu chaperones as well as two membrane transporters have human homologs that are functional when they replace the natural yeast protein: yeast Atx1/human Hah1 (7), yeast Lys7/human Ccs1 (4), yeast Cox17/human Cox17 (8), yeast Ccc2/human Wilson disease protein (9), and yeast Ctr1/human Ctr1 (10). In addition, the multicopper oxidase Fet3 is homologous to human ceruloplasmin (6). These findings suggest strongly that many aspects of the mechanisms of Cu and iron homeostasis in yeast and humans will prove to be very similar.

The Cu transport mechanisms described in the figure are high-affinity pathways, active in conditions of low Cu concentration, and some of them can be entirely bypassed when there are high concentrations of Cu salts in the medium. Thus, yeast strains missing the gene for Cox17 cannot respire in normal growth media because CCO is Cu deficient, but are rescued when the medium is made 0.4% CuSO4 (2). Likewise, increasing the Cu concentration in the medium allows Cu to be delivered to Fet3 in yeast strains missing the gene for Atx1 (1). These results indicate that neither Cox17 nor Atx1 is required for proper Cu trafficking when Cu levels are high and that their presence is not absolutely required to detoxify Cu. The observation that high levels of Cu in the growth medium can under some circumstances be beneficial to yeast is counterintuitive but reminiscent of the ability of similarly high levels of Cu to enable strains lacking CuZnSOD to grow well in air, as long as metallothionein is present (11). Metallothionein is a key player in Cu detoxification for yeast, [HN13] probably acting as a Cu buffer to keep intracellular “free” Cu concentrations low; its synthesis is induced by Cu.

Multiple Cu-binding equilibria must be present in the cell, since Cu-binding proteins such as metallothionein, Atx1, Cox17, and Lys7 are all cytosolic and must presumably compete for available Cu. It will be interesting to learn to what extent this competition regulates cellular Cu distribution, and whether competitive success is determined by kinetic or thermodynamic factors. Although many questions remain, the convergence of chemistry, biochemistry, and genetics on this problem has set the stage for a complete understanding of cellular copper and iron metabolism, from the molecular details of the intermolecular transfer reactions to the genetic control of the relevant proteins in both yeast and humans.

HyperNotes Related Resources on the World Wide Web

General Hypernotes

The Dictionary of Cell Biology (London, Academic Press, 1995) defines some of the terms used in this article.

The MIT Biology Hypertextbook, developed by the Experimental Study Group at the Massachusetts Institute of Technology, provides background information on the biology of cells organelles, membranes, and proteins. Structure and Function of Organelles provides descriptions and diagrams of mitochondria, the Golgi apparatus, and other organelles. Membrane Transport Mechanisms describes the movement of materials across cell membranes.

The World Wide Web Virtual Library: Biosciences points to virtual library pages for Biomolecules, Biochemistry and Molecular Biology, and Yeast. Each of these pages presents a long list of Web resources. The World Wide Web Virtual Library: Biomolecules covers molecular sequence and structure databases, metabolic pathway databases, and other lists of Web resources. The World Wide Web Virtual Library: Biochemistry and Molecular Biology is a list of resources listed by provider. The World Wide Web Virtual Library: Yeast provides a description of yeast and a list of Web resources for molecular biology.

CSUBIOWEB, the California State University Biological Sciences Web server, provides links to other Web sites on cell biology and molecular biology.

Numbered Hypernotes

1. Joan S. Valentine's Web page describes her research.

2. Introduction to Cell Biology, a chapter of The WWW Cell Biology Course by Mark Dalton, lists the chemical elements and their biological roles.

3. What is Plant Nutrition? by Dorothy Morgan outlines the elements of plant nutrition and describes the importance of copper to plants.

4. The Florida Agriculture Information Retrieval System (FAIRS) presents Soils and Plant Nutrition by Jimmy J. Street and Gerald Kidder, a discussion of plant nutrition that outlines the importance of copper.

5. Copper in the Environment by Dave Brown presents data on the toxicity of copper in aquatic ecosystems.

6. The Saccharomyces Genome Database (SGD) offers a searchable list of yeast genes including Atx1, Cox17, and Lys7. References to literature are provided, and in some cases the function of the gene is described.

7. Global View of Biological Electron-Transfer presents an explanation of oxidation and reduction and a discussion of their importance in biological systems.

8. Cytochrome Oxidase provides an introduction to cytochrome c oxidase and other cytochrome oxidases. Images and links to other resources are included.

9. Protein Data Bank (PDB) provides sequence and structure data for cytochrome c oxidase.

10. Cytochrome c Oxidase by Craig Martin includes an image of the molecule that can be rotated.

11. The Cytochrome Oxidase Home Page is a forum for disseminating information about this enzyme. The page presents general information, images, lists of recent publications on cytochrome c oxidase, and a directory of researchers who study cytochrome oxidases.

12. Menkes disease, Wilson disease, ceruloplasmin, and metallothionein 1A are described in Online Mendelian Inheritance in Man (OMIM), an online catalog of human genes and genetic disorders.

13. Interactions With Other Metals describes the role of metallothioneins in metal detoxification. This page is a part of Hamilton Harbour: Metals and their Effects on Life.

14. Department of Chemistry and Biochemistry, University of California at Los Angeles.


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