Mutants of Saccharomyces cerevisiae lacking a functional SOD1 gene encoding Cu/Zn superoxide dismutase (SOD) are sensitive to atmospheric levels of oxygen and are auxotrophic for lysine and methionine when grown in air. We have previously shown that these defects of SOD-deficient yeast cells can be overcome through mutations in either the BSD1 or BSD2 (bypass SOD defects) gene. In this study, the wild-type allele of BSD1 was cloned by functional complementation and was physically mapped to the left arm of chromosome VII. BSD1 is identical to PMR1, encoding a member of the P-type ATPase family that localizes to the Golgi apparatus. PMR1 is thought to function in calcium metabolism, and we provide evidence that PMR1 also participates in the homeostasis of manganese ions. Cells lacking a functional PMR1 gene accumulate elevated levels of intracellular manganese and are also extremely sensitive to manganese ion toxicity. We demonstrate that mutations in PMR1 bypass SOD deficiency through a mechanism that depends on extracellular manganese. Collectively, these findings indicate that oxidative damage in a eukaryotic cell can be prevented through alterations in manganese homeostasis.

The baker's yeast Saccharomyces cerevisiae expresses three homologues of the Nramp family of metal transporters: Smf1p, Smf2p, and Smf3p, encoded by SMF1, SMF2, and SMF3, respectively. Here we report a comparative analysis of the yeast Smf proteins at the levels of localization, regulation, and function of the corresponding metal transporters. Smf1p and Smf2p function in cellular accumulation of manganese, and the two proteins are coregulated by manganese ions and the BSD2 gene product. Under manganese-replete conditions, Bsd2p facilitates trafficking of Smf1p and Smf2p to the vacuole, where these transport proteins are degraded. However, Smf1p and Smf2p localize to distinct cellular compartments under metal starvation: Smf1p accumulates at the cell surface, while Smf2p is restricted to intracellular vesicles. The third Nramp homologue, Smf3p, is quite distinctive. Smf3p is not regulated by Bsd2p or by manganese ions and is not degraded in the vacuole. Instead, Smf3p is down-regulated by iron through a mechanism that does not involve transcription or protein stability. Smf3p localizes to the vacuolar membrane independently of metal treatment, and yeast cells lacking Smf3p show symptoms of iron starvation. We propose that Smf3p helps to mobilize vacuolar stores of iron.Nramp (for natural resistance-associated macrophage protein) represents a family of evolutionarily conserved membrane proteins that facilitate the transport of heavy metal ions (5,6,15,17,31). Members of the Nramp family have been found in mammals, birds, insects, plants, fungi, and bacteria (3,5,9,15,16,22,40). Among the best studied are the Nramp1 and Nramp2 transporters of rodents. Although these proteins share 61% homology at the amino acid level, they exhibit distinct functions. Mouse Nramp1 plays an important role in the control of infection against intracellular parasites and is exclusively expressed in monocytes/macrophages and polymorphonuclear leukocytes (2, 15). Nramp2 (also known as DCT1 or DMT1) is more ubiquitously expressed in most tissues (17) and acts as a divalent metal transporter capable of transporting iron, manganese, copper, zinc, cadmium, and lead (17). Mutations in Nramp2 have been associated with defects in duodenal iron uptake and cellular iron utilization in the mk mouse and the Belgrade rat models of anemia (13,14).The baker's yeast Saccharomyces cerevisiae expresses three closely related Nramp homologues, Smf1p, Smf2p, and Smf3p, encoded by SMF1, SMF2, and SMF3, respectively (6,7,29,43). Like mammalian DMT1, the Smf proteins exhibit a somewhat broad substrate specificity. Smf1p was originally defined as a high-affinity manganese transporter (39) and was later shown to contribute to cellular accumulation of cadmium and copper (28). Smf2p can affect cobalt levels in yeast (28) and may also participate in manganese trafficking (43). In more recent studies, Chen and coworkers demonstrated that both Smf1p and Smf2p can stimulate iron uptake into Xenopus oocytes (7). The role of Smf3p in metal homeostasis has not been define...

The Saccharomyces cerevisiae SMF1 gene encodes a member of the well conserved family of Nramp metal transport proteins. Previously, we determined that heavy metal uptake by Smf1p was down-regulated by the product of the S. cerevisiae BSD2 gene. We now demonstrate that this regulation occurs at the level of protein stability. In wild type strains, the bulk of Smf1p is normally directed to the vacuole and is rapidly degraded by vacuolar proteases in a PEP4-dependent manner. In bsd2⌬ mutants, Smf1p fails to enter the vacuole, and the Nramp protein is stabilized. Metal ions themselves play an important role in the post-translational regulation of Smf1p. The depletion of heavy metals from the growth medium effects stabilization of Smf1p and additionally results in accumulation of this transporter at the cell surface. Supplementation of manganese alone is sufficient to trigger rapid degradation of Smf1p in a Bsd2p-dependent manner. Together the action of Bsd2p and metal ions provide a rapid and effective means for controlling Nramp metal transport in response to environmental changes.The Nramp family of polypeptides (for natural resistance associated macrophage protein) consists of a group of highly conserved integral membrane proteins thought to play an important role in heavy metal transport. Homologues to Nramp have been identified in animals, plants, and fungi, as well as in certain bacteria (1, 2). Among the most studied are the Nramp1 and Nramp2 genes of rodents. Nramp1 is believed to control the phagosomal accumulation of redox active iron or manganese ions, thereby contributing to an oxygen radical defense against parasitic infection (3-7). Nramp2 is expressed in all tissues and is needed for proper iron absorption and utilization (7-9). In rats, the Nramp2 isoform (DCT1) is induced following iron starvation and exhibits a broad substrate range including essential metals such as zinc, iron, manganese, and copper, as well as the nonessential metals cadmium and lead (10). Transporters such as DCT1/Nramp that act on both essential and toxic metals are expected to fall under tight cellular control.The bakers' yeast Saccharomyces cerevisiae provides an excellent model system in which to study the function and regulation of eukaryotic metal transporters. The high affinity uptake of copper, iron, and zinc in yeast is accomplished by the action of the CTR1, FTR1, and ZTR1 gene products, respectively (11-13). Each of these transport systems is induced under metal-starvation conditions and correspondingly repressed at physiological metal concentrations, and this regulation occurs at the level of CTR1, FTR1, or ZTR1 gene transcription (13)(14)(15)(16)(17)(18)(19) We have previously shown that metal transport by Smf1p is suppressed in yeast by a process involving the product of the BSD2 gene (22). When BSD2 is inactivated by mutation, the transport of copper and cadmium by Smf1p greatly increases, and cells accumulate toxic levels of the metals (22). Bsd2p exhibits an endoplasmic reticulum (ER) 1 localization (22), yet th...

We have previously shown that mutations in the Saccharomyces cerevisiae BSD2 gene suppress oxidative damage in cells lacking superoxide dismutase and also lead to hyperaccumulation of copper ions. We demonstrate here that bsd2 mutant cells additionally accumulate high levels of cadmium and cobalt. By biochemical fractionation and immunofluorescence microscopy, BSD2 exhibited localization to the endoplasmic reticulum, suggesting that BSD2 acts at a distance to inhibit metal uptake from the growth medium. This BSD2 control of ion transport occurs independently of the CTR1 and FET4 metal transport systems. Genetic suppressor analysis revealed that hyperaccumulation of copper and cadmium in bsd2 mutants is mediated through SMF1, previously shown to encode a plasma membrane transporter for manganese. A nonsense mutation removing the carboxyl-terminal hydrophobic domain of SMF1 was found to mimic a smf1 gene deletion by eliminating the copper and cadmium toxicity of bsd2 mutants and also by precluding the bsd2 suppression of superoxide dismutase deficiency. However, inactivation of SMF1 did not eliminate the elevated cobalt levels in bsd2 mutants. Instead, this cobalt accumulation was found to be specifically mediated through the SMF1 homologue, SMF2. Hence, BSD2 prevents metal hyperaccumulation by exerting negative control over the SMF1 and SMF2 metal transport systems.Transition metals such as copper and manganese serve as essential cofactors for a variety of enzymatic reactions and play important structural and functional roles in cell metabolism. However, these same ions can be toxic when present at elevated levels. One mechanism of toxicity is believed to involve the metal-catalyzed generation of hydroxyl radicals in the Fenton reaction (1). Nonessential metals such as cadmium have no known biological function and are highly toxic at relatively low concentrations. To balance the stimulatory and inhibitory effects of essential ions and to counteract the toxicity of nonessential metals, all organisms possess homeostatic mechanisms that properly control the cellular accumulation, distribution, and detoxification of metals.The bakers' yeast Saccharomyces cerevisiae provides an ideal system in which to study the factors controlling metal homeostasis. A number of heavy metal transport proteins have been identified in yeast that function in transporting and delivering the ions to cellular targets. For example, the plasma membrane protein CTR1 is required for high affinity copper uptake (2). Copper uptake is also controlled by the S. cerevisiae CTR3 gene, although this gene is inactivated in most laboratory strains of yeast through insertion of a transposable element (3). In addition to these pathways for the high affinity uptake of copper, a low affinity divalent metal transporter encoded by the FET4 gene has been identified in yeast (4). FET4 exhibits specificity for a subset of divalent metal ions including cobalt, cadmium, and iron (4). Another gene that directly participates in metal ion uptake is SMF1. SMF1 was ori...

The human ECT2 protooncogene encodes a guanine nucleotide exchange factor for the Rho GTPases and regulates cytokinesis. Although the oncogenic form of ECT2 contains an N-terminal truncation, it is not clear how the structural abnormality of ECT2 causes malignant transformation. Here we show that both the removal of the negative regulatory domain and alteration of subcellular localization are required to induce the oncogenic activity of ECT2. The transforming activity of oncogenic ECT2 was strongly inhibited by dominant negative Rho GTPases, suggesting the involvement of Rho GTPases in ECT2 transformation. Although deletion of the N-terminal cell cycle regulator-related domain (N) of ECT2 did not activate its transforming activity, removal of the small central domain (S), which contains two nuclear localization signals (NLSs), significantly induced the activity. The ECT2 N domain interacted with the catalytic domain and significantly inhibited the focus formation by oncogenic ECT2. Interestingly, the introduction of the NLS mutations in the S domain of N-terminally truncated ECT2 dramatically induced the transforming activity of this otherwise nononcogenic derivative. Among the known Rho GTPases expressed in NIH 3T3 cells, RhoA was predominantly activated by oncogenic ECT2 in vivo. Therefore, the mislocalization of structurally altered ECT2 might cause the untimely activation of cytoplasmic Rho GTPases leading to the malignant transformation.The ECT2 oncogene has been isolated in a search for mitogenic signal transducers in epithelial cells, where a murine keratinocyte expression cDNA library was introduced into fibroblasts to induce foci of morphologically transformed cells (1). The ECT2 transfectants exhibit anchorage-independent cell growth and efficient tumor formation in nude mice. The transforming ECT2 cDNA encodes the C-terminal half of the full-length protein containing Dbl-homology (DH) 1 and pleckstrin homology (PH) domains, which are now found in a number of molecules involved in regulation of the Rho family GTPases. The N-terminal half of ECT2 contains domains related to cell cycle control and repair proteins, including Clb6 and Rad4/Cut5 (2, 3). CLB6 encodes a B-type cyclin of the budding yeast, which promotes the transition from G 1 into S phase (4). Fission yeast cut5, which is identical to the repair gene rad4, is required for both the onset of S phase and the restraint of M phase before the completion of S phase (5). The Cut5-related domain of ECT2 consists of two repeats (6, 7), designated BRCT (BRCA1 C-terminal) repeats, which are widespread in a number of cell-cycle checkpoint control and DNA repair proteins (7). These cell-cycle regulator-related domains of ECT2 play essential roles on the regulation of cytokinesis (2, 3).ECT2 catalyzes guanine nucleotide exchange in vitro on three representative Rho GTPases; RhoA, Rac1, and Cdc42 (2). The Rho family of small GTPases function as molecular switches of diverse biological functions, including cytoplasmic actin reorganization, cell motility, and...