ions exert their toxic effects on cellular metabolism in the ER rather than in the cytosol.Ions of heavy metals such as iron, copper, zinc, cobalt, or nickel are essential micronutrients, required for function of a large number of proteins. At supraoptimal concentrations, however, these metal ions can be detrimental. Furthermore, living organisms can be exposed to the highly toxic ions of cadmium, lead, mercury, and other metals that are generally considered non-essential. Consequently, a complex network of transport, chelation, and sequestration processes has evolved that functions to maintain the concentrations of essential metal ions in different cellular compartments within the narrow physiological range and to minimize the damage caused by the entry of non-essential metal ions into the cytosol (1, 2). The exquisitely tight control of free ion concentrations has been demonstrated for copper (3). Most recent evidence suggests a similar degree of control also for zinc (4). The intracellular mechanisms of storage and cellular distribution, however, are largely unknown.Proteins belonging to the cation diffusion facilitator family (CDF) 1 (5, 6) could potentially play a major role in metal homeostasis. They have been shown in bacteria and budding yeast to confer tolerance of Zn 2ϩ , Co 2ϩ , or Cd 2ϩ ions (7-10). ZRC1 and COT1 in Saccharomyces cerevisiae localize to the vacuolar membrane and are hypothesized to contribute to the storage of Zn 2ϩ and Co 2ϩ ions, respectively (11, 12). Mammalian members of the CDF family appear to be involved mainly in the removal of Zn 2ϩ ions from the cytosol either through the plasma membrane (Zn-T1) (13) or into endosomal vesicles (Zn-T2) (14).We are interested in the physiological role of CDFs and other putative metal transporters for cellular metal homeostasis, tolerance, and accumulation in organisms that express phytochelatin synthases, using fission yeast as the most suitable model system. The synthesis of phytochelatins (PCs), small metal-binding peptides derived from glutathione (15, 16), represents one of the main metal chelation and detoxification mechanisms in plants, fungi, marine diatoms, and also certain animals (17-19). Here we report on the functional characterization of a Schizosaccharomyces pombe CDF (named Zhf, for zinc homeostasis factor), whose function affects tolerance to a range of metal ions in drastically different fashion; disruption of the gene renders S. pombe cells Zn 2ϩ -and Co 2ϩ -hypersensitive yet significantly enhances tolerance toward Cd 2ϩ and Ni 2ϩ . Electron microscopic protein and zinc localization indicate Zhf-dependent zinc accumulation in the ER. Our findings represent novel evidence for the role of the respective compartment in metal homeostasis and identify a major pathway of zinc storage in the ER. Furthermore, the data provide new insights into the still poorly understood cellular mechanisms of cadmium toxicity.
EXPERIMENTAL PROCEDURESS. pombe Strains and Media-The S. pombe strains employed in this study were derived from FY261 (h ϩ ...