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 ...

The ZAT1p zinc transporter from Arabidopsis thaliana (L.) Heynh. is a member of the cation diffusion facilitator (CDF) protein family. When heterologously expressed in Escherichia coli, ZAT1p bound zinc in a metal blot. Binding of zinc occurred mainly to the hydrophilic amino acid region from H182 to H232. A ZAT1p/ZAT1p*Delta(M1-I25) protein mixture was purified and reconstituted into proteoliposomes. Uptake of zinc into the proteoliposomes did not require a proton gradient across the liposomal membrane. ZAT1p did not transport cobalt, and transported cadmium at only 1% of the zinc transport rate. ZAT1p functioned as an uptake system for 65Zn2+ in two strains of the Gram-negative bacterium Ralstonia metallidurans, which were different in their content of zinc-efflux systems. The ZAT1 gene did not rescue increased zinc sensitivity of a Delta ZRC1single-mutant strain or of a Delta ZRC1 Delta COT1 double-mutant strain of Saccharomyces cerevisiae, but ZAT1 complemented this phenotype in a Delta SpZRC1 mutant strain of Schizosaccharomyces pombe.

Introduction: Assessing intraspecific variation in plant volatile organic compounds (VOCs) involves pitfalls that may bias biological interpretation, particularly when several laboratories collaborate on joint projects. Comparative, inter-laboratory ring trials can inform on the reproducibility of such analyses. Objectives: In a ring trial involving five laboratories, we investigated the reproducibility of VOC collections with polydimethylsiloxane (PDMS) and analyses by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). As model plant we used Tanacetum vulgare, which shows a remarkable diversity in terpenoids, forming so-called chemotypes. We performed our ring-trial with two chemotypes to examine the sources of technical variation in plant VOC measurements during pre-analytical, analytical, and post-analytical steps. Methods: Monoclonal root cuttings were generated in one laboratory and distributed to five laboratories, in which plants were grown under laboratory-specific conditions. VOCs were collected on PDMS tubes from all plants before and after a jasmonic acid (JA) treatment. Thereafter, each laboratory (donors) sent a subset of tubes to four of the other laboratories (recipients), which performed TD-GC-MS with their own established procedures. Results: Chemotype-specific differences in VOC profiles were detected but with an overall high variation both across donor and recipient laboratories. JA-induced changes in VOC profiles were not reproducible. Laboratory-specific growth conditions led to phenotypic variation that affected the resulting VOC profiles. Conclusion: Our ring trial shows that despite large efforts to standardise each VOC measurement step, the outcomes differed both qualitatively and quantitatively. Our results reveal sources of variation in plant VOC research and may help to avoid systematic errors in similar experiments.

Introduction Assessing intraspecific variation in plant volatile organic compounds (VOCs) involves pitfalls that may bias biological interpretation, particularly when several laboratories collaborate on joint projects. Comparative, inter-laboratory ring trials can inform on the reproducibility of such analyses. Objectives In a ring trial involving five laboratories, we investigated the reproducibility of VOC collections with polydimethylsiloxane (PDMS) and analyses by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). As model plant we used Tanacetum vulgare, which shows a remarkable diversity in terpenoids, forming so-called chemotypes. We performed our ring-trial with two chemotypes to examine the sources of technical variation in plant VOC measurements during pre-analytical, analytical, and post-analytical steps. Methods Monoclonal root cuttings were generated in one laboratory and distributed to five laboratories, in which plants were grown under laboratory-specific conditions. VOCs were collected on PDMS tubes from all plants before and after a jasmonic acid (JA) treatment. Thereafter, each laboratory (donors) sent a subset of tubes to four of the other laboratories (recipients), which performed TD-GC-MS with their own established procedures. Results Chemotype-specific differences in VOC profiles were detected but with an overall high variation both across donor and recipient laboratories. JA-induced changes in VOC profiles were not reproducible. Laboratory-specific growth conditions led to phenotypic variation that affected the resulting VOC profiles. Conclusion Our ring trial shows that despite large efforts to standardise each VOC measurement step, the outcomes differed both qualitatively and quantitatively. Our results reveal sources of variation in plant VOC research and may help to avoid systematic errors in similar experiments.