Direct Comparison of Manganese Detoxification/Efflux Proteins and Molecular Characterization of ZnT10 as a Manganese Transporter

Manganese (Mn) homeostasis involves coordinated regulation of specific proteins involved in Mn influx and efflux. However, the proteins that are involved in detoxification/efflux have not been completely resolved, nor has the basis by which they select their metal substrate. Here, we compared six proteins, which were reported to be involved in Mn detoxification/efflux, by evaluating their ability to reduce Mn toxicity in chicken DT40 cells, finding that human ZnT10 (hZnT10) was the most significant contributor. A domain swapping and substitution analysis between hZnT10 and a zinc-specific transporter hZnT1 showed that residue N43, which corresponds to the His residue constituting the potential intramembranous zinc coordination site in other ZnT transporters, is necessary to impart hZnT10's unique Mn mobilization activity; residues C52 and L242 in transmembrane domains II and V play a subtler role in controlling the metal specificity of hZnT10. Interestingly, the H->N reversion mutant in hZnT1 conferred Mn transport activity and loss of zinc transport activity. These results provide important information about Mn detoxification/efflux mechanisms in vertebrate cells as well as the molecular characterization of hZnT10 as a Mn transporter

Functional characterization of BjCET3 and BjCET4, two new cation-efflux transporters from Brassica juncea L.

Brassica juncea is promising for metal phytoremediation, but little is known about the functional role of most metal transporters in this plant. The functional characterization of two B. juncea cation-efflux family proteins BjCET3 and BjCET4 is reported here. The two proteins are closely related to each other in amino acid sequence, and are members of Group III of the cation-efflux transporters. Heterologous expression of BjCET3 and BjCET4 in yeast confirmed their functions in exporting Zn, and possibly Cd, Co, and Ni. Yeast transformed with BjCET4 showed higher metal resistance than did BjCET3 transformed. The two BjCET–GFP fusion proteins were localized to the plasma membrane in the roots when expressed in tobacco, and significantly enhanced the plants’ Cd tolerance ability. Under Cd stress, tobacco plants transformed with BjCET3 accumulated significant amounts of Cd in shoots, while maintaining similar shoot biomass production with vector-control subjects. Transformed BjCET4 tobacco plants showed significantly enhanced shoot biomass production with markedly decreased shoot Cd content. The two transporter genes have a lower basal transcript expression in B. juncea seedling tissues when grown in normal conditions than under metal-stress, however, their transcripts levels could be substantially increased by Zn, Cd, NaCl or PEG, suggesting that BjCET3 and BjCET4 may play roles in several stress conditions, roles which appear to be different from those of previous characterized cation-efflux transporters, for example, AtMTP1, BjCET2, and BjMTP1

Comparative molecular biological analysis of membrane transport genes in organisms

Comparative analyses of membrane transport genes revealed many differences in the features of transport homeostasis in eight diverse organisms, ranging from bacteria to animals and plants. In bacteria, membrane-transport systems depend mainly on single genes encoding proteins involved in an ATP-dependent pump and secondary transport proteins that use H+ as a co-transport molecule. Animals are especially divergent in their channel genes, and plants have larger numbers of P-type ATPase and secondary active transporters than do other organisms. The secondary transporter genes have diverged evolutionarily in both animals and plants for different co-transporter molecules. Animals use Na+ ions for the formation of concentration gradients across plasma membranes, dependent on secondary active transporters and on membrane voltages that in turn are dependent on ion transport regulation systems. Plants use H+ ions pooled in vacuoles and the apoplast to transport various substances; these proton gradients are also dependent on secondary active transporters. We also compared the numbers of membrane transporter genes in Arabidopsis and rice. Although many transporter genes are similar in these plants, Arabidopsis has a more diverse array of genes for multi-efflux transport and for response to stress signals, and rice has more secondary transporter genes for carbohydrate and nutrient transport

Molecular identification of differentially expressed Zinc related genes in cultivated bread wheat

Zinc (Zn) is an essential micronutrient required for adequate growth of plant species. Zinc is particularly needed for structural and functional integrity of enzymes and biological membranes and directly involved in synthesis of protein. Consequently, Zn deficiency results in severe decreases in growth and yield. Among the crop species, wheat is very sensitive to Zn deficiency. There is very limited information on the molecular mechanisms affecting expression of high Zn deficiency tolerance. Our objective in this study is, therefore, to identify the differentially expressed cDNA fragments in response to varying levels of Zn applications in a tolerant cultivated wheat genotype. For this purpose, we performed a screening experiment by using a number of cultivated bread wheat genotypes which displayed a considerable variation in response to Zn deficiency. Among various modern bread wheat genotypes tested, Bezostaja was selected as the most tolerant genotype. mRNA differential display method has been used to study the expression profile of Bezostaja genotype exposed to different Zn treatments. We observed 20 differentially expressed cDNA bands by using mRNA differential display. Out of 20 cDNA fragments that were isolated, cloned and sequenced, 14 cDNAs displayed similarity with previously identified metal Zn binding proteins and enzymes such as; alcohol dehydrogenase, cystathionine gamma synthase, and cation diffusion facilitator family transporter containing protein