A novel mitochondrial carrier protein Mme1 acts as a yeast mitochondrial magnesium exporter.

The homeostasis of magnesium (Mg2+), an abundant divalent cation indispensable for many biological processes including mitochondrial functions, is underexplored. Previously, two mitochondrial Mg2+ importers, Mrs2 and Lpe10, were characterized for mitochondrial Mg2+ uptake. We now show that mitochondrial Mg2+ homeostasis is accurately controlled through the combined effects of previously known importers and a novel exporter, Mme1 (mitochondrial magnesium exporter 1). Mme1 belongs to the mitochondrial carrier family and was isolated for its mutation that is able to suppress the mrs2Δ respiration defect. Deletion of MME1 significantly increased steady-state mitochondrial Mg2+ concentration, while overexpression decreased it. Measurements of Mg2+ exit from proteoliposomes reconstituted with purified Mme1 provided definite evidence for Mme1 as an Mg2+ exporter. Our studies identified, for the first time, a mitochondrial Mg2+ exporter that works together with mitochondrial importers to ensure the precise control of mitochondrial Mg2+ homeostasis.

Huntington disease arises from a combinatory toxicity of polyglutamine and copper binding.

Huntington disease (HD) is a progressive neurodegenerative disorder caused by dominant polyglutamine (polyQ) expansion within the N terminus of huntingtin (Htt) protein. Abnormal metal accumulation in the striatum of HD patients has been reported for many years, but a causative relationship has not yet been established. Furthermore, if metal is indeed involved in HD, the underlying mechanism needs to be explored. Here using a Drosophila model of HD, wherein Htt exon1 with expanded polyQ (Htt exon1-polyQ) is introduced, we show that altered expression of genes involved in copper metabolism significantly modulates the HD progression. Intervention of dietary copper levels also modifies HD phenotypes in the fly. Copper reduction to a large extent decreases the level of oligomerized and aggregated Htt. Strikingly, substitution of two potential copper-binding residues of Htt, Met8 and His82, completely dissociates the copper-intensifying toxicity of Htt exon1-polyQ. Our results therefore indicate HD entails two levels of toxicity: the copper-facilitated protein aggregation as conferred by a direct copper binding in the exon1 and the copper-independent polyQ toxicity. The existence of these two parallel pathways converging into Htt toxicity also suggests that an ideal HD therapy would be a multipronged approach that takes both these actions into consideration.

Genetic inhibition of solute-linked carrier 39 family transporter 1 ameliorates aß pathology in a Drosophila model of Alzheimer's disease.

The aggregation or oligomerization of amyloid-ß (Aß) peptide is thought to be the primary causative event in the pathogenesis of Alzheimer's disease (AD). Considerable in vitro evidence indicates that the aggregation/oligomerization of Aß is promoted in the presence of Zn; however, the functional role of Zn in AD pathogenesis is still not well clarified in vivo. Zn is imported into the brain mainly through the solute-linked carrier (Slc) 39 family transporters. Using a genetically tractable Drosophila model, we found that the expression of dZip1, the orthologue of human Slc39 family transporter hZip1 in Drosophila, was altered in the brains of Aß42-expressing flies, and Zn homeostasis could be modulated by forcible dZip1 expression changes. An array of phenotypes associated with Aß expression could be modified by altering dZip1 expression. Importantly, Aß42 fibril deposits as well as its SDS-soluble form were dramatically reduced upon dZip1 inhibition, resulting in less neurodegeneration, significantly improved cognitive performance, and prolonged lifespan of the Aß42-transgenic flies. These findings suggest that zinc contributes significantly to the Aß pathology, and manipulation of zinc transporters in AD brains may provide a novel therapeutic strategy.

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.

The metal transporter ZIP13 supplies iron into the secretory pathway in Drosophila melanogaster.

The intracellular iron transfer process is not well understood, and the identity of the iron transporter responsible for iron delivery to the secretory compartments remains elusive. In this study, we show Drosophila ZIP13 (Slc39a13), a presumed zinc importer, fulfills the iron effluxing role. Interfering with dZIP13 expression causes iron-rescuable iron absorption defect, simultaneous iron increase in the cytosol and decrease in the secretory compartments, failure of ferritin iron loading, and abnormal collagen secretion. dZIP13 expression in E. coli confers upon the host iron-dependent growth and iron resistance. Importantly, time-coursed transport assays using an iron isotope indicated a potent iron exporting activity of dZIP13. The identification of dZIP13 as an iron transporter suggests that the spondylocheiro dysplastic form of Ehlers-Danlos syndrome, in which hZIP13 is defective, is likely due to a failure of iron delivery to the secretory compartments. Our results also broaden our knowledge of the scope of defects from iron dyshomeostasis.

Inhibition of human high-affinity copper importer Ctr1 orthologous in the nervous system of Drosophila ameliorates Aß42-induced Alzheimer's disease-like symptoms.

Disruption of copper homeostasis has been implicated in Alzheimer's disease (AD) during the last 2 decades; however, whether copper is a friend or a foe is controversial. Within a genetically tractable Drosophila AD model, we manipulated the expression of human high-affinity copper importer orthologous in Drosophila to explore the in vivo roles of copper ions in the development of AD. We found that inhibition of Ctr1C expression by RNAi in Aß-expressing flies significantly reduced copper accumulation in the brains of the flies as well as ameliorating neurodegeneration, enhancing climbing ability, and prolonging lifespan. Interestingly, Ctr1C inhibition led to a significant increase in higher-molecular-weight Aß42 forms in brain lysates, whereas it was accompanied by a trend of decreased expression of amyloid-ß degradation proteases (including NEP1-3 and IDE) with age and reduced Cu-Aß interaction-induced oxidative stress in Ctr1C RNAi flies. Similar results were obtained from inhibiting another copper importer Ctr1B and overexpressing a copper exporter DmATP7 in the nervous system of AD flies. These results imply that copper may play a causative role in developing AD, as either Aß oligomers or aggregates were less toxic in a reduced copper environment or one with less copper binding. Early manipulation of brain copper uptake can have a great effect on Aß pathology.

Artemisinin directly targets malarial mitochondria through its specific mitochondrial activation.

The biological mode of action of artemisinin, a potent antimalarial, has long been controversial. Previously we established a yeast model addressing its mechanism of action and found mitochondria the key in executing artemisinin's action. Here we present data showing that artemisinin directly acts on mitochondria and it inhibits malaria in a similar way as yeast. Specifically, artemisinin and its homologues exhibit correlated activities against malaria and yeast, with the peroxide bridge playing a key role for their inhibitory action in both organisms. In addition, we showed that artemisinins are distributed to malarial mitochondria and directly impair their functions when isolated mitochondria were tested. In efforts to explore how the action specificity of artemisinin is achieved, we found strikingly rapid and dramatic reactive oxygen species (ROS) production is induced with artemisinin in isolated yeast and malarial but not mammalian mitochondria, and ROS scavengers can ameliorate the effects of artemisinin. Deoxyartemisinin, which lacks an endoperoxide bridge, has no effect on membrane potential or ROS production in malarial mitochondria. OZ209, a distantly related antimalarial endoperoxide, also causes ROS production and depolarization in isolated malarial mitochondria. Finally, interference of mitochondrial electron transport chain (ETC) can alter the sensitivity of the parasite towards artemisinin. Addition of iron chelator desferrioxamine drastically reduces ETC activity as well as mitigates artemisinin-induced ROS production. Taken together, our results indicate that mitochondrion is an important direct target, if not the sole one, in the antimalarial action of artemisinins. We suggest that fundamental differences among mitochondria from different species delineate the action specificity of this class of drugs, and differing from many other drugs, the action specificity of artemisinins originates from their activation mechanism.