Role of zinc in health and disease.

This review provides a concise overview of the cellular and clinical aspects of the role of zinc, an essential micronutrient, in human physiology and discusses zinc-related pathological states. Zinc cannot be stored in significant amounts, so regular dietary intake is essential. ZIP4 and/or ZnT5B transport dietary zinc ions from the duodenum into the enterocyte, ZnT1 transports zinc ions from the enterocyte into the circulation, and ZnT5B (bidirectional zinc transporter) facilitates endogenous zinc secretion into the intestinal lumen. Putative promoters of zinc absorption that increase its bioavailability include amino acids released from protein digestion and citrate, whereas dietary phytates, casein and calcium can reduce zinc bioavailability. In circulation, 70% of zinc is bound to albumin, and the majority in the body is found in skeletal muscle and bone. Zinc excretion is via faeces (predominantly), urine, sweat, menstrual flow and semen. Excessive zinc intake can inhibit the absorption of copper and iron, leading to copper deficiency and anaemia, respectively. Zinc toxicity can adversely affect the lipid profile and immune system, and its treatment depends on the mode of zinc acquisition. Acquired zinc deficiency usually presents later in life alongside risk factors like malabsorption syndromes, but medications like diuretics and angiotensin-receptor blockers can also cause zinc deficiency. Inherited zinc deficiency condition acrodermatitis enteropathica, which occurs due to mutation in the SLC39A4 gene (encoding ZIP4), presents from birth. Treatment involves zinc supplementation via zinc gluconate, zinc sulphate or zinc chloride. Notably, oral zinc supplementation may decrease the absorption of drugs like ciprofloxacin, doxycycline and risedronate.

Zinc transporters and their functional integration in mammalian cells.

Zinc is a ubiquitous biological metal in all living organisms. The spatiotemporal zinc dynamics in cells provide crucial cellular signaling opportunities, but also challenges for intracellular zinc homeostasis with broad disease implications. Zinc transporters play a central role in regulating cellular zinc balance and subcellular zinc distributions. The discoveries of two complementary families of mammalian zinc transporters (ZnTs and ZIPs) in the mid-1990s spurred much speculation on their metal selectivity and cellular functions. After two decades of research, we have arrived at a biochemical description of zinc transport. However, in vitro functions are fundamentally different from those in living cells, where mammalian zinc transporters are directed to specific subcellular locations, engaged in dedicated macromolecular machineries, and connected with diverse cellular processes. Hence, the molecular functions of individual zinc transporters are reshaped and deeply integrated in cells to promote the utilization of zinc chemistry to perform enzymatic reactions, tune cellular responsiveness to pathophysiologic signals, and safeguard cellular homeostasis. At present, the underlying mechanisms driving the functional integration of mammalian zinc transporters are largely unknown. This knowledge gap has motivated a shift of the research focus from in vitro studies of purified zinc transporters to in cell studies of mammalian zinc transporters in the context of their subcellular locations and protein interactions. In this review, we will outline how knowledge of zinc transporters has been accumulated from in-test-tube to in-cell studies, highlighting new insights and paradigm shifts in our understanding of the molecular and cellular basis of mammalian zinc transporter functions.

Buckwheat FeNramp5 Mediates High Manganese Uptake in Roots.

Manganese (Mn) is an essential element for plant growth and development, but transporters required for Mn uptake have only been identified in a few plant species. Here, we functionally characterized a member of the natural resistance-associated macrophage proteins (Nramps) family, FeNramp5 in buckwheat (Fagopyrum esculentum Moench), which is known as a species well adapted to acidic soils. FeNramp5 was mainly expressed in the roots, and its expression was upregulated by the deficiency of Mn and Fe. Furthermore, spatial and tissue-specific expression analysis showed that FeNramp5 was expressed in all tissues of the basal root regions. FeNramp5-GFP protein was localized to the plasma membrane when transiently expressed in buckwheat leaf protoplast. FeNramp5 showed the transport activity for Mn2+ and Cd2+ but not for Fe2+ when expressed in yeast. Furthermore, the transport activity for Mn2+ was higher in yeast expressing FeNramp5 than in yeast expressing AtNramp1. FeNramp5 was also able to complement the phenotype of Arabidopsis atnramp1 mutant in terms of the growth and accumulation of Mn and Cd. The absolute expression level of AtNramp1 was comparable to that of FeNramp5 in the roots, but buckwheat accumulated higher Mn than Arabidopsis when grown under the same condition. Further analysis showed that at least motif B in FeNramp5 seems important for its high transport activity for Mn. These results indicate that FeNramp5 is a transporter for the uptake of Mn and Cd and its higher transport activity for Mn is probably associated with higher Mn accumulation in buckwheat.

Functional analysis of whether the glycine residue of the GMN motif of the Arabidopsis MRS2/MGT/CorA-type Mg2+ channel protein AtMRS2-11 is critical for Mg2+ transport activity.

Magnesium (Mg2+) plays a critical role in many physiological processes. The AtMRS2/MGT family, which contains nine Arabidopsis genes (and two pseudogenes), belongs to a eukaryotic subset of the CorA superfamily of divalent cation transporters. AtMRS2-11/MGT10 possesses the signature GlyMetAsn sequence (the GMN motif) conserved in the CorA superfamily; however, little is known about the role of the GMN motif in AtMRS2. Direct measurement using the fluorescent dye mag-fura-2 revealed that reconstituted AtMRS2-11 mediated rapid Mg2+ uptake into proteoliposomes at extraliposomal Mg2+ concentrations of 10 and 20 mM. Mutations in the GMN motif, G417 to A, S or V, did not show a significant change in Mg2+ uptake relative to the wild-type protein. The G417W mutant exhibited a significant increase in Mg2+ uptake. The functional complementation assay in Escherichia coli strain TM2 showed that E. coli cells expressing AtMRS2-11 with mutations in G of the GMN motif did not grow in LB medium without Mg2+ supplementation, while growth was observed in LB medium supplemented with 0.5 mM Mg2+; no difference was observed between the growth of TM2 cells expressing the AtMRS2-11 G417W mutant and that of cells expressing wild-type AtMRS2-11. These results suggested that the Mg2+ transport activity of the AtMRS2-11 GMN-motif mutants was low at low physiological Mg2+ concentrations; thus, the Gly residue is critical for Mg2+ transport, and the Mg2+ transport activity of the GMN-motif mutants was increased at high Mg2+ concentrations.

Molecular model of the ferroportin intracellular gate and implications for the human iron transport cycle and hemochromatosis type 4A.

Ferroportin 1 (FPN1) is a major facilitator superfamily transporter that is essential for proper maintenance of human iron homeostasis at the systemic and cellular level. FPN1 dysfunction leads to the progressive accumulation of iron in reticuloendothelial cells, causing hemochromatosis type 4A (or ferroportin disease), an autosomal dominant disorder that displays large phenotypic heterogeneity. Although crystal structures have unveiled the outward- and inward-facing conformations of the bacterial homolog Bdellovibrio bacteriovorus Fpn (or Bd2019) and calcium has recently been identified as an essential cofactor, our molecular understanding of the iron transport mechanism remains incomplete. Here, we used a combination of molecular modeling, molecular dynamics simulations, and Ala site-directed mutagenesis, followed by complementary in vitro functional analyses, to explore the structural architecture of the human FPN1 intracellular gate. We reveal an interdomain network that involves 5 key amino acids and is likely very important for stability of the iron exporter facing the extracellular milieu. We also identify inter- and intradomain interactions that rely on the 2 Asp84 and Asn174 critical residues and do not exist in the bacterial homolog. These interactions are thought to play an important role in the modulation of conformational changes during the transport cycle. We interpret these results in the context of hemochromatosis type 4A, reinforcing the idea that different categories of loss-of-function mutations exist. Our findings provide an unprecedented view of the human FPN1 outward-facing structure and the particular function of the so-called "gating residues" in the mechanism of iron export.-Guellec, J., Elbahnsi, A., Le Tertre, M., Uguen, K., Gourlaouen, I., Férec, C., Ka, C., Callebaut, I., Le Gac, G. Molecular model of the ferroportin intracellular gate and implications for the human iron transport cycle and hemochromatosis type 4A.

Cu(II) Binding to the N-Terminal Model Peptide of the Human Ctr2 Transporter at Lysosomal and Extracellular pH.

It was shown that His3 of human copper transporter 1 (hCtr1) prompts the ATCUN-like Cu(II) coordination for model peptides of the hCtr1 N-terminus. Its high Cu(II) affinity is a potential driving force for the transfer of Cu(II) from extracellular Cu(II) carriers to hCtr1. Having a sequence similar to that of hCtr1, hCtr2 has been proposed as another human copper transporter. However, the N-terminal domain of hCtr2 is much shorter than that of hCtr1, with different copper binding motifs at its N-terminus. Employing a model peptide of the hCtr2 N-terminus, MAMHF-am, we demonstrated that His4 provides a unique pattern of Cu(II) complexes, involving Met sulfurs in their Cu(II) coordination sphere. The affinity of Cu(II) for MAMHF-am is a few orders of magnitude lower than that reported for the hCtr1 model peptides at the extracellular pH of 7.4, suggesting a maximal complementary role of Cu(II) binding to hCtr2 in the import of copper from the extracellular space to the cytoplasm. On the other hand, the ability of the hCtr2 model peptide to capture Cu(II) from amino acids and short peptides (potential degradation products of proteins) at pH 5.0 and the known predominant lysosomal localization of hCtr2 support an important potential role of the Cu(II)-hCtr2 interaction in the recovery of copper from lysosomes.

Crystal structure of plant vacuolar iron transporter VIT1.

The iron ion is an essential cofactor in several vital enzymatic reactions, such as DNA replication, oxygen transport, and respiratory and photosynthetic electron transfer chains, but its excess accumulation induces oxidative stress in cells. Vacuolar iron transporter 1 (VIT1) is important for iron homeostasis in plants, by transporting cytoplasmic ferrous ions into vacuoles. Modification of the VIT1 gene leads to increased iron content in crops, which could be used for the treatment of human iron deficiency diseases. Furthermore, a VIT1 from the malaria-causing parasite Plasmodium is considered as a potential drug target for malaria. Here we report the crystal structure of VIT1 from rose gum Eucalyptus grandis, which probably functions as a H+-dependent antiporter for Fe2+ and other transition metal ions. VIT1 adopts a novel protein fold forming a dimer of five membrane-spanning domains, with an ion-translocating pathway constituted by the conserved methionine and carboxylate residues at the dimer interface. The second transmembrane helix protrudes from the lipid membrane by about 40 Å and connects to a three-helical bundle, triangular cytoplasmic domain, which binds to the substrate metal ions and stabilizes their soluble form, thus playing an essential role in their transport. These mechanistic insights will provide useful information for the further design of genetically modified crops and the development of anti-malaria drugs.

Spore Germination Requires Ferrichrome Biosynthesis and the Siderophore Transporter Str1 in Schizosaccharomyces pombe.

Spore germination is a process whereby spores exit dormancy to become competent for mitotic cell division. In Schizosaccharomyces pombe, one critical step of germination is the formation of a germ tube that hatches out the spore wall in a stage called outgrowth. Here, we show that iron deficiency blocks the outgrowth of germinating spores. The siderophore synthetase Sib1 and the ornithine N5-oxygenase Sib2 participate in ferrichrome biosynthesis, whereas Str1 functions as a ferrichrome transporter. Expression profiles of sib1+ , sib2+ , and str1+ transcripts reveal that they are induced shortly after induction of germination and their expression remains upregulated throughout the germination program under low-iron conditions. sib1Δ sib2Δ mutant spores are unable to form a germ tube under iron-poor conditions. Supplementation with exogenous ferrichrome suppresses this phenotype when str1+ is present. Str1 localizes at the contour of swollen spores 4 hr after induction of germination. At the onset of outgrowth, localization of Str1 changes and it moves away from the mother spore to primarily localize at the periphery of the new daughter cell. Two conserved Tyr residues (Tyr553 and Tyr567) are predicted to be located in the last extracellular loop region of Str1. Results show that these amino acid residues are critical to ensure timely completion of the outgrowth phase of spores in response to exogenous ferrichrome. Taken together, the results reveal the essential requirement of ferrichrome biosynthesis to promote outgrowth, as well as the necessity to take up ferrichrome from an external source via Str1 when ferrichrome biosynthesis is blocked.

Zinc Transporter Protein (tzn-1) May Also Play a Role in Conidiation Pathway of Neurospora crassa: An Insight Using Proteogenomic Approach.

BACKGROUND: Zinc transporter (tzn-1) of Neurospora crassa plays a crucial role in conidiation pathway, as its removal results in aconidiation which was reported in our earlier studies. OBJECTIVES: The main objective of this study was to analyze the role of tzn-1 in conidiation process, by comparing knockout (KO) mutants zinc transporter KO (Δtzn-1) and aconidiating gene KO (Δacon-3) with wild oak ridge (OR) 74 'A' strain by 'Proteo-genomic' approach. METHODS: To identify the commonly expressed protein spots in knockout (KO) mutants zinc transporter KO (Δtzn-1) and aconidiating gene KO (Δacon-3) by comparing with wild oak ridge (OR) 74 'A' strain. Two sets (Δtzn-1 to wild and Δacon-3 to wild) were analyzed by combining 2- Dimensional gel electrophoresis (2DE) with Matrix Associated Laser Desortion/Ionization mass spectrometry -Peptide Mass Fingerprint (MALDI-PMF). Then, the peptide sequences which were obtained by MASCOT (database software) were identified by FGSC BLASTp search analysis. Finally, to evaluate the expression of the KO mutants zinc transporter KO (Δtzn-1) and aconidiating gene KO (Δacon-3) in comparison to wild (OR) 74 'A' type was analyzed by Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) studies. RESULTS: 2DE and MALDI-PMF has shown the nine commonly overexpressed protein spots from the two sets (Δtzn-1 to wild and Δacon-3 to wild). Peptide sequences were obtained by MASCOT (database software) analysis and peptide sequences were identified by FGSC BLASTp search. Eight sequences have shown the similarities with the genes involved during the early stages of conidial and sexual development. Our qRT-PCR analysis has shown that tzn-1 gene was upregulated in contrast to acon-3 gene in absence of iron concentration and down regulated with increase in iron concentrations in wild samples. With increase in zinc supplements, the tzn-1 gene is normally regulated and shown contrasting feature in absence of zinc and acon-3 gene is normally regulated both in presence and absence of zinc. At regular time intervals, declined growth rate was observed after 18hours of induction. CONCLUSION: Thus, we conclude that tzn-1 and acon-3 genes were actively participating in early stages of conidial process and metal ions play some crucial role in the development of the organism.

Identification of a rice metal tolerance protein OsMTP11 as a manganese transporter.

Metal tolerance proteins (MTPs) are a gene family of cation efflux transporters that occur widely in plants and might serve an essential role in metal homeostasis and tolerance. Our research describes the identification, characterization, and localization of OsMTP11, a member of the MTP family from rice. OsMTP11 was expressed constitutively and universally in different tissues in rice plant. Heterologous expression in yeast showed that OsMTP11 complemented the hypersensitivity of mutant strains to Mn, and also complemented yeast mutants to other metals, including Co and Ni. Real time RT-PCR analysis demonstrated OsMTP11 expression was substantially enhanced following 4 h under Cd, Zn, Ni, and Mn treatments, suggesting possible roles of OsMTP11 involvement in heavy metal stress responses. Promoter analysis by transgenic assays with GUS as a reporter gene and mRNA in situ hybridization experiments showed that OsMTP11 was expressed specifically in conducting tissues in rice. DNA methylation assays of genomic DNA in rice treated with Cd, Zn, Ni, and Mn revealed that decreased DNA methylation levels were present in the OsMTP11 promoter region, which was consistent with OsMTP11 induced-expression patterns resulting from heavy metal stress. This result suggested that DNA methylation is one of major factors regulating expression of OsMTP11 through epigenetic mechanisms. OsMTP11 fused to green fluorescent protein (GFP) localized to the entire onion epidermal cell cytoplasm, while vacuolar membrane exhibited increased GFP signals, consistent with an OsMTP11 function in cation sequestration. Our results indicated that OsMTP11 might play vital roles in Mn and other heavy metal transportation in rice.

Investigating the Relationship between Transverse Relaxation Rate (R2) and Interecho Time in MagA-Expressing, Iron-Labeled Cells.

Reporter gene-based labeling of cells with iron is an emerging method of providing magnetic resonance imaging contrast for long-term cell tracking and monitoring cellular activities. This report investigates 9.4 T nuclear magnetic resonance properties of mammalian cells overexpressing MagA, a putative iron transport protein from magnetotactic bacteria. MagA-expressing MDA-MB-435 cells were cultured in the presence and absence of iron supplementation and compared to the untransfected control. The relationship between the transverse relaxation rate (R2) and interecho time was investigated using the Carr-Purcell-Meiboom-Gill sequence. This relationship was analyzed using a model based on water diffusion in weak magnetic field inhomogeneities (Jensen-Chandra model) as well as a fast-exchange model (Luz-Meiboom model). Increases in R2 with increasing interecho time were larger in the iron-supplemented, MagA-expressing cells compared to other cells. The dependence of R2 on interecho time in these iron-supplemented, MagA-expressing cells was better represented by the Jensen-Chandra model compared to the Luz-Meiboom model, whereas the Luz-Meiboom model performed better for the remaining cell types. Our findings provide an estimate of the distance scale of microscopic magnetic field variations in MagA-expressing cells, which is thought to be related to the size of iron-containing vesicles.

Unresolved questions in human copper pump mechanisms.

Copper (Cu) is an essential transition metal providing activity to key enzymes in the human body. To regulate the levels and avoid toxicity, cells have developed elaborate systems for loading these enzymes with Cu. Most Cu-dependent enzymes obtain the metal from the membrane-bound Cu pumps ATP7A/B in the Golgi network. ATP7A/B receives Cu from the cytoplasmic Cu chaperone Atox1 that acts as the cytoplasmic shuttle between the cell membrane Cu importer, Ctr1 and ATP7A/B. Biological, genetic and structural efforts have provided a tremendous amount of information for how the proteins in this pathway work. Nonetheless, basic mechanistic-biophysical questions (such as how and where ATP7A/B receives Cu, how ATP7A/B conformational changes and domain-domain interactions facilitate Cu movement through the membrane, and, finally, how target polypeptides are loaded with Cu in the Golgi) remain elusive. In this perspective, unresolved inquiries regarding ATP7A/B mechanism will be highlighted. The answers are important from a fundamental view, since mechanistic aspects may be common to other metal transport systems, and for medical purposes, since many diseases appear related to Cu transport dysregulation.

Cloning and characterization of a Ca(2+)/H(+) exchanger from the halophyte Salicornia europaea L.

The calcium ion (Ca(2+)), which functions as a second messenger, plays an important role in plants' responses to various abiotic stresses, and Ca(2+)/H(+) exchangers (CAXs) are an important part of this process. In this study, we isolated and characterized a putative Ca(2+)/H(+) exchanger gene (SeCAX3) from Salicornia europaea L., a succulent, leafless euhalophyte. The SeCAX3 open reading frame was 1368 bp long and encoded a 455-amino-acid polypeptide that showed 67.9% similarity to AtCAX3. SeCAX3 was expressed in the shoots and roots of S. europaea. Expression of SeCAX3 was up-regulated by Ca(2+), Na(+), sorbitol, Li(+), abscisic acid, and cold treatments in shoots, but down-regulated by Ca(2+), sorbitol, abscisic acid, and cold treatments in roots. When SeCAX3 was transformed into a Ca-sensitive yeast strain, the transformed cells were able to grow in the presence of 200 mM Ca(2+). Furthermore, SeCAX3 conferred drought, salt, and cold tolerance in yeast. Compared with the control strains, the yeast transformants expressing SeCAX3 were able to grow well in the presence of 30 mM Li(+), 150 mM Mg(2+), or 6 mM Ba(2+). These results showed that the expression of SeCAX3 in yeast suppressed its Ca(2+) hypersensitivity and conferred tolerance to Mg(2+) and Ba(2+). Together, these findings suggest that SeCAX3 might be a Ca(2+) transporter that plays a role in regulating cation tolerance and the responses of S. europaea to various abiotic stresses.

Multiple cellular roles of Neurospora crassa plc-1, splA2, and cpe-1 in regulation of cytosolic free calcium, carotenoid accumulation, stress responses, and acquisition of thermotolerance.

Phospholipase C1 (PLC1), secretory phospholipase A2 (sPLA2) and Ca(2+)/H(+) exchanger proteins regulate calcium signaling and homeostasis in eukaryotes. In this study, we investigate functions for phospholipase C1 (plc-1), sPLA2 (splA2) and a Ca(2+)/H(+) exchanger (cpe-1) in the filamentous fungus Neurospora crassa. The Δplc-1, ΔsplA2, and Δcpe-1 mutants exhibited a growth defect on medium supplemented with the divalent ionophore A23187, suggesting that these genes might play a role in regulation of cytosolic free Ca(2+) concentration ([Ca(2+)](c)) in N. crassa. The strains lacking plc-1, splA2, and cpe-1 possessed higher carotenoid content than wild type at 8°C, 22°C, and 30°C, and showed increased ultraviolet (UV)-survival under conditions that induced carotenoid accumulation. Moreover, Δplc-1, ΔsplA2, and Δcpe-1 mutants showed reduced survival rate under hydrogen peroxide-induced oxidative stress and induced thermotolerance after exposure to heat shock temperatures. Thus, this study revealed multiple cellular roles for plc-1, splA2, and cpe-1 genes in regulation of [Ca(2+)](c), carotenoid accumulation, survival under stress conditions, and acquisition of thermotolerance induced by heat shock.

Pseudo-peptides based on methyl cysteine or methionine inspired from Mets motifs found in the copper transporter Ctr1.

Most proteins involved in Cu homeostasis bind to intracellular Cu(I) in stable Cu(S-Cys)x environments, thanks to well-conserved cysteine-rich sequences. Similarly, the Cu(I) transport protein Ctr1, responsible for copper acquisition, binds Cu(I) in Cu(S-Met)3 environments in conserved methionine-rich MXMXXM sequences, referred as Mets motifs. Pseudo-peptides based on a nitrilotriacetic acid scaffold and functionalized with three amino acids bearing thioether side chains, either methyl cysteine in T(1) or methionine in T(2), were synthesized as mimics of the Mets sequences found in Ctr1. These two ligands were obtained with good overall yields from commercial amino acids and demonstrate efficient chelating ability for Cu(I). Only one species, the mononuclear [CuT(1,2)](+) complex, was evidenced by electrospray ionization-mass spectroscopy (ESI-MS) and the circular dichroism signature obtained for the most constrained CuT(1) complex having the shortest side chains showed reorganization of the pseudo-peptide scaffold upon Cu(I) complexation. Considering that thioether functions are neutral sulfur donors, the stability constants measured by competition with ferrozine are quite large: log K ≈ 10.2-10.3. The CuT(1,2) complexes are significantly more stable that those formed with linear peptides, mimicking isolated Mets motifs MXMXXM of the Cu transport protein Ctr1 (log K ≈ 5-6). This may be attributed to the preorganized pseudo-peptide scaffold, which arranges the three neutral sulfur donors toward the metal center. Such moderate affinity Cu(I) chelators are interesting for applications in chelation therapy, for instance, to induce minimum disturbance of Cu homeostasis in Wilson's disease treatments.

Mössbauer, EPR, and modeling study of iron trafficking and regulation in Δccc1 and CCC1-up Saccharomyces cerevisiae.

Strains lacking and overexpressing the vacuolar iron (Fe) importer CCC1 were characterized using Mössbauer and EPR spectroscopies. Vacuolar Fe import is impeded in Δccc1 cells and enhanced in CCC1-up cells, causing vacuolar Fe in these strains to decline and accumulate, respectively, relative to WT cells. Cytosolic Fe levels should behave oppositely. The Fe content of Δccc1 cells grown under low-Fe conditions was similar to that in WT cells. Most Fe was mitochondrial with some nonheme high spin (NHHS) Fe(II) present. Δccc1 cells grown with increasing Fe concentration in the medium contained less total Fe, less vacuolar HS Fe(III), and more NHHS Fe(II) than in comparable WT cells. As the Fe concentration in the growth medium increased, the concentration of HS Fe(III) in Δccc1 cells increased to just 60% of WT levels, while NHHS Fe(II) increased to twice WT levels, suggesting that the NHHS Fe(II) was cytosolic. Δccc1 cells suffered more oxidative damage than WT cells, suggesting that the accumulated NHHS Fe(II) promoted Fenton chemistry. The Fe concentration in CCC1-up cells was higher than in WT cells; the extra Fe was present as NHHS Fe(II) and Fe(III) and as Fe(III) oxyhydroxide nanoparticles. These cells contained less mitochondrial Fe and exhibited less ROS damage than Δccc1 cells. CCC1-up cells were adenine-deficient on minimal medium; supplementing with adenine caused a decline of NHHS Fe(II) suggesting that some of the NHHS Fe(II) that accumulated in these cells was associated with adenine deficiency rather than the overexpression of CCC1. A mathematical model was developed that simulated changes in Fe distributions. Simulations suggested that only a modest proportion of the observed NHHS Fe(II) in both strains was the cytosolic form of Fe that is sensed by the Fe import regulatory system. The remainder is probably generated by the reduction of the vacuolar NHHS Fe(III) species.

Role of protons in the pump cycle of KdpFABC investigated by time-resolved kinetic experiments.

The time-resolved kinetics of the KdpFABC complex solubilized in Aminoxide WS-35 was investigated by ATP concentration jump experiments. ATP was photoreleased from its inactive precursor, caged ATP, and charge movements in the membrane domain of the KdpFABC were detected by the electrochromic dye RH421. At low ATP concentrations, the ATP binding step became rate-limiting with an apparent, pH-independent ATP binding affinity of ~70 µM. At saturating ATP concentrations, the rate-limiting step is the conformational transition (E1-P → P-E2) with a rate constant of ~1.7 s(-1) at 20 °C that was independent of K(+) concentration. This observation together with the detected fluorescence decrease indicates that K(+) (or another positive ion) is bound in the membrane domain after enzyme phosphorylation and the conformational transition to the P-E2 state. pH dependence experiments revealed different roles of H(+) in the transport mechanism. Two different functions of protons for the ion pump must be distinguished. On one hand, there are electrogenically bound "functional" protons, which are not transported but prerequisite for the performance of the ATP-driven half-cycle. On the other hand, protons bind to the transport sites, acting as weak congeners of K(+). There possibly are noncompetitively bound protons, affecting the enzyme activity and/or coupling between KdpA and KdpB subunits. Finally, the recently proposed Post-Albers model for the KdpFABC complex was supplemented with stoichiometry factors of 2 for K(+) and 3 for H(+), and additional inhibitory side reactions controlled by H(+) were introduced, which are relevant at pH <6.5 and/or in the absence of K(+).

Functional analysis of the C-terminal region of the vacuolar cadmium-transporting rice OsHMA3.

Rice OsHMA3 is a vacuolar cadmium (Cd) transporter belonging to the P1B-ATPase family and has a long (273aa) C-terminal region. We analyzed the function of the region related to Cd using the transgenic Arabidopsis Col-0 ecotype, which is sensitive to Cd. The OsHMA3 variant containing a truncated (58aa) C-terminal region did not confer Cd tolerance, whereas an OsHMA3 variant containing a longer truncated (105aa) C-terminal region conferred Cd tolerance to transgenic Arabidopsis. We conclude that the C-terminal region, particularly the region containing the first 105aa, has an important role in OsHMA3 activity.

The TORC1 effector kinase Npr1 fine tunes the inherent activity of the Mep2 ammonium transport protein.

The TORC1 complex controls cell growth upon integrating nutritional signals including amino-acid availability. TORC1 notably adapts the plasma membrane protein content by regulating arrestin-mediated endocytosis of amino-acid transporters. Here we demonstrate that TORC1 further fine tunes the inherent activity of the ammonium transport protein, Mep2, a yeast homologue of mammalian Rhesus factors, independently of arrestin-mediated endocytosis. The TORC1 effector kinase Npr1 and the upstream TORC1 regulator Npr2 control Mep2 transport activity by phospho-silencing a carboxy-terminal autoinhibitory domain. Under poor nitrogen supply, Npr1 enables Mep2 S457 phosphorylation and thus ammonium transport activity. Supplementation of the preferred nitrogen source glutamine leads to Mep2 inactivation and instant S457 dephosphorylation via plasma membrane Psr1 and Psr2 redundant phosphatases. This study underscores that TORC1 also adjusts nutrient permeability to regulate cell growth in a fast and flexible response to environmental perturbation, establishing a hierarchy in the transporters to be degraded, inactivated or maintained active at the plasma membrane.

In situ dimerization of multiple wild type and mutant zinc transporters in live cells using bimolecular fluorescence complementation.

Zinc transporters (ZnTs) facilitate zinc efflux and zinc compartmentalization, thereby playing a key role in multiple physiological processes and pathological disorders, presumed to be modulated by transporter dimerization. We recently proposed that ZnT2 homodimerization is the underlying basis for the dominant negative effect of a novel heterozygous G87R mutation identified in women producing zinc-deficient milk. To provide direct visual evidence for the in situ dimerization and function of multiple normal and mutant ZnTs, we applied here the bimolecular fluorescence complementation (BiFC) technique, which enables direct visualization of specific protein-protein interactions. BiFC is based upon reconstitution of an intact fluorescent protein including YFP when its two complementary, non-fluorescent N- and C-terminal fragments (termed YN and YC) are brought together by a pair of specifically interacting proteins. Homodimerization of ZnT1, -2, -3, -4, and -7 was revealed by high subcellular fluorescence observed upon co-transfection of non-fluorescent ZnT-YC and ZnT-YN; this homodimer fluorescence localized in the characteristic compartments of each ZnT. The validity of the BiFC assay in ZnT dimerization was further corroborated when high fluorescence was obtained upon co-transfection of ZnT5-YC and ZnT6-YN, which are known to form heterodimers. We further show that BiFC recapitulated the pathogenic role that ZnT mutations play in transient neonatal zinc deficiency. Zinquin, a fluorescent zinc probe applied along with BiFC, revealed the in situ functionality of ZnT dimers. Hence, the current BiFC-Zinquin technique provides the first in situ evidence for the dimerization and function of wild type and mutant ZnTs in live cells.