Drosophila multicopper oxidase 3 is a potential ferroxidase involved in iron homeostasis

Multicopper oxidases (MCOs) are a specific group of enzymes that contain multiple copper centers through which different substrates are oxidized. Main members of MCO family include ferroxidases, ascorbate oxidases, and laccases. MCO type of ferroxidases is key to iron transport across the plasma membrane. In Drosophila, there are four potential multicopper oxidases, MCO1–4. No convincing evidence has been presented so far to indicate any of these, or even any insect multicopper oxidase, to be a ferroxidase. Here we show Drosophila MCO3 (dMCO3) is highly likely a bona fide ferroxidase. In vitro activity assay with insect-cell-expressed dMCO3 demonstrated it has potent ferroxidase activity. Meanwhile, the ascorbate oxidase and laccase activities of dMCO3 are much less significant. dMCO3 expression in vivo, albeit at low levels, appears mostly extracellular, reminiscent of mammalian ceruloplasmin in the serum. A null dMCO3 mutant, generated by CRISPR/Cas9 technology, showed disrupted iron homeostasis, evidenced by increased iron level and reduced metal importer Mvl expression. Notably, dMCO3-null flies phenotypically are largely normal at normal or iron stressed-conditions. We speculate the likely existence of a similar iron efflux apparatus as the mammalian ferroportin/ferroxidase in Drosophila. However, its importance to fly iron homeostasis is greatly minimized, which is instead dominated by another iron efflux avenue mediated by the ZIP13-ferritin axis along the ER/Golgi secretion pathway.     

Effects of iron deficiency on iron binding and internalization into acidic vacuoles in Dunaliella salina

Uptake of iron in the halotolerant alga Dunaliella salina is mediated by a transferrin-like protein (TTf), which binds and internalizes Fe(3+) ions. Recently, we found that iron deficiency induces a large enhancement of iron binding, which is associated with accumulation of three other plasma membrane proteins that associate with TTf. In this study, we characterized the kinetic properties of iron binding and internalization and identified the site of iron internalization. Iron deficiency induces a 4-fold increase in Fe binding, but only 50% enhancement in the rate of iron uptake and also increases the affinity for iron and bicarbonate, a coligand for iron binding. These results indicate that iron deprivation leads to accumulation and modification of iron-binding sites. Iron uptake in iron-sufficient cells is preceded by an apparent time lag, resulting from prebound iron, which can be eliminated by unloading iron-binding sites. Iron is tightly bound to surface-exposed sites and hardly exchanges with medium iron. All bound iron is subsequently internalized. Accumulation of iron inhibits further iron binding and internalization. The vacuolar inhibitor bafilomycin inhibits iron uptake and internalization. Internalized iron was localized by electron microscopy within vacuolar structures that were identified as acidic vacuoles. Iron internalization is accompanied by endocytosis of surface proteins into these acidic vacuoles. A novel kinetic mechanism for iron uptake is proposed, which includes two pools of bound/compartmentalized iron separated by a rate-limiting internalization stage. The major parameter that is modulated by iron deficiency is the iron-binding capacity. We propose that excessive iron binding in iron-deficient cells serves as a temporary reservoir for iron that is subsequently internalized. This mechanism is particularly suitable for organisms that are exposed to large fluctuations in iron availability.     

The multicopper oxidase of Pseudomonas aeruginosa is a ferroxidase with a central role in iron acquisition

Recently it has been observed that multicopper oxidases are present in a number of microbial genomes, raising the question of their function in prokaryotes. Here we describe the analysis of an mco mutant from the opportunistic pathogen Pseudomonas aeruginosa. Unlike wild-type Pseudomonas aeruginosa, the mco mutant was unable to grow aerobically on minimal media with Fe(II) as sole iron source. In contrast, both the wild-type and mutant strain were able to grow either anaerobically via denitrification with Fe(II) or aerobically with Fe(III). Analysis of iron uptake showed that the mco mutant was impaired in Fe(II) uptake but unaffected in Fe(III) uptake. Purification and analysis of the MCO protein confirmed ferroxidase activity. Taken together, these data show that the mco gene encodes a multicopper oxidase that is involved in the oxidation of Fe(II) to Fe(III) subsequent to its acquisition by the cell. In view of the widespread distribution of the mco gene in bacteria, it is suggested that an iron acquisition mechanism involving multicopper oxidases may be an important and hitherto unrecognized feature of bacterial pathogenicity.     

A transferrin-like protein that does not bind iron is induced by iron deficiency in the alga Dunaliella salina

Iron deficiency induces two major transferrin-like proteins in the plasma membrane (Pm) of the halotolerant alga Dunaliella salina. TTf, a 150-kDa protein, previously identified as a salt-induced triplicated transferrin, having iron-binding characteristics resembling animal transferrins, and a 100-kDa protein designated idi-100 (for iron-deficiency-induced 100 kDa protein). According to the predicted amino acid sequence of idi-100, it is only 30% identical to TTf and differs from it in having two, rather than three, homologous internal repeats and in a lower conservation of canonical iron/bicarbonate binding residues. Both are localized in the outer surface of the membrane; however, TTf can be dissociated from the membrane by treatment with EDTA, whereas release of idi-100 requires detergents. The accumulation of idi-100 under iron deficiency lags behind that of TTf and in contrast to TTf, it is not induced by high salinity, suggesting that induction of idi-100 requires lower Fe threshold levels than that of TTf. In contrast to TTf, idi-100 does not bind Fe; however, there are indications for interactions with bicarbonate ions. These results suggest that despite their common resemblance to transferrins, their similar subcellular localization and their induction by iron deficiency, idi-100 and TTf fulfill different functions.     

Physiological responses of Dunaliella salina and Dunaliella tertiolecta to copper toxicity

Species differences in heavy metal tolerance were investigated by comparing the responses of Dunaliella tertiolecta and Dunaliella salina to elevated concentrations of CuCl2. Although both species showed reduced cell number ml(-1) of algal culture, D. salina was more affected by increase in CuCl2. This reflects higher sensitivity of D. salina to CuCl2 compared to D. tertiolecta. Total chlorophyll in terms of microg ml(-1) was higher in D. tertiolecta at all tested CuCl2 levels, but in terms of microg cell(-1) no significant difference was observed between the two species. Total carotenoids in microg cell(-1) increased with increase in CuCl2 in both species and it was about five times higher in D. salina at all CuCl2 concentrations. While both species showed significant increase in lipid peroxidation at elevated CuCl2, the malondialdehyde content of D. salina cells was about three times higher at most CuCl2 concentrations. Although ascorbate peroxidase (APX) activity increased with increase in CuCl2 levels in both species, higher activity was observed in D. tertiolecta at all tested CuCl2 concentrations. Cu content of D. salina cells was higher than D. tertiolecta which may be due to larger volume of D. salina cells. In conclusion, since hydroxyl radical (HO*) produced from H2O2 by Cu2+ (Haber-Weiss cycle) is involved in lipid peroxidation, higher ascorbate peroxidase activity in D. tertiolecta may partly account for lower sensitivity of this species to CuCl2 compared to D. salina.     

Salt-Induced Metabolic Changes in Dunaliella salina

An increase of medium NaCl concentration induces Dunaliella cells to evolve O₂ photosynthetically even in the absence of CO₂. This NaCl-induced O₂ evolution may reflect the induced conversion of reserve carbohydrate to glycerol. The quantum yield for the NaCl-induced O₂ evolution, in the absence of CO₂, is 1.5-fold higher than that obtained for CO₂ fixation. Since the synthesis of glycerol from reserve carbohydrate in the absence of CO₂ requires only 0.5 ATP/NADPH, whereas photosynthesis requires at least 1.3 ATP/NADPH, it is concluded that the ATP/2e⁻ ratio coupled to NADP reduction in Dunaliella is lower than required for CO₂ fixation.     

Competitive binding of iron by transferrins from different vertebrates.

1. A competitive dialysis technique has been used to study the relative affinities of the two iron-binding sites on transferrin molecules and the relative binding strengths of transferrins isolated from plasma of different species. 2. The comparisons were extended to include desialylated human transferrin, ovotransferrin, and a cyanogen bromide fragment of the latter. 3. Although the results of bilateral experiments could generally be accounted for in terms of the theory of independent sites, there were some exceptions, and cyclic comparisons were inconsistent. 4. All the comparisons made were compatible with a model in which site-interaction occurred, but it was not possible to decide whether the sites were intrinsically identical or not. For most species this corresponded to positive cooperativity, but for rabbit it was negative. 5. The average affinity of transferrin for iron depended on species, but the variation was never more than about one order of magnitude. 6. No effect on the binding constants for human transferrin could be detected when the sialic acid residues were removed. 7. The fragment of ovotransferrin competed fairly effectively with the native molecule for iron, although the average relative affinity was only about 1:15. 8. The relative binding of iron by ovotranferrin and human transferrin was affected little when bicarbonate anion was replaced by oxalate, although the ratio of the two binding constants for ovotranferrin increased.     

盐生杜氏藻对盐度改变的生理响应

以盐生杜氏藻为实验材料,采用f/2培养基,设置了8个盐度(15、20、25、30、50、70、90、110)处理,分盐度改变前(A)和盐度改变后(B)两个实验阶段,研究了盐生杜氏藻在不同盐度处理下的生长情况,测定了藻液的OD值、叶绿素a、β-胡萝卜素、可溶性蛋白质和可溶性糖含量等指标。结果表明,A阶段,几个较低盐度(15、20、25和30)处理生长状况较好,其中又以盐度20的处理最好;余下的处理,盐度越高,其生长所受的影响越大。B阶段,盐生杜氏藻的生长进入平台期后,50、70、90、110几个盐度较高处理的细胞密度、叶绿素a、β-胡萝卜素含量均显著超过了作为对照的盐度20的处理。且B阶段末期,先前盐度15的处理蛋白质、糖的积累量,与A阶段末期相比都有了不同程度的增加,而其余盐度处理组的蛋白质、糖含量则分别产生了不同程度的下降。     

Subcellular Adaptation to Salinity and Irradiance in Dunaliella salina

Dunaliella salina V-63 was cultivated in different concentrations of NaCl (0.5, 1.0, 2.5, 3.0, or 4.0 M) and at two irradiances (170 or 220 μmol m⁻²s⁻¹). Concentration-dependent suppression of growth was observed above 1 M NaCl, and elevated salinity induced formation of salt-containing vacuoles. However, the changes in the chloroplast ultrastructure following changes in salinity and irradiance (increase of invaginations and protuberances, numerous grana with low number of thylakoids, less number of starch grains, etc.) appeared to be of primary importance. This revised version was published online in July 2006 with corrections to the Cover Date.     

Calmodulin binding to platelet plasma membranes

Calmodulin copurifies with platelet plasma membranes isolated by glycerol-induced lysis and density gradient centrifugation. These membranes also bind ¹²⁵I-labeled calmodulin in vitro in the presence of Ca²⁺. Binding is largely reduced by replacing Ca²⁺ by Mg²⁺ or by addition of an excess unlabeled calmodulin. The specific component of binding is saturable, with an apparent K_d of 27 nM and a maximum of 15.9 pmol binding sites per mg of membrane protein. This is equivalent to approx. 4100 binding sites per platelet. Binding was inhibited by addition of phenothiazines, a group of calmodulin antagonists. Half-maximal inhibition was attained with approx. 20 μM trifluoperazine or 50 μM chlorpromazine. In contrast, chlorpromazine-sulfoxide which is inactive towards calmodulin, did not affect the binding. Calmodulin binding polypeptides of the plasma membrane were identified by a gel-overlay technique. A major calmodulin-binding component of molecular weight 149 000 was detected. Binding to this band was Ca²⁺-dependent and inhibited by chlorpromazine. The molecular weight of this polypeptide is similar to that of glycoprotein I and also that of the red cell (Ca²⁺ + Mg²⁺)-stimulated ATPase, which is known to bind calmodulin. The possible role of calmodulin in platelet activation is analysed.     

Localization of glycollate dehydrogenase in Dunaliella salina

Glycollate dehydrogenase of the halotolerant green alga Dunaliella salina, isolated from a brine pond, was found associated with the membrane fraction which exhibited complete photosynthetic activity. Highest enzyme activity was found in cells grown in the presence of 5% NaCl. Any increase in NaCl concentration led to a decrease in specific enzyme activity.Abbreviations PSI(II) photosystem I(II)     

盐藻对除草剂草丁膦的抗性研究

对分离出的单藻落盐藻进行除草剂草丁膦的抗性实验,液体培养结果显示,野生盐藻对除草剂草丁膦敏感,3.0mg/L剂量的草丁膦能完全抑制盐藻的生长和增殖。     

Effect of osmotic shock on the redox system in plasma membrane of Dunaliella salina

The unicellular halotolerant alga Dunaliella salina had the ability to oxidize NADH and reduce Fe(CN)6^3-.The redox reactions were to some extent stimulated by slight hyperosmotic shock (2.0mol/L→2.6mol/L NaCl),but markably inhibited by abrupt hyperosmotic shock (2.0mol/L→3.5mol/L NaCl) and hypoosmotic shock (2.0mol/L→1.0mol/L NaCl;2.0mol/L→0.67mol/L NaCl).With the adaptation of algal cells to osmotic shock by accumulating or degraging intracellular grycerol,the plasmalemma redox activities were also restored.The O2 uptake stimulated by NADH could be promoted by FA and SHAM.Hypoosmotic shock increases the basal respiration rate of alga cells,but weakened the stimulating effects of NADH,FA and SHAM on O2 uptake.On the other hand,hyperosmotic shock reduced the basal respiration rate,but relatively enhanced the above effects of NADH,FA and SHAM.H^ extrusion of alga cells was inhibited by NADH and stimulated by Fe(CN)6^3-.Vanadate and DES could inhibit H^ efflux,but had little effect in the presence of NADH and Fe(CN)6^3-.Both hyperand hypoosmotic shock stimulated H^ extrusion.This effect could be totally inhibited by vanadate and DES,but almost unaffected by 8-hydroxyquinoline.It was suggested that H^ -ATPase probably played a more important role in H^ extrusion and osmoregulation under the conditions of osmotic shock.     

Photoinhibition and repair in Dunaliella salina acclimated to different growth irradiances

The light-dependent rate of photosystem-II (PSII) damage and repair was measured in photoautotrophic cultures of Dunaliella salina Teod. grown at different irradiances in the range 50–3000 mol photons · m^–2· s^–1. Rates of cell growth increased in the range of 50–800 mol photons·m^–2·s^–1, remained constant at a maximum in the range of 800–1,500 mol photons·m^–2 ·s^–1, and declined due to photoinhibition in the range of 1500–3000 mol photons·m^–2·s^–1. Western blot analyses, upon addition of lincomycin to the cultures, revealed first-order kinetics for the loss of the PSII reaction-center protein (D1) from the 32-kDa position, occurring as a result of photodamage. The rate constant of this 32-kDa protein loss was a linear function of cell growth irradiance. In the presence of lincomycin, loss of the other PSII reaction-center protein (D2) from the 34-kDa position was also observed, occurring with kinetics similar to those of the 32-kDa form of D1. Increasing rates of photodamage as a function of irradiance were accompanied by an increase in the steady-state level of a higher-molecular-weight protein complex ( 160-kDa) that cross-reacted with D1 antibodies. The steady-state level of the 160-kDa complex in thylakoids was also a linear function of cell growth irradiance. These observations suggest that photodamage to D1 converts stoichiometric amounts of D1 and D2 (i.e., the D1/D2 heterodimer) into a 160-kDa complex. This complex may help to stabilize the reaction-center proteins until degradation and replacement of D1 can occur. The results indicated an intrinsic half-time of about 60 min for the repair of individual PSII units, supporting the idea that degradation of D1 after photodamage is the rate-limiting step in the PSII repair process.Abbreviations Chl chlorophyll - PSI photosystem I - PSII photosystem II - D1 the 32-kDa reaction-center protein of PSII, encoded by the chloroplast psbA gene - D2 the 34-kDa reactioncenter protein of PSII, encoded by the chloroplast psbD gene - Q_A primary electron-accepting plastoquinone of PSII The work was supported by grant 94-37100-7529 from the US Department of Agriculture, National Research Initiative Competitive Grants Program.     

Salt-induced redox-independent phosphorylation of light harvesting chlorophyll a/b proteins in Dunaliella salina thylakoid membranes

This study investigated the regulation of the major light harvesting chlorophyll a/b protein (LHCII) phosphorylation in Dunaliella salina thylakoid membranes. We found that both light and NaCl could induce LHCII phosphorylation in D. salina thylakoid membranes. Treatments with oxidants (ferredoxin and NADP) or photosynthetic electron flow inhibitors (DCMU, DBMIB, and stigmatellin) inhibited LHCII phosphorylation induced by light but not that induced by NaCl. Furthermore, neither addition of CuCl(2), an inhibitor of cytochrome b(6)f complex reduction, nor oxidizing treatment with ferricyanide inhibited light- or NaCl-induced LHCII phosphorylation, and both salts even induced LHCII phosphorylation in dark-adapted D. salina thylakoid membranes as other salts did. Together, these results indicate that the redox state of the cytochrome b(6)f complex is likely involved in light- but not salt-induced LHCII phosphorylation in D. salina thylakoid membranes.     

Somatostatin binding to pituitary plasma membranes.

A method has been developed for the study of somatostatin binding to anterior pituitary plasma membranes. When 5×10^?9M [¹²⁵I]Tyr¹-somatostatin (SA 18 Ci/mmol) was incubated with isolated pituitary plasma membranes (protein = 100 μg), 13.6% of total radioactivity was bound excluding nonspecific binding. The Scatchard plot could be resolved into two distinct components and analyzed to yield: K₁diss = 3.3×10^?8M and K₂diss = 7.7×10^?6M. This binding was shown to be specific for somatostatin.     

甘油对杜氏盐藻电击转化的影响

通过在HEPES电击缓冲液中添加不同浓度的甘油,讨论了甘油对电转前细胞存活率的影响;通过在盐藻培养基中添加不同浓度的甘油,讨论了甘油对盐藻细胞生长的影响;使用含有不同浓度甘油的HEPES缓冲液介导质粒载体转入盐藻细胞,比较了甘油对于转化率的影响。实验结果表明,在电击缓冲液中添加0.5mol/L甘油能有效提高细胞存活率,促进转化细胞恢复生长,从而获得最佳转化效果。因此,0.5mol/L甘油可作为杜氏盐藻电击转化过程中一种良好的稳渗剂。