Crystal Structure of a Yeast Aquaporin at 1.15 ? Reveals a Novel Gating Mechanism

Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 Å resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.     

RIN4 Functions with Plasma Membrane H+-ATPases to Regulate Stomatal Apertures during Pathogen Attack

Pathogen perception by the plant innate immune system is of central importance to plant survival and productivity. The Arabidopsis protein RIN4 is a negative regulator of plant immunity. In order to identify additional proteins involved in RIN4-mediated immune signal transduction, we purified components of the RIN4 protein complex. We identified six novel proteins that had not previously been implicated in RIN4 signaling, including the plasma membrane (PM) H⁺-ATPases AHA1 and/or AHA2. RIN4 interacts with AHA1 and AHA2 both in vitro and in vivo. RIN4 overexpression and knockout lines exhibit differential PM H⁺-ATPase activity. PM H⁺-ATPase activation induces stomatal opening, enabling bacteria to gain entry into the plant leaf; inactivation induces stomatal closure thus restricting bacterial invasion. The rin4 knockout line exhibited reduced PM H⁺-ATPase activity and, importantly, its stomata could not be re-opened by virulent Pseudomonas syringae. We also demonstrate that RIN4 is expressed in guard cells, highlighting the importance of this cell type in innate immunity. These results indicate that the Arabidopsis protein RIN4 functions with the PM H⁺-ATPase to regulate stomatal apertures, inhibiting the entry of bacterial pathogens into the plant leaf during infection.     

Roles of plasma membrane proton ATPases AHA2 and AHA7 in normal growth of roots and root hairs in Arabidopsis thaliana

Plasma membrane H⁺‐ATPase pumps build up the electrochemical H⁺ gradients that energize most other transport processes into and out of plant cells through channel proteins and secondary active carriers. In Arabidopsis thaliana, the AUTOINHIBITED PLASMA MEMBRANE H⁺‐ATPases AHA1, AHA2 and AHA7 are predominant in root epidermal cells. In contrast to other H⁺‐ATPases, we find that AHA7 is autoinhibited by a sequence present in the extracellular loop between transmembrane segments 7 and 8. Autoinhibition of pump activity was regulated by extracellular pH, suggesting negative feedback regulation of AHA7 during establishment of an H⁺ gradient. Due to genetic redundancy, it has proven difficult to test the role of AHA2 and AHA7, and mutant phenotypes have previously only been observed under nutrient stress conditions. Here, we investigated root and root hair growth under normal conditions in single and double mutants of AHA2 and AHA7. We find that AHA2 drives root cell expansion during growth but that, unexpectedly, restriction of root hair elongation is dependent on AHA2 and AHA7, with each having different roles in this process.     

蚕豆叶片发育与衰老过程中超氧物歧化酶活性与丙二醛含量变化

蚕豆植株叶片随茎节自上而下表现出明显的发育与衰老顺序,可作为衰老特征的是叶绿素和蛋白质含量明显下降。蚕豆叶中SOD活性主要定位于12 000× g离心后所得的上清液和叶绿体组分。衰老叶片的SOD总活性和叶绿体组分的相对活性都有所下降,SOD同工酶谱也发生了改变。O_2~ 产生速率随叶龄增大而稍上升;而MDA含量在叶片外观表现枯黄衰老征兆前就急剧上升。可能因为衰老叶片过氧化氢酶活性大幅度下降与SOD之间的不平衡,致使O_2~ 代谢中间产物累积而引起膜的损伤.     

Substrate stabilization of lysophosphatidylcholine-solubilized plasma membrane H+-ATPase from oat roots

Plasma membrane vesicles with H⁺-ATPase activity were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots using an aqueous polymer two-phase system. Of several detergents tested, only lysophosphatidylcholine solubilized the H⁺-ATPase in an active form. Solubilization of the H⁺-ATPase with lysophosphatidylcholine was possible in the absence of glycerol, but the ATPase activity decreased about 4–5 times as rapidly in the absence as in the presence of 30% (w/v) glycerol. The solubilized enzyme was further stabilized by ATP and protons. Addition of 1 m M ATP to the plasma membranes halted inactivation of the H⁺-ATPase. Even in the absence of polyol compounds and ATP, the enzyme was stable for hours at relatively low pH with an optimum around pH 6.7 at room temperature. The curve for the stability of soluble H⁺-ATPase as a function of pH closely resembles the pH curve for the activity of the H⁺-ATPase. This suggests that binding of protons to transport sites may stabilize the soluble H⁺-ATPase in an enzymatically active form.     

Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

Monoclonal antibody to dengue capsid protein: Its application in dengue studies

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection.Key words: monoclonal antibodies, capsid protein, dengue virus, diagnosis, immunoassays     

Zinc biofortification of cereals: problems and solutions

The goal of biofortification is to develop plants that have an increased content of bioavailable nutrients in their edible parts. Cereals serve as the main staple food for a large proportion of the world population but have the shortcoming, from a nutrition perspective, of being low in zinc and other essential nutrients. Major bottlenecks in plant biofortification appear to be the root-shoot barrier and - in cereals - the process of grain filling. New findings demonstrate that the root-shoot distribution of zinc is controlled mainly by heavy metal transporting P(1B)-ATPases and the metal tolerance protein (MTP) family. A greater understanding of zinc transport is important to improve crop quality and also to help alleviate accumulation of any toxic metals.