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Doganli C.,Center for Membrane Pumps in Cells and Disease in | Doganli C.,University of Aarhus | Beck H.C.,University of Southern Denmark | Ribera A.B.,University of Colorado at Denver | And 3 more authors.
Journal of Biological Chemistry

Na+/K+-ATPases are transmembrane ion pumps that maintain ion gradients across the basolateral plasma membrane in all animal cells to facilitate essential biological functions. Mutations in the Na +/K+-ATPase α3 subunit gene (ATP1A3) cause rapid-onset dystonia-parkinsonism, a rare movement disorder characterized by sudden onset of dystonic spasms and slow movements. In the brain, ATP1A3 is principally expressed in neurons. In zebrafish, the transcripts of the two ATP1A3 orthologs, Atp1a3a and Atp1a3b, show distinct expression in the brain. Surprisingly, targeted knockdown of either Atp1a3a or Atp1a3b leads to brain ventricle dilation, a likely consequence of ion imbalances across the plasma membrane that cause accumulation of cerebrospinal fluid in the ventricle. The brain ventricle dilation is accompanied by a depolarization of spinal Rohon-Beard neurons in Atp1a3a knockdown embryos, suggesting impaired neuronal excitability. This is further supported by Atp1a3a or Atp1a3b knockdown results where altered responses to tactile stimuli as well as abnormal motility were observed. Finally, proteomic analysis identified several protein candidates highlighting proteome changes associated with the knockdown of Atp1a3a or Atp1a3b. Our data thus strongly support the role of α3Na +/K+-ATPase in zebrafish motility and brain development, associating for the first time theα3Na+/K +-ATPase deficiency with brain ventricle dilation. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Source

Olsen L.I.,Center for Membrane Pumps in Cells and Disease in | Olsen L.I.,Copenhagen University | Hansen T.H.,Copenhagen University | Larue C.,Ruhr University Bochum | And 16 more authors.
Nature Plants

Insufficient intake of zinc and iron from a cereal-based diet is one of the causes of 'hidden hunger' (micronutrient deficiency), which affects some two billion people1,2. Identifying a limiting factor in the molecular mechanism of zinc loading into seeds is an important step towards determining the genetic basis for variation of grain micronutrient content and developing breeding strategies to improve this trait3. Nutrients are translocated to developing seeds at a rate that is regulated by transport processes in source leaves, in the phloem vascular pathway, and at seed sinks. Nutrients are released from a symplasmic maternal seed domain into the seed apoplasm surrounding the endosperm and embryo by poorly understood membrane transport processes4'6. Plants are unique among eukaryotes in having specific P1B-ATPase pumps for the cellular export of zinc7. In Arabidopsis, we show that two zinc transporting P1B-ATPases actively export zinc from the mother plant to the filial tissues. Mutant plants that lack both zinc pumps accumulate zinc in the seed coat and consequently have vastly reduced amounts of zinc inside the seed. Blockage of zinc transport was observed at both high and low external zinc supplies. The phenotype was determined by the mother plant and is thus due to a lack of zinc pump activity in the seed coat and not in the filial tissues. The finding that P1B-ATPases are one of the limiting factors controlling the amount of zinc inside a seed is an important step towards combating nutritional zinc deficiency worldwide. © 2016 Macmillan Publishers Limited. All rights reserved. Source

Pedersen B.P.,Center for Membrane Pumps in Cells and Disease in | Pedersen B.P.,University of Aarhus | Ifrim G.,Insight Centre for Data Analytics | Liboriussen P.,Center for Membrane Pumps in Cells and Disease in | And 10 more authors.

Background: Structured Logistic Regression (SLR) is a newly developed machine learning tool first proposed in the context of text categorization. Current availability of extensive protein sequence databases calls for an automated method to reliably classify sequences and SLR seems well-suited for this task. The classification of P-type ATPases, a large family of ATPdriven membrane pumps transporting essential cations, was selected as a test-case that would generate important biological information as well as provide a proof-of-concept for the application of SLR to a large scale bioinformatics problem. Results: Using SLR, we have built classifiers to identify and automatically categorize P-type ATPases into one of 11 predefined classes. The SLR-classifiers are compared to a Hidden Markov Model approach and shown to be highly accurate and scalable. Representing the bulk of currently known sequences, we analysed 9.3 million sequences in the UniProtKB and attempted to classify a large number of P-type ATPases. To examine the distribution of pumps on organisms, we also applied SLR to 1,123 complete genomes from the Entrez genome database. Finally, we analysed the predicted membrane topology of the identified P-type ATPases. Conclusions: Using the SLR-based classification tool we are able to run a large scale study of P-type ATPases. This study provides proof-of-concept for the application of SLR to a bioinformatics problem and the analysis of P-type ATPases pinpoints new and interesting targets for further biochemical characterization and structural analysis. © 2014 Pedersen et al. Source

Justesen B.H.,Center for Membrane Pumps in Cells and Disease in | Justesen B.H.,Copenhagen University | Hansen R.W.,Copenhagen University | Martens H.J.,Copenhagen University | And 9 more authors.
Journal of Biological Chemistry

Background: The plasma membrane H+-ATPase generates electrochemical gradients in plants and fungi. The minimal subunit organization required for activity is not known. Results: We developed a protocol for reconstitution of active H+-ATPase in nanodiscs. Conclusion: The minimal functional unit of the H+-ATPase is a monomer. Significance: The plasma membrane H+-ATPase functions like well characterized cation pumping P-type ATPases. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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