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      ATP increases within the lumen of the endoplasmic reticulum upon intracellular Ca 2+ release

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          Abstract

          Real-time recordings of ER ATP fluxes in single cells using an ER-targeted, genetically encoded ATP sensor within the lumen of the ER reveal a local Ca 2+-controlled ATP signal that is restored during energy stress. The data highlight a novel Ca 2+-controlled process that supplies the ER with additional energy upon cell stimulation.

          Abstract

          Multiple functions of the endoplasmic reticulum (ER) essentially depend on ATP within this organelle. However, little is known about ER ATP dynamics and the regulation of ER ATP import. Here we describe real-time recordings of ER ATP fluxes in single cells using an ER-targeted, genetically encoded ATP sensor. In vitro experiments prove that the ATP sensor is both Ca 2+ and redox insensitive, which makes it possible to monitor Ca 2+-coupled ER ATP dynamics specifically. The approach uncovers a cell type–specific regulation of ER ATP homeostasis in different cell types. Moreover, we show that intracellular Ca 2+ release is coupled to an increase of ATP within the ER. The Ca 2+-coupled ER ATP increase is independent of the mode of Ca 2+ mobilization and controlled by the rate of ATP biosynthesis. Furthermore, the energy stress sensor, AMP-activated protein kinase, is essential for the ATP increase that occurs in response to Ca 2+ depletion of the organelle. Our data highlight a novel Ca 2+-controlled process that supplies the ER with additional energy upon cell stimulation.

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          Most cited references49

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          Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.

          Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.
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            Regulation of mitochondrial dehydrogenases by calcium ions.

            Studies in Bristol in the 1960s and 1970s, led to the recognition that four mitochondrial dehydrogenases are activated by calcium ions. These are FAD-glycerol phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol phosphate dehydrogenase is located on the outer surface of the inner mitochondrial membrane and is influenced by changes in cytoplasmic calcium ion concentration. The other three enzymes are located within mitochondria and are regulated by changes in mitochondrial matrix calcium ion concentration. These and subsequent studies on purified enzymes, mitochondria and intact cell preparations have led to the widely accepted view that the activation of these enzymes is important in the stimulation of the respiratory chain and hence ATP supply under conditions of increased ATP demand in many stimulated mammalian cells. The effects of calcium ions on FAD-isocitrate dehydrogenase involve binding to an EF-hand binding motif within this enzyme but the binding sites involved in the effects of calcium ions on the three intramitochondrial dehydrogenases remain to be fully established. It is also emphasised in this article that these three dehydrogenases appear only to be regulated by calcium ions in vertebrates and that this raises some interesting and potentially important developmental issues.
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              PROSITE: a documented database using patterns and profiles as motif descriptors.

              Among the various databases dedicated to the identification of protein families and domains, PROSITE is the first one created and has continuously evolved since. PROSITE currently consists of a large collection of biologically meaningful motifs that are described as patterns or profiles, and linked to documentation briefly describing the protein family or domain they are designed to detect. The close relationship of PROSITE with the SWISS-PROT protein database allows the evaluation of the sensitivity and specificity of the PROSITE motifs and their periodic reviewing. In return, PROSITE is used to help annotate SWISS-PROT entries. The main characteristics and the techniques of family and domain identification used by PROSITE are reviewed in this paper.
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                Author and article information

                Contributors
                Role: Monitoring Editor
                Journal
                Mol Biol Cell
                Mol. Biol. Cell
                molbiolcell
                mbc
                Mol. Bio. Cell
                Molecular Biology of the Cell
                The American Society for Cell Biology
                1059-1524
                1939-4586
                01 February 2014
                : 25
                : 3
                : 368-379
                Affiliations
                [1] aInstitute of Molecular Biology and Biochemistry, Center of Physiological Medicine, Medical University of Graz, 8010 Graz, Austria
                [2] bInstitute of Physiological Chemistry, Center of Physiological Medicine, Medical University of Graz, 8010 Graz, Austria
                [3] cPrecursory Research for Embryonic Science, Japan Science and Technology Agency, Tokyo 102-0075, Japan
                Carnegie Mellon University
                Author notes
                1Address correspondence to: Roland Malli ( roland.malli@ 123456medunigraz.at ).
                Article
                E13-07-0433
                10.1091/mbc.E13-07-0433
                3907277
                24307679
                856f0dc5-3f9b-491d-95f0-333ed7eacf4d
                © 2014 Vishnu et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License ( http://creativecommons.org/licenses/by-nc-sa/3.0).

                “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology.

                History
                : 01 August 2013
                : 19 November 2013
                : 22 November 2013
                Categories
                Articles
                Cell Physiology

                Molecular biology
                Molecular biology

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