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      Overexpression of CALNUC (Nucleobindin) Increases Agonist and Thapsigargin Releasable Ca 2+ Storage in the Golgi

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          Abstract

          We previously demonstrated that CALNUC, a Ca 2+-binding protein with two EF-hands, is the major Ca 2+-binding protein in the Golgi by 45Ca 2+ overlay (Lin, P., H. Le-Niculescu, R. Hofmeister, J.M. McCaffery, M. Jin, H. Henneman, T. McQuistan, L. De Vries, and M. Farquhar. 1998. J. Cell Biol. 141:1515–1527). In this study we investigated CALNUC's properties and the Golgi Ca 2+ storage pool in vivo. CALNUC was found to be a highly abundant Golgi protein (3.8 μg CALNUC/mg Golgi protein, 2.5 × 10 5 CALNUC molecules/NRK cell) and to have a single high affinity, low capacity Ca 2+-binding site ( K d = 6.6 μM, binding capacity = 1.1 μmol Ca 2+/μmol CALNUC). 45Ca 2+ storage was increased by 2.5- and 3-fold, respectively, in HeLa cells transiently overexpressing CALNUC-GFP and in EcR-CHO cells stably overexpressing CALNUC. Deletion of the first EF-hand α helix from CALNUC completely abolished its Ca 2+-binding capability. CALNUC was correctly targeted to the Golgi in transfected cells as it colocalized and cosedimented with the Golgi marker, α-mannosidase II (Man II). Approximately 70% of the 45Ca 2+ taken up by HeLa and CHO cells overexpressing CALNUC was released by treatment with thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) (Ca 2+ pump) blocker. Stimulation of transfected cells with the agonist ATP or IP 3 alone (permeabilized cells) also resulted in a significant increase in Ca 2+ release from Golgi stores. By immunofluorescence, the IP 3 receptor type 1 (IP 3R-1) was distributed over the endoplasmic reticulum and codistributed with CALNUC in the Golgi. These results provide direct evidence that CALNUC binds Ca 2+ in vivo and together with SERCA and IP 3R is involved in establishment of the agonist-mobilizable Golgi Ca 2+ store.

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          Store depletion and calcium influx.

          Calcium influx in nonexcitable cells regulates such diverse processes as exocytosis, contraction, enzyme control, gene regulation, cell proliferation, and apoptosis. The dominant Ca2+ entry pathway in these cells is the store-operated one, in which Ca2+ entry is governed by the Ca2+ content of the agonist-sensitive intracellular Ca2+ stores. Only recently has a Ca2+ current been described that is activated by store depletion. The properties of this new current, called Ca2+ release-activated Ca2+ current (ICRAC), have been investigated in detail using the patch-clamp technique. Despite intense research, the nature of the signal that couples Ca2+ store content to the Ca2+ channels in the plasma membrane has remained elusive. Although ICRAC appears to be the most effective and widespread influx pathway, other store-operated currents have also been observed. Although the Ca2+ release-activated Ca2+ channel has not yet been cloned, evidence continues to accumulate that the Drosophila trp gene might encode a store-operated Ca2+ channel. In this review, we describe the historical development of the field of Ca2+ signaling and the discovery of store-operated Ca2+ currents. We focus on the electrophysiological properties of the prototype store-operated current ICRAC, discuss the regulatory mechanisms that control it, and finally consider recent advances toward the identification of molecular mechanisms involved in this ubiquitous and important Ca2+ entry pathway.
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            Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins.

            Many cellular events depend on a tightly compartmentalized distribution of H+ ions across membrane-bound organelles. However, measurements of organelle pH in living cells have been scarce. Several mutants of the Aequorea victoria green fluorescent protein (GFP) displayed a pH-dependent absorbance and fluorescent emission, with apparent pKa values ranging from 6.15 (mutations F64L/S65T/H231L) and 6.4 (K26R/F64L/S65T/Y66W/N146I/M153T/ V163A/N164H/H231L) to a remarkable 7.1 (S65G/S72A/T203Y/H231L). We have targeted these GFPs to the cytosol plus nucleus, the medial/trans-Golgi by fusion with galactosyltransferase, and the mitochondrial matrix by using the targeting signal from subunit IV of cytochrome c oxidase. Cells in culture transfected with these cDNAs displayed the expected subcellular localization by light and electron microscopy and reported local pH that was calibrated in situ with ionophores. We monitored cytosolic and nuclear pH of HeLa cells, and mitochondrial matrix pH in HeLa cells and in rat neonatal cardiomyocytes. The pH of the medial/trans-Golgi was measured at steady-state (calibrated to be 6.58 in HeLa cells) and after various manipulations. These demonstrated that the Golgi membrane in intact cells is relatively permeable to H+, and that Cl- serves as a counter-ion for H+ transport and likely helps to maintain electroneutrality. The amenability to engineer GFPs to specific subcellular locations or tissue targets using gene fusion and transfer techniques should allow us to examine pH at sites previously inaccessible.
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              Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400.

              Cloning and expression of functional P400 protein from cerebellar Purkinje neurons shows that this protein is a receptor for inositol 1,4,5-trisphosphate, a second messenger that mediates the release of intracellular calcium.
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                Author and article information

                Journal
                J Cell Biol
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                19 April 1999
                : 145
                : 2
                : 279-289
                Affiliations
                [* ]Division of Cellular and Molecular Medicine, []Department of Pathology, []Department of Pharmacology, and [§ ]Howard Hughes Medical Institute, University of California San Diego, La Jolla, California 92093-0651
                Author notes

                Address correspondence to Marilyn G. Farquhar, Ph.D., Division of Cellular and Molecular Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0651. Tel.: (619) 534-7711. Fax: (619) 534-8549. E-mail: mfarquhar@ 123456ucsd.edu

                Article
                2133108
                10209024
                af370956-1fce-4d01-ba55-897447cfdcd9
                Copyright @ 1999
                History
                : 15 October 1998
                : 17 March 1999
                Categories
                Regular Articles

                Cell biology
                golgi resident calcium-binding protein,ef-hand,ip3 receptor,serca,nucleobindin
                Cell biology
                golgi resident calcium-binding protein, ef-hand, ip3 receptor, serca, nucleobindin

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