Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Full Text Full Text (PDF, 405K) PPT slides of all figures Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JCB Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Lin, P. Articles by Farquhar, M. G. Search for Related Content PubMed PubMed Citation Articles by Lin, P. Articles by Farquhar, M. G. Social Bookmarking                            What's this?

© The Rockefeller University Press, 0021-9525/1999//279 $5.00
The Journal of Cell Biology, Volume 145, Number 2, , 1999 279-289

Overexpression of CALNUC (Nucleobindin) Increases Agonist and Thapsigargin Releasable Ca²⁺ Storage in the Golgi

Ping Lin^*, Yong Yao, Robert Hofmeister^*, Roger Y. Tsien^,, and Marilyn Gist Farquhar^*,||

^* 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

We previously demonstrated that CALNUC, a Ca²⁺-binding protein with two EF-hands, is the major Ca²⁺-binding protein in the Golgi by ⁴⁵Ca²⁺ 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 Ca²⁺ storage pool in vivo. CALNUC was found to be a highly abundant Golgi protein (3.8 µg CALNUC/mg Golgi protein, 2.5 x 10⁵ CALNUC molecules/NRK cell) and to have a single high affinity, low capacity Ca²⁺-binding site (K_d = 6.6 µM, binding capacity = 1.1 µmol Ca²⁺/µmol CALNUC). ⁴⁵Ca²⁺ 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 helix from CALNUC completely abolished its Ca²⁺-binding capability. CALNUC was correctly targeted to the Golgi in transfected cells as it colocalized and cosedimented with the Golgi marker, -mannosidase II (Man II). Approximately 70% of the ⁴⁵Ca²⁺ taken up by HeLa and CHO cells overexpressing CALNUC was released by treatment with thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) (Ca²⁺ pump) blocker. Stimulation of transfected cells with the agonist ATP or IP₃ alone (permeabilized cells) also resulted in a significant increase in Ca²⁺ release from Golgi stores. By immunofluorescence, the IP₃ receptor type 1 (IP₃R-1) was distributed over the endoplasmic reticulum and codistributed with CALNUC in the Golgi. These results provide direct evidence that CALNUC binds Ca²⁺ in vivo and together with SERCA and IP₃R is involved in establishment of the agonist-mobilizable Golgi Ca²⁺ store.

Key Words: Golgi resident calcium-binding protein • EF-hand • IP₃ receptor • SERCA • nucleobindin

Abbreviations used in this paper: CaM, calmodulin; CRT, calreticulin; GFP, green fluorescent protein; IP₃R, inositol 1, 4, 5 trisphosphate receptor; Man II, -mannosidase II; PLC, phospholipase C; SERCA, sarcoplasmic/ER calcium ATPase; Tg, thapsigargin.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Facebook

Reddit

Technorati

Twitter

This article has been cited by other articles:

• Tsukumo, Y., Tsukahara, S., Saito, S., Tsuruo, T., Tomida, A. (2009). A Novel Endoplasmic Reticulum Export Signal: PROLINE AT THE +2-POSITION FROM THE SIGNAL PEPTIDE CLEAVAGE SITE. J. Biol. Chem. 284: 27500-27510 [Abstract] [Full Text]  
• Tsukumo, Y., Tomida, A., Kitahara, O., Nakamura, Y., Asada, S., Mori, K., Tsuruo, T. (2007). Nucleobindin 1 Controls the Unfolded Protein Response by Inhibiting ATF6 Activation. J. Biol. Chem. 282: 29264-29272 [Abstract] [Full Text]  
• Lara-Lemus, R., Liu, M., Turner, M. D., Scherer, P., Stenbeck, G., Iyengar, P., Arvan, P. (2006). Lumenal protein sorting to the constitutive secretory pathway of a regulated secretory cell. J. Cell Sci. 119: 1833-1842 [Abstract] [Full Text]  
• Le-Niculescu, H., Niesman, I., Fischer, T., DeVries, L., Farquhar, M. G. (2005). Identification and Characterization of GIV, a Novel G{alpha}i/s -interacting Protein Found on COPI, Endoplasmic Reticulum-Golgi Transport Vesicles. J. Biol. Chem. 280: 22012-22020 [Abstract] [Full Text]  
• Ito, D., Walker, J. R., Thompson, C. S., Moroz, I., Lin, W., Veselits, M. L., Hakim, A. M., Fienberg, A. A., Thinakaran, G. (2004). Characterization of Stanniocalcin 2, a Novel Target of the Mammalian Unfolded Protein Response with Cytoprotective Properties. Mol. Cell. Biol. 24: 9456-9469 [Abstract] [Full Text]  
• Reinhardt, T. A., Horst, R. L., Waters, W. R. (2004). Characterization of Cos-7 cells overexpressing the rat secretory pathway Ca2+-ATPase. Am. J. Physiol. Cell Physiol. 286: C164-C169 [Abstract] [Full Text]  
• Pattingre, S., De Vries, L., Bauvy, C., Chantret, I., Cluzeaud, F., Ogier-Denis, E., Vandewalle, A., Codogno, P. (2003). The G-protein Regulator AGS3 Controls an Early Event during Macroautophagy in Human Intestinal HT-29 Cells. J. Biol. Chem. 278: 20995-21002 [Abstract] [Full Text]  
• Lavoie, C., Meerloo, T., Lin, P., Farquhar, M. G. (2002). Calnuc, an EF-Hand Ca2+-Binding Protein, Is Stored and Processed in the Golgi and Secreted by the Constitutive-Like Pathway in AtT20 Cells. Mol. Endocrinol. 16: 2462-2474 [Abstract] [Full Text]  
• Srinivas, S. P., Ong, A., Goon, L., Goon, L., Bonanno, J. A. (2002). Lysosomal Ca2+ Stores in Bovine Corneal Endothelium. IOVS 43: 2341-2350 [Abstract] [Full Text]  
• Missiaen, L., Vanoevelen, J., Parys, J. B., Raeymaekers, L., De Smedt, H., Callewaert, G., Erneux, C., Wuytack, F. (2002). Ca2+ Uptake and Release Properties of a Thapsigargin-insensitive Nonmitochondrial Ca2+ Store in A7r5 and 16HBE14o- Cells. J. Biol. Chem. 277: 6898-6902 [Abstract] [Full Text]  
• Weiss, T. S., Chamberlain, C. E., Takeda, T., Lin, P., Hahn, K. M., Farquhar, M. G. (2001). Galpha i3 binding to calnuc on Golgi membranes in living cells monitored by fluorescence resonance energy transfer of green fluorescent protein fusion proteins. Proc. Natl. Acad. Sci. USA 98: 14961-14966 [Abstract] [Full Text]  
• Saez, E., Nelson, M. C., Eshelman, B., Banayo, E., Koder, A., Cho, G. J., Evans, R. M. (2000). Identification of ligands and coligands for the ecdysone-regulated gene switch. Proc. Natl. Acad. Sci. USA 10.1073/pnas.260499497v1 [Abstract] [Full Text]  
• Reinhardt, T. A., Filoteo, A. G., Penniston, J. T., Horst, R. L. (2000). Ca2+-ATPase protein expression in mammary tissue. Am. J. Physiol. Cell Physiol. 279: C1595-C1602 [Abstract] [Full Text]  
• Jaconi, M., Bony, C., Richards, S. M., Terzic, A., Arnaudeau, S., Vassort, G., Pucéat, M. (2000). Inositol 1,4,5-Trisphosphate Directs Ca2+ Flow between Mitochondria and the Endoplasmic/Sarcoplasmic Reticulum: A Role in Regulating Cardiac Autonomic Ca2+ Spiking. Mol. Biol. Cell 11: 1845-1858 [Abstract] [Full Text]  
• Lin, P., Fischer, T., Weiss, T., Farquhar, M. G. (2000). Calnuc, an EF-Hand Ca2+ binding protein, specifically interacts with the C-terminal alpha 5-helix of Galpha i3. Proc. Natl. Acad. Sci. USA 97: 674-679 [Abstract] [Full Text]  
• Porat, A., Elazar, Z. (2000). Regulation of Intra-Golgi Membrane Transport by Calcium. J. Biol. Chem. 275: 29233-29237 [Abstract] [Full Text]  
• Van Baelen, K., Vanoevelen, J., Missiaen, L., Raeymaekers, L., Wuytack, F. (2001). The Golgi PMR1 P-type ATPase of Caenorhabditis elegans. IDENTIFICATION OF THE GENE AND DEMONSTRATION OF CALCIUM AND MANGANESE TRANSPORT. J. Biol. Chem. 276: 10683-10691 [Abstract] [Full Text]  
• Numata, M., Orlowski, J. (2001). Molecular Cloning and Characterization of a Novel (Na+,K+)/H+ Exchanger Localized to the trans-Golgi Network. J. Biol. Chem. 276: 17387-17394 [Abstract] [Full Text]  
• Ahluwalia, J. P., Topp, J. D., Weirather, K., Zimmerman, M., Stamnes, M. (2001). A Role for Calcium in Stabilizing Transport Vesicle Coats. J. Biol. Chem. 276: 34148-34155 [Abstract] [Full Text]  
• Saez, E., Nelson, M. C., Eshelman, B., Banayo, E., Koder, A., Cho, G. J., Evans, R. M. (2000). Identification of ligands and coligands for the ecdysone-regulated gene switch. Proc. Natl. Acad. Sci. USA 97: 14512-14517 [Abstract] [Full Text]  
• Wang, X., Kaetzel, M. A., Yoo, S. E., Kim, P. S., Dedman, J. R. (2002). Ligand-regulated secretion of recombinant annexin V from cultured thyroid epithelial cells. Am. J. Physiol. Cell Physiol. 282: C1313-C1321 [Abstract] [Full Text]  

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents