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© The Rockefeller University Press, 0021-9525/1998//1515 $5.00
The Journal of Cell Biology, Volume 141, Number 7, , 1998 1515-1527


Articles

The Mammalian Calcium-binding Protein, Nucleobindin (CALNUC), Is a Golgi Resident Protein



Ping Lin, Helen Le-Niculescu, Robert Hofmeister, J. Michael McCaffery, Mingjie Jin, Hanjo Hennemann, Tammie McQuistan, Luc De Vries, and Marilyn Gist Farquhar

Division of Cellular and Molecular Medicine and Department of Pathology, University of California, San Diego, La Jolla, California 92093-0651

We have identified CALNUC, an EF-hand, Ca2+-binding protein, as a Golgi resident protein. CALNUC corresponds to a previously identified EF-hand/calcium-binding protein known as nucleobindin. CALNUC interacts with G{alpha}i3 subunits in the yeast two-hybrid system and in GST-CALNUC pull-down assays. Analysis of deletion mutants demonstrated that the EF-hand and intervening acidic regions are the site of CALNUC's interaction with G{alpha}i3. CALNUC is found in both cytosolic and membrane fractions. The membrane pool is tightly associated with the luminal surface of Golgi membranes. CALNUC is widely expressed, as it is detected by immunofluorescence in the Golgi region of all tissues and cell lines examined. By immunoelectron microscopy, CALNUC is localized to cis-Golgi cisternae and the cis-Golgi network (CGN). CALNUC is the major Ca2+-binding protein detected by 45Ca2+-binding assay on Golgi fractions. The properties of CALNUC and its high homology to calreticulin suggest that it may play a key role in calcium homeostasis in the CGN and cis-Golgi cisternae.


Abbreviations used in this paper: {alpha}-Man II, {alpha}-Mannosidase II; β-gal, β-galactosidase; aa, amino acids; CGN, cis-Golgi network; CHX, cycloheximide, CRT, calreticulin; CV, carrier vesicle fraction; Cyt, cytosol; EELS, electron energy loss spectroscopy; ERGIC, ER–Golgi intermediate compartment; GAP, GTPase-activating protein; GH, Golgi heavy; GL, Golgi light; GDT, glutathione-S-transferase; NLS, nuclear localization signal; PFA, paraformaldehyde; PK, proteinase K; PNS, postnuclear supernatant; PVDF, polyvinylidene difluoride; RM, residual microsomes; TBS-T, TBS–0.05% Tween; TM, total membranes.

Helen Le-Niculescu is a graduate student in the Molecular Pathology Graduate Program at UCSD and is a Markey Foundation Fellow. This work was supported by National Institutes of Health grants DK 17780 and CA 58689 to Marilyn G. Farquhar.

P. Lin and H. Le-Niculescu contributed equally to this work.

Dr. Hennemann's present address is the Institute of Cell Biology, Cancer Research, University of Essen, Virchowstr. 173, D-45122 Essen, Germany.



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