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

Abstract

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 × 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 α 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, α-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.

Molecular Cloning and Characterization of a Novel (Na+,K+)/H+ Exchanger Localized to the trans-Golgi Network

“…To assess NHE7 HA activity, we adapted procedures that had been used previously for measuring 45 Ca 2ϩ uptake and release from the Golgi (45). The plasma membrane was selectively permeabilized with saponin, and 22 Na ϩ uptake into intact endomembrane compartments was compared with and without ponasterone A-induced overexpression of NHE7 HA .…”

Identification of ligands and coligands for the ecdysone-regulated gene switch

“…Deliberate manipulation of gene activity is also an indispensable tool for clinical applications such as gene therapy. The ecdysone-inducible system has proved useful for studying a multitude of processes, such as apoptosis (32)(33)(34)(35)(36), cancer and cell-cycle regulation (37)(38)(39)(40), embryonic development (41,42), signal transduction (43)(44)(45), lipid metabolism (46), and neuronal function (47)(48)(49)(50). The strengths of the system appear to be its tight regulation, its dose responsiveness, and the favorable uptake and clearance kinetics of the steroid inducer, which results in rapid gene switching.…”

Organelles Containing Inositol Trisphosphate Receptor Type 2 in Adrenal Medullary Cells

“…These results suggest that SERCA pumps are responsible for Ca 2+ uptake into the InsP 3 -sensitive Ca 2+ store sites. SERCA pumps are firmly established to be present in the SR in the muscles [2][3][4], but in other cells [24,29] they were also very likely to reside in other organelles, such as the Golgi. Thus we studied whether SERCA pumps were located in the ER.…”