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

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.