Overexpression of CALNUC ( Nucleobindin ) Increases Agonist and Thapsigargin Releasable Ca 2 1 Storage in the Golgi

We previously demonstrated that CALNUC, a Ca 2 1 -binding protein with two EF-hands, is the major Ca 2 1 -binding protein in the Golgi by 45 Ca 2 1 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 1 storage pool in vivo. CALNUC was found to be a highly abundant Golgi protein (3.8 m g CALNUC/mg Golgi protein, 2.5 3 10 5 CALNUC molecules/NRK cell) and to have a single high affinity, low capacity Ca 2 1 -binding site ( K d 5 6.6 m M, binding capacity 5 1.1 m mol Ca 2 1 / m mol CALNUC). 45 Ca 2 1 storage was increased by 2.5and 3-fold, respectively, in HeLa cells transiently overexpressing CALNUC-GFP and in EcR-CHO cells stably overexpressing CALNUC. Deletion of the first EF-hand a helix from CALNUC completely abolished its Ca 2 1 -binding capability. CALNUC was correctly targeted to the Golgi in transfected cells as it colocalized and cosedimented with the Golgi marker, a -mannosidase II (Man II). Approximately 70% of the 45 Ca 2 1 taken up by HeLa and CHO cells overexpressing CALNUC was released by treatment with thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) (Ca 2 1 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 1 release from Golgi stores. By immunofluorescence, the IP 3 receptor type 1 (IP 3 R-1) was distributed over the endoplasmic reticulum and codistributed with CALNUC in the Golgi. These results provide direct evidence that CALNUC binds Ca 2 1 in vivo and together with SERCA and IP 3 R is involved in establishment of the agonist-mobilizable Golgi Ca 2 1 store.