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 microg CALNUC/mg Golgi protein, 2.5 x 10(5) CALNUC molecules/NRK cell) and to have a single high affinity, low capacity Ca2+-binding site (Kd = 6.6 microM, binding capacity = 1.1 micromol Ca2+/micromol 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 alpha 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, alpha-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.
Lin
P
P
Division of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093-0651, USA.
Yao
Y
Y
Hofmeister
R
R
Tsien
R Y
RY
Farquhar
M G
MG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D006133:Growth Substances / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019284:Thapsigargin / Q000494:pharmacology*; D014162:Transfection
D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D006133:Growth Substances; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium