Overexpression of Calreticulin Increases the Ca 2 ÷ Capacity of Rapidly

A molecularly tagged form of calreticulin (CR), a low affinity-high capacity Ca 2+ binding protein that resides in the ER lumen, was transiently transfected into HeLa cells to specifically modify the Ca 2÷ buffering capacity of the intracellular Ca 2÷ stores. Fluorescence and confocal microscope immunocytochemistry revealed the tagged protein to be expressed by over 40% of the cells and to overlap in its distribution the endogenous CR yielding a delicate cytoplasmic network, i.e., the typical pattern of ER. In contrast, no signal was observed associated with the plasmalemma (marked by ConA) and within the nucleus. Oneand two-dimensional Western blots revealed the transfected to exceed the endogenous CR of ~3.5-fold and to maintain its Ca 2+ binding ability, whereas the expression of other ER proteins was unchanged. Ca 2÷ homeostasis in the transfected cells was investigated by three parallel approaches: (a) 45Ca equilibrium loading of cell populations; (b) [Ca2+]c measurement with fura2 followed by quantitative immunocytochemistry of single cells and iii) [Ca2+]c measurement of cell population upon cotransfection with the Ca2+-sensitive photoprotein, aequorin. The three approaches revealed different aspects of Ca 2+ homeostasis, yielding results which were largely complementary. In particular, the following conclusions were established: (a) both endogenous and transfected CR participate in Ca 2÷ buffering within the IP3-sensitive, rapidly exchanging, Ca 2÷ stores; the other pools of the cells were in contrast unaffected by CR transfection; (b) the Ca 2+ capacity of the stores is not the main limiting factor of individual IP3mediated Ca 2+ release responses triggered by receptor agonists; (c) in control cells, the contribution of CR to Ca 2+ buffering within the IP3-sensitive stores accounts for ~45 % of the total, the rest being probably contributed by the other lumenal (and also membrane) Ca 2÷ binding proteins; (d) the free [Ca z÷] within the lumen of the IP3-sensitive stores, revealed by the degree of Ca 2÷ binding to the transfected CR protein, amounts to values in (or approaching) the millimolar range; and (e) Ca 2+ influx across the plasmalemma activated by depletion of the stores is directly dependent on the lumenal [Ca2+]. T HE intracellular rapidly exchanging stores of Ca 2+ have attracted increasing interest during the last several years (for reviews see Carafoli, 1987; Berridge, 1993; Pozzan et al., 1994). These structures are responsible for a fundamental step in many types of cell activation, the release of Ca 2+ to the cytosol taking place not at the surface but at multiple sites within the cytoplasm. Among cytoplasmic organelles, the ER is commonly identified as the cytological counterpart of the stores. Results in various cell types have however indicated that not the entire ER, but discrete areas (e.g., the sarcoplasmic reticuAddress correspondence to Prof. T. Pozzan, Dept. of Biomedical Sciences, University of Padova, Via Trieste, 75, 35121 Padova, Italy. Ph.: 3949-8286568. Fax: 39-49-8286576. lum of striated and smooth muscle fibers) are specialized in Ca 2÷ uptake and release (for review see Pozzan et al., 1994). Moreover, although many of the various molecular actors of the stores have been identified (a family of Ca 2÷ pumps, the sarcoplasmic-endoplasmic reticulum Ca 2+ ATPases, [SERCAs], 1 for uptake [MacLennan, 1990]; a group of lumenal Ca 2÷ binding proteins characterized by low affinity and high capacity, for storage [Lytton and Ni1. Abbreviations used in this paper. Ab, antibody; [Ca2+]c and [Ca2+]er, concentration of Ca 2÷ in the cytosol and in the lumen of rapidly exchanging Ca 2÷ stores; CR, tCR, and tCR cells, calreticulin, tagged calreticulin, and cells transfected with tagged calreticulin; IP3, inositol 1,4,5-trisphosphate; PDI, protein disulfide isornerase; SERCA, sarcoplasmic-endoplasmic reticulum Ca 2+ ATPase; Tg, thapsigargin. © The Rockefeller University Press, 0021-9525/95/08/847/9 $2.00 The Journal of Cell Biology, Volume 130, Number 4, August 1995 847-855 847 on F ebuary 1, 2013 jcb.rress.org D ow nladed fom Published August 15, 1995