Characterization of Cos-7 cells overexpressing the rat secretory pathway Ca²⁺-ATPase

Abstract

On the basis of sequence similarities to the yeast PMR1 and hSPCA gene, the rat alternatively spliced mRNA has been suggested to be a Golgi secretory pathway Ca2+-ATPase (SPCA). Data in this report lend further support for this hypothesis in that sucrose gradient fractionation of rat liver microsomes resulted in SPCA comigrating with the Golgi calcium binding protein CALNUC, which was well resolved from the endoplasmic reticulum marker calreticulin. Also, in PC-12 cells, antibody to SPCA colocalized with an antibody to the Golgi marker α-mannosidase II. To study the biological effects of SPCA expression, we performed stable overexpression of SPCA in COS-7 cells. Seven clones were selected for further comparison with COS-7 cells containing an empty expression vector. Overexpression of SPCA resulted in a significant reduction of plasma membrane Ca2+-ATPase, sarco(endo)plasmic reticulum Ca2+-ATPase, and calreticulin expression in these clones. In contrast, the expression of the Golgi calcium-binding protein CALNUC increased significantly. The phosphoenzyme intermediate formed using membranes from clone G11/5 was calcium dependent, significantly more intense than in COS-7 cells, and not affected by La3+ treatment. Calcium uptake by G11/5 microsomes was ATP dependent and significantly greater than in microsomes from parent COS-7 cells. The overexpression of SPCA significantly increased the growth rate of these cells compared with COS-7 cells containing only the empty vector. These data demonstrate that overexpression of the rat SPCA results in significant changes in the expression of calcium transport and storage proteins in COS-7 cells.

Activity of the hSPCA1 Golgi Ca2+ pump is essential for Ca2+-mediated Ca2+ response and cell viability in Darier disease

“…Thus, this report adds to the growing body of information demonstrating that this previously obscure (to mammalian cell biologists) Ca 2+ -ATPase in fact plays an essential role in mammalian cell Ca 2+ signaling. For example, Reinhardt and co-workers observed that overexpression of rat SPCA in COS-7 cells caused significant alterations in several of the cell Ca 2+ transport molecules and dramatically increased the cell division rate (Reinhardt et al, 2004 Taken together, the yeast and human data strongly suggest that SPCA is the mammalian Golgi Ca 2+ -ATPase. The opposing argument put forth by Taylor and co-workers (Taylor et al, 1997) is that the Golgi complex does not contain a unique resident Ca 2+ -ATPase.…”

Deficiency of ATP2C1, a Golgi Ion Pump, Induces Secretory Pathway Defects in Endoplasmic Reticulum (ER)-associated Degradation and Sensitivity to ER Stress

“…A-treated cells) as has been reported previously (17,20,30,31). It was therefore of interest to examine endogenous expression of ATP2C1 in keratinocytes relative to other cells, as patients with ATP2C1 haploinsufficiency exhibit a keratinocyte cell adhesion defect (32).…”

The Golgi Apparatus: Panel Point of Cytosolic Ca2+Regulation

“…These locations were also proven by experiments of C. elegans; SPCA1 was heterologously expressed on COS-1 cells [31] and the human SPCA1a on CHO cells [44]. SPCA1 separated from rat liver cosedimented with the Golgi-localized Ca 2+ binding protein CALNUC [45]. Fluorescence microscopy of the endogenous expression of SPCA1 in HeLa [46] and PC12 cells [47] also revealed its Golgi-like distribution.…”

“…Much earlier research showed that CALNUC could increase Ca 2+ concentration in the GA cisternae, which was independent of agonist or thapsigargin [7]. Studies on the relationship between CALNUC content and SPCA1 expression indicated that overexpression of SPCA1 raised the level of CALNUC [45]. Associated with Ca 2+ elevation in GA lumens triggered by its overexpression, CALNUC probably cooperated with SPCA1 to control the agonist-induced calcium signal.…”