The contribution of the SPCA1 Ca2+ pump to the Ca2+ accumulation in the Golgi apparatus of HeLa cells assessed via RNA-mediated interference

Author: Van Baelen, K.; Vanoevelen, J.; Callewaert, G.; Parys, J.B.; De Smedt, H.; Raeymaekers, L.; Rizzuto, R.; Missiaen, L.; Wuytack, F.

Description: The secretory-pathway Ca(2+)-ATPase SPCA1 is a thapsigargin-insensitive intracellular Ca(2+) pump found mostly in the Golgi compartment. We have explored the contribution of this Ca(2+) pump to cytosolic Ca(2+) signaling in HeLa cells by using RNA-mediated interference to disrupt its expression. Removal of SPCA1 was confirmed by immunofluorescence with specific anti-SPCA1 antibodies. Measurements of the free Ca(2+) concentration in the lumen of the Golgi apparatus by specifically targeting the Ca(2+)-sensitive luminescent protein aequorin to this organelle revealed that endogenous SPCA1 was responsible for Ca(2+) uptake in a subfraction of the Golgi apparatus. HeLa cells lacking SPCA1 could still set up baseline Ca(2+) spiking when stimulated with histamine, indicating that the SPCA1-containing Ca(2+) store was not absolutely needed to set up these oscillations. However, baseline Ca(2+) oscillations occurred less frequently than in control cells, pointing to a contribution of SPCA1 in the shaping of the cytosolic Ca(2+) signal in HeLa cells.

Subject headings: Calcium/metabolism; Calcium-Transporting ATPases/chemistry/metabolism/physiology; Cell Membrane/metabolism; Endoplasmic Reticulum/metabolism; Enzyme Inhibitors/pharmacology; Genetic Vectors; Golgi Apparatus/metabolism; HeLa Cells; Humans; Immunoblotting; Immunohistochemistry; Kinetics; Microscopy, Fluorescence; RNA Interference; Recombinant Proteins/metabolism; Thapsigargin/pharmacology; Transfection

Publication year: 2003

Journal or book title: Biochemical and Biophysical Research Communications

Volume: 306

Issue: 2

Pages: 430-436

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Type: Journal Article

Serial number: 120