Molecular characterization of a sarco(endo)plasmic reticulum Ca²⁺-ATPase gene from Paramecium tetraurelia and localization of its gene product to sub-plasmalemmal calcium stores

Abstract

A cDNA encoding the gene for a sarco(endo)plasmic reticulum-type Ca2+-ATPase (SERCA) was isolated from a cDNA library of Paramecium tetraurelia by using degenerated primers according to conserved domains of SERCA-type ATPases. The identified nucleotide sequence (PtSERCA) is 3114 nucleotides in length with an open reading frame of 1037 amino acids. An intron of only 22 nucleotides occurs. Homology searches for the deduced amino acid sequence revealed 38-49% similarity to SERCA-type ATPases from organisms ranging from protozoans to mammals, with no more similarity to some parasitic protozoa of the same phylum. The calculated molecular mass of the encoded protein is 114.7 kDa. It contains the typical 10 transmembrane domains of SERCA-type ATPases and other conserved domains, such as the phosphorylation site and the ATP binding site. However, there are no binding sites for phospholamban and thapsigargin present in the PtSERCA. Antibodies raised against a cytoplasmic loop peptide between the phosphorylation site and the ATP binding site recognize on Western blots a protein of 106 kDa, exclusively in the fraction of sub-plasmalemmal calcium stores ('alveolar sacs'). In immunofluorescence studies the antibodies show labelling exclusively in the cell cortex of permeabilized cells in a pattern characteristic of the arrangement of alveolar sacs. When alveolar sacs where tested for phosphoenzyme-intermediate formation a phosphoprotein of the same molecular mass (106 kDa) could be identified.