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()

A cDNA encoding the gene for a sarco(endo)plasmic reticulum-type Ca2+- ATPase (SERCA) was isolated from a cDNA library of Paramecium tetraure lia by using degenerated primers according to conserved domains of SER CA-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 ded uced amino acid sequence revealed 38-49% similarity to SERCA-type ATPa ses from organisms ranging from protozoans to mammals, with no more si milarity to some parasitic protozoa of the same phylum. The calculated molecular mass of the encoded protein is 114.7 kDa. It contains the t ypical 10 transmembrane domains of SERCA-type ATPases and other conser ved domains, such as the phosphorylation site and the ATP binding site . However, there are no binding sites for phospholamban and thapsigarg in present in the PtSERCA. Antibodies raised against a cytoplasmic loo p peptide between the phosphorylation site and the ATP binding site re cognize on Western blots a protein of 106 kDa, exclusively in the frac tion of sub-plasmalemmal calcium stores ('alveolar sacs'). In immunofl uorescence studies the antibodies show labelling exclusively in the ce ll cortex of permeabilized cells in a pattern characteristic of the ar rangement of alveolar sacs. When alveolar sacs where tested for phosph oenzyme-intermediate formation a phosphoprotein of the same molecular mass (106 kDa) could be identified.