Overexpression and characterization of a gene for a Ca(2+)-ATPase of the endoplasmic reticulum in Trypanosoma brucei.
Procyclic forms of Trypanosoma brucei were stably transformed with an expression vector containing a gene for a P-type ATPase (tba1) cloned from T. brucei genomic DNA. Transformation with this gene resulted specifically in a 4-5-fold increase in the cellular Ca(2+)-ATPase activity. Subcellular fractionation studies revealed this increase to be enriched in the microsomal fraction. There was no detectable change in the plasma membrane Ca(2+)-ATPase activity of the transformants. Western blot analysis of subcellular fractions using antibodies raised against the recombinant tba1 gene product also demonstrated a significant enrichment of a protein with a M(r) of 115,000 in the microsomal fraction of transformed cells. This protein was not detected in purified plasma membranes. Significantly, the increased Ca(2+)-ATPase activity possessed a high affinity for Ca2+. The activity was sensitive to the classical P-type ATPase inhibitor vanadate, anti-tba1 antibodies, as well as low concentrations of thapsigargin, a specific inhibitor of endoplasmic reticulum Ca(2+)-ATPases. Taken together, these data demonstrated that the tba1 gene codes for a high affinity Ca(2+)-ATPase of the endoplasmic reticulum, with properties similar to those reported for the sarcoplasmic/endoplasmic reticulum family of Ca2+ pumps from higher eukaryotes. In addition, these results have identified the tba1 gene product as potentially important element, in conjunction with the mitochondrial membrane potential and the plasma membrane Ca2+ pump, in the pathways of cellular Ca2+ homeostasis in these protozoans.
Nolan
D P
DP
Department of Molecular Biology, University of Brussels.
Reverlard
P
P
Pays
E
E
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000032:analysis / Q000235:genetics*; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D017125:Genes, Protozoan; D008274:Magnesium / Q000494:pharmacology; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics*
D000252:Calcium-Transporting ATPases; D008274:Magnesium