OVEREXPRESSION AND CHARACTERIZATION OF A GENE FOR A CA2-ATPASE OF THEENDOPLASMIC-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 spe cifically in a 4-5-fold increase in the cellular Ca2+-ATPase activity. Subcellular fractionation studies revealed this increase to be enrich ed in the microsomal fraction. There was no detectable change in the p lasma membrane Ca2+-ATPase activity of the transformants. Western blot analysis of subcellular fractions using antibodies raised against the recombinant tba1 gene product also demonstrated a significant enrichm ent 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 me mbranes. Significantly, the increased Ca2+-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 re ticulum Ca2+-ATPases. Taken together, these data demonstrated that the tba1 gene codes for a high affinity Ca2+-ATPase of the endoplasmic re ticulum, with properties similar to those reported for the sarcoplasmi c/endoplasmic reticulum family of Ca2+ pumps from higher eukaryotes. I n addition, these results have identified the tba1 gene product as pot entially important element, in conjunction with the mitochondrial memb rane potential and the plasma membrane Ca2+ pump, in the pathways of c ellular Ca2+ homeostasis in these protozoans.