TcSCA Complements Yeast Mutants Defective in Ca2+ Pumps and Encode a Ca2+ -ATPase That Localizes to the Endoplasmic Reticulum of Trypanosoma cruzi

Abstract

Intraeellular Ca2+ in Trypanosoma cruzi is mainly located in an acidic compartment named the acidocalcisome, which among other pumps and exchangers possesses a plasma membrane-type Ca2+ -ATPase. Evidence for an endoplasmic reticulum-located Ca2+ uptake has been more elusive and based on indirect results. Here we report the cloning and sequencing of a gene encoding a sarcoplasmic-endoplasmic reticulum-type Ca 2+ -ATPase from T. cruzi. The protein (TcSCA) predicted from the nucleotide sequence of the gene has 1006 amino acids and a molecular mass of 109.7 kDa. Several sequence motifs found in sarcoplasmic-endoplasmic reticulum-type Ca2+ -ATPases were present in TcSCA. Expression of TcSCA in yeast mutants deficient in the Golgi and vacuolar Ca2+ pumps (pmr1 pmc1 cnb 1) restored growth on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with TcSCA, and it was found that the TcSCA polypeptide formed a Ca2+ -dependent and hydroxylamine-sensitive 32P-labeled phosphoprotein of 110 kDa in the presence of [γ-32P]ATP. Cyclopiazonic acid, but not thapsigargin, blocked this phosphoprotein formation. Transgenic parasites expressing constructs of TcSCA with green fluorescent protein exhibited co-localization of TcSCA with the endoplasmic reticulum proteins BiP and calreticulin. An endoplasmic reticulum location was also found in amastigotes and trypomastigotes using a polyclonal antibody against a COOH-terminal region of the protein. The ability of TcSCA to restore growth of mutant pmr1 pmc1 cnb1 on medium containing Mn2+ suggests that TcSCA may also regulate Mn2+ homeostasis by pumping Mn2+ into the endoplasmic reticulum of T. cruzi.