TcSCA complements yeast mutants defective in Ca2+ pumps and encodes a Ca2+-ATPase that localizes to the endoplasmic reticulum of Trypanosoma cruzi.
Intracellular Ca(2+) in Trypanosoma cruzi is mainly located in an acidic compartment named the acidocalcisome, which among other pumps and exchangers possesses a plasma membrane-type Ca(2+)-ATPase. Evidence for an endoplasmic reticulum-located Ca(2+) 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 Ca(2+)-ATPases were present in TcSCA. Expression of TcSCA in yeast mutants deficient in the Golgi and vacuolar Ca(2+) 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 Ca(2+)-dependent and hydroxylamine-sensitive (32)P-labeled phosphoprotein of 110 kDa in the presence of [gamma-(32)P]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 cnb 1 on medium containing Mn(2+) suggests that TcSCA may also regulate Mn(2+) homeostasis by pumping Mn(2+) into the endoplasmic reticulum of T. cruzi.
Furuya
T
T
Laboratory of Molecular Parasitology, Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802, USA.
Okura
M
M
Ruiz
F A
FA
Scott
D A
DA
Docampo
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics*
D012333:RNA, Messenger; D011994:Recombinant Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium