A new adenine nucleotide transporter located in the ER is essential for maintaining the growth of Toxoplasma gondii

Author summaryThe secretory protein of T. gondii is essential for its invasion and normal growth in host cells, all the secretory proteins are synthesized in the ER before being destined for these distinct organelles, such as apicoplast, microneme, dense granule and rhoptry. ER ATP is demanded to support secretory protein folding and trafficking, and the level of ER ATP determines which proteins are able to be directed to the distinct organelles. In theory, the supply of ATP in the ER is necessary for T. gondii. However, the transport mechanism and importance of the ER ATP in T. gondii are still unclear. In our study, we identified an ATP/ADP transporter (TgANT) located in the ER and verified its function through various methods. Unlike the ER ATP/ADP transporter in mammals, we proved that TgANT is functionally specific; the deletion of TgANT caused the interruption of the supply of ATP in the ER, which leads to fatal phenotypic defects of T. gondii. Our research further expands the understanding of the growth regulation in T. gondii. The lumen of the endoplasmic reticulum (ER) is the subcellular site where secretory protein folding, glycosylation and sulfation of membrane-bound proteins, proteoglycans, and lipids occur. The protein folding and degradation in the lumen of the ER require high levels of energy in the form of ATP. Biochemical and genetic approaches show that ATP must first be translocated across ER membrane by particular transporters before serving as substrates and energy sources in the lumenal reactions. Here we describe an ATP/ADP transporter residing in the ER membranes of T.gondii. Immunofluorescence (IFA) assay in transgenic TgANT1-HA tag revealed that TgANT1 is a protein specifically expressed in the ER. In vitro assays, functional integration of TgANT in the cytoplasmic membrane of intact E. coli cells reveals high specificity for an ATP/ADP antiport. The depletion of TgANT leads to fatal growth defects in T.gondii, including a significant slowdown in replication, no visible plaque formation, and reduced ability to invade. We also found that the amino acid mutations in two domains of TgANT lead to the complete loss of its function. Since these two domains are conserved in multiple species, they may share the same transport mechanism. Our results indicate that TgANT is the only ATP/ADP transporter in the ER of T. gondii, and the lack of ATP in the ER is the cause of the death of T. gondii.