Additional remarks phenotype | Mutant/mutation
The mutant expresses a C-terminal HA-tagged version of DMT1
Protein (function)
Divalent metal transporter (DMT1) is a critical iron transporter with 12 transmembrane structures responsible for iron uptake in many organisms.
Through bioinformatics analysis, we found a homolog of human Dmt1 in the malaria parasite genome, which we named PyDMT1 in P. yoelii. Bioinformatic analysis shows that all Plasmodium genomes encode one gene product with homology to Saccharomyces cerevisiae (the yeast) Smf1-3, Homo sapiens Nramp1 and Dmt1 (Nramp2). PyDMT1 shares 29.53% amino-acid sequence identity with Homo sapiens Dmt1, and 24.94% with S. cerevisiae Smf3.
Phenotype
Confocal analysis after immunostaining with HA antibody showed that PyDMT-HA surrounded the hemozoin in the blood stage, implying that PyDMT1 is likely localized on the membrane of the food vacuole.
Additional information:
The unsuccessful attempts to disrupt DMT1 indicate an essential function during asexual blood stage growth/multiplication (see RMgm-5515)
To test whether PyDMT1 acts as an iron importer in P. yoelii, a mutant was generated with a partial loss of function allele of PyDMT1 by a single crossover to the regulatory region of Pydmt1. This mutation caused a partial loss of PyDMT1 expression but resulted in severe defects, rescued by iron supplementation (RMgm-5517).
From the paper: 'Plasmodium CRT (chloroquine-resistant transport) is known to localize on the vacuolar membrane, responsible for chloroquine resistance. To further confirm that PyDMT1 is localized on the food vacuole membrane, we fused a FLAG tag at the C-terminal of CRT by single crossover to label the food vacuole. Confocal imaging revealed that PyDMT1 indeed mainly colocalized with CRT at all blood stages, and slightly with the Golgi marker Rab6, but completely not with the endoplasmic reticulum marker PMV. During the schizont stage, a small amount of PyDMT1 signal is found in the merozoites, mainly in the hemozoin region. These data strongly suggest that PyDMT1 mainly localizes on the digestive vacuole membrane'.
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