Additional remarks phenotype | Mutant/mutation
The mutant contains a mutated sams gene where the sams gene has been fused to the destabilizing domain (DD) and a HA tag.
Protein (function)
SAMS is the first and rate-limiting enzyme of the methionine cycle. Methionine, among the twenty amino acids, plays an integral role in protein biosynthesis and in regulation of translation and global gene expression - as it is coded by the translation initiation codon. Met is metabolized into S-adenosylmethionine (SAM), the cellular methylation currency, via an ATP-dependent process which is catalyzed by the Plasmodium SAMS enzyme.
Phenotype
Upon removal of the stabilizing ligand trimethoprim, PbSAMS expression was reduced by 60% in pbsams-dd-ha blood stages and resulted in a two-fold increase in sexual commitment (see also below)
Additional information
Stabilization of PbSAMS-DD fusion protein throughout infection was achieved in vivo, by administration of trimethoprim (TMP) to mice (0.25 mg/ml of TMP in drinking water), 2 days prior to infection.
Earlier studies found that, unlike in P. falciparum, supplementation with P-cho precursors had little to no effect on sexual commitment in the rodent malaria parasite Plasmodium berghei. In the context of our proposed model, where the consumption of SAM by (phosphoethanolamine methyltransferase) PMT provides the link between P-cho precursors and histone methylation, this observation is readily explained by the fact that the PMT ortholog was lost in the rodent malaria parasite lineage (Dechamps et al., J. Lipid Res. 51, 81–96 (2010), thereby decoupling P-cho availability from SAM and SAH abundance in rodent parasites. However, since heterochromatin-mediated silencing of the ap2-g locus also controls sexual commitment in rodent parasites, we hypothesized that commitment in P. berghei would never-the-less remain sensitive to changes in SAM availability. To test this, we generated SAMS knockdown parasites in P. berghei by creating a C-terminal fusion of the endogenous coding sequence with the ecDHFR-based destabilization domain (pbsams-dd-ha). Upon removal of the stabilizing ligand trimethoprim, PbSAMS expression was reduced by 60% in pbsams-dd-ha blood-stages and resulted in a two-fold increase in sexual commitment. This demonstrates that SAM availability regulates the rate of sexual commitment even when decoupled from PtdCho metabolism.
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