Taken together, these data suggest the essentiality of PfI2 for the survival of blood stage parasites. Effect of PfI2 on Phosphatase activity of PfPP1 Ne t, we assayed PfI2 for its potential capacity to regulate PfPP1 activity. As previously described, PfPP1 produced as a recombinant protein dephosphorylates the pNPP sub strate, is sensitive to known PP1 inhibitors and its activity is Mn2 dependent. Using a concentration of recom binant PfPP1 within a range producing linear release of phosphate, the effect of wild type recombinant, deleted or mutant recombinant PfI2 proteins was evalu ated as described in Methods. Deleted or mutated PfI2 versions presented in Figure 4A were produced as recom binant proteins and used in the functional assay.

Results showed a strong decrease in the phosphatase activity when PfPP1 was pre incubated with PfI2WT. As PfI2 contains the 2 main motifs, 12 known to be essential for the function of Inhibitor 2, we e plored the impact of these motifs on PfI2 function in terms of PP1 inhibition. The deletion of either the Nt or Ct portion containing the RV F and HYNE motifs of PfI2 respectively abolished its inhibitory function almost completely. When the PfI2W16A mutant protein was tested, we observed that this mutation led to an almost complete loss of function of PfI2, whatever the concentration of PfI2W16A used. The PfPP1 activity detected was identical to the control. In the case of the PfI2Y103A mutant protein, a loss of function was observed at the lowest concentration, however, at higher concentrations of PfI2Y103A a decrease of up to 50% of PfPP1 activity was observed, suggesting that this mutation only partially affected the function of PfI2.

These data suggest that the RV F motif is the major contributor for the func tion of PfI2. Study of PfI2 PfPP1 interaction Anacetrapib and mapping of binding motifs The loss of function of deleted mutated PfI2 observed above may be related to its failure to interact with PfPP1. Hence, the binding capacity of wild type, deleted and mutated PfI2 with PfPP1 was assessed using the yeast two hybrid system. The interaction between PfPP1 Gal4 BD and PfI2 Gal4 AD can be evidenced by growing diploid strains on SD media lacking Leucine, Tryptophan, Histidine or SD LWHA. Mating assays between different strains are summarized in Figure 5A, in cluding those with control constructs. All mated strains were shown to be able to grow on SD LW, indi cating that they contained the PfI2 and PfPP1 constructs. Western blot analysis showed the e pression of tagged PfPP1 and the e pres sion of PfI2.