I2

I2 (PF3D7_0320000) is an essential regulator of the Plasmodium falciparum type-1 protein phosphatase catalytic subunit (PfPP1c), binding PfPP1c and inhibiting its phosphatase activity in vitro and in vivo (PMID:23837822). Binding occurs through a two-site mechanism: an N-terminal RVxF-like element within the 12KTISW16 motif together with the 102HYNE105 region (PMID:23837822), and a second PP1-binding site between residues D94 and T117 containing an 'FxxR/KxR/K' motif mapped by NMR, such that mutation of both motifs is required to fully abolish the PfI2–PfPP1 interaction while mutation of either motif alone abrogates I2 function (PMID:25132288). Through this PP1 inhibition I2 is linked to cell cycle control, overriding the G2/M checkpoint to induce germinal vesicle breakdown when microinjected into Xenopus oocytes (PMID:23837822). Attempted gene disruption failed to yield viable parasites, establishing I2 as essential for blood-stage survival (PMID:23837822). In P. berghei, affinity pulldown-mass spectrometry of tagged I2 across asexual and sexual stages identified nuclear export mediator factor and a peptidyl-prolyl cis-trans isomerase as candidate targets of the PP1c/I2 complex (PMID:35162991). Beyond these findings, the downstream substrates and cellular pathways controlled by the I2/PP1c complex have not been characterized in the available corpus.

1. 2013 High

   Establishing whether P. falciparum encodes a functional PP1 inhibitor, this work showed that I2 directly binds and inhibits the PfPP1 catalytic subunit through defined sequence motifs.

   Evidence In vitro phosphatase inhibition and binding assays with site-directed mutagenesis of the 12KTISW16 and 102HYNE105 motifs

   PMID:23837822

   Open questions at the time

   • Native substrates dephosphorylated by PP1c that are affected by I2 not identified
   • Structural basis of inhibition not resolved at this stage
2. 2013 Medium

   To test physiological importance, gene disruption was attempted and failed, defining I2 as essential for blood-stage parasite survival.

   Evidence Attempted targeted gene disruption by transfection in P. falciparum with assessment of viable mutants

   PMID:23837822

   Open questions at the time

   • Single method (failed disruption) does not distinguish essentiality from locus inaccessibility
   • Conditional knockdown to define the essential function not performed
3. 2013 Medium

   To connect I2-mediated PP1 inhibition to a cellular process, I2 was shown to override the G2/M checkpoint, linking it to cell cycle regulation.

   Evidence Xenopus oocyte microinjection and germinal vesicle breakdown (GVBD) assay

   PMID:23837822

   Open questions at the time

   • Cell cycle role demonstrated only in a heterologous oocyte system
   • Direct role in parasite cell cycle progression not shown
4. 2014 High

   Resolving how I2 engages PP1, NMR mapping revealed a second binding site (D94–T117, FxxR/KxR/K motif), establishing a two-site binding mechanism required to fully abolish the interaction.

   Evidence NMR spectroscopy with site-directed mutagenesis of both binding motifs and interaction assays

   PMID:25132288

   Open questions at the time

   • No co-complex crystal structure of I2 bound to PfPP1c
   • Whether the two sites bind sequentially or cooperatively not defined
5. 2014 Medium

   Establishing functional requirement of each site, mutation of either the RVxF or FxxR/KxR/K motif alone abolished I2's ability to trigger GVBD, showing tight two-site association is necessary for activity.

   Evidence Site-directed mutagenesis of individual motifs followed by Xenopus oocyte GVBD assay

   PMID:25132288

   Open questions at the time

   • Functional readout limited to heterologous oocyte assay
   • Contribution of each motif to inhibition in the parasite not tested
6. 2022 Medium

   To identify downstream targets of the complex, interactome mapping of tagged I2 across asexual and sexual stages nominated nuclear export mediator factor and a peptidyl-prolyl cis-trans isomerase as candidate PP1c/I2 partners.

   Evidence mCherry-tagged transgenic P. berghei schizont and gametocyte lines, affinity pulldown and mass spectrometry

   PMID:35162991

   Open questions at the time

   • Candidate partners not functionally validated as I2-specific interactors
   • Whether these are direct I2 contacts or bridged through PP1c not resolved
   • Shown in P. berghei, not confirmed in P. falciparum

• The endogenous substrates dephosphorylated by the I2/PP1c complex and the pathway by which I2 controls parasite cell cycle progression in vivo remain undefined.
• No parasite substrate directly linked to I2-regulated PP1 activity
• In-parasite cell cycle function inferred only from heterologous assays
• Regulation of I2 activity itself uncharacterized