ISP3

ISP3 (PF3D7_1460600) is a dual-acylated inner membrane complex (IMC) sub-compartment protein that organizes apical polarity and tethers the subpellicular cytoskeleton during Plasmodium morphogenesis (PMID:24244852, PMID:32395856). It localizes to the anterior apical end of the parasite where IMC assembly is initiated, is expressed across developmental stages with enrichment in invasive stages, and its disruption in P. berghei is non-lethal but mislocalizes ISP1 in ookinetes and sporozoites, establishing ISP3 as a determinant of proper apical polarity (PMID:24244852). Membrane recruitment depends on an N-terminal acylation motif: ISP3 is both N-myristoylated and palmitoylated, and its N-terminal sequence requirements govern IMC targeting (PMID:24244852, PMID:25425642). In P. berghei, palmitoylation of N-terminal cysteines by the IMC-resident palmitoyl-S-acyltransferase DHHC2 is required for IMC localization and for normal zygote-to-ookinete differentiation, while in P. falciparum schizogony PfISP3 co-distributes with the IMC-localized PAT PfDHHC1 (PMID:25425642, PMID:32395856). Once anchored at the IMC, ISP3 physically interacts with β-tubulin, functioning as a tether that maintains subpellicular microtubule structure during zygote elongation (PMID:32395856). Beyond these acylation, localization, and cytoskeletal-tethering roles, no further mechanistic detail for ISP3 has been characterized in the available corpus.

1. 2012 Low

   Before ISP3 was functionally distinguished, it was unknown how IMC sub-compartment proteins were spatially organized across motile stages; surveying the IMC family placed ISP3 among structurally distinct members with stage-specific localization patterns during sexual-stage development.

   Evidence Fluorescence colocalization with centromere/spindle markers and live imaging across life-cycle stages in P. falciparum

   PMID:22389454

   Open questions at the time

   • ISP3-specific function not separated from other IMC proteins
   • no functional disruption of ISP3 in this study
   • colocalization-only, no mechanism
2. 2013 Medium

   It was unknown whether ISP3 contributes to apical organization; gene disruption showed ISP3 is dispensable for viability but required to correctly localize ISP1, establishing it as an organizer of apical polarity in invasive stages.

   Evidence Reverse genetics (gene disruption), live cell imaging and localization in P. berghei

   PMID:24244852

   Open questions at the time

   • molecular basis of ISP1 mislocalization not defined
   • role of myristoylation vs phosphorylation in membrane binding not dissected
   • no binding partners identified
3. 2014 Medium

   How ISP3 achieves IMC membrane association was unresolved; mapping its dual-acylation motif and co-distribution with the IMC PAT PfDHHC1 during schizogony showed that N-terminal acylation and specific sequence requirements drive IMC recruitment.

   Evidence Phylogenetic analysis, subcellular co-localization with PfDHHC1, acylation-motif mutagenesis and IMC fractionation in P. falciparum

   PMID:25425642

   Open questions at the time

   • direct enzyme-substrate relationship with PfDHHC1 not demonstrated
   • functional consequence of acylation in P. falciparum not tested
   • co-localization does not prove catalysis
4. 2020 High

   The enzyme responsible for ISP3 palmitoylation and the downstream cytoskeletal role were unknown; DHHC2 was identified as the PAT palmitoylating ISP3 N-terminal cysteines, and IMC-anchored ISP3 was shown to bind β-tubulin to maintain subpellicular microtubule integrity during ookinete morphogenesis.

   Evidence In vivo palmitoylation assay, ISP3–β-tubulin co-immunoprecipitation, conditional knockouts and live morphogenesis imaging in P. berghei

   PMID:32395856

   Open questions at the time

   • structural basis of the ISP3–β-tubulin interaction not defined
   • whether the P. falciparum ortholog uses the same DHHC2/β-tubulin mechanism not tested
   • reciprocal validation across stages beyond ookinete not shown

• It remains unknown how ISP3 mechanistically coordinates ISP1 apical positioning with microtubule tethering, and whether its P. falciparum functions mirror the P. berghei cytoskeletal role.
• no structural model of ISP3 or its complexes
• mechanism linking apical polarity organization to β-tubulin tethering unresolved
• P. falciparum functional phenotype not established