ISP1

ISP1 is a Toxoplasma gondii inner membrane complex (IMC) sub-compartment protein that organizes the apical cap region and establishes a hierarchical protein-targeting system for the IMC (PMID:20844581). It is targeted to the apical cap membrane through its N-terminal region, whose predicted coordinated myristoylation and palmitoylation are necessary and sufficient for apical cap localization (PMID:20844581); the palmitoylation depends on the palmitoyl acyltransferase TgDHHC2, whose depletion abolishes ISP1 membrane association (PMID:32329212). Beyond membrane anchoring, ISP1 functions as a gate-keeper: its C-terminal region actively excludes the paralogues ISP2 and ISP3 from the apical cap, and loss of ISP1 relocalizes both paralogues into this sub-compartment (PMID:20844581). Structurally, ISP1 adopts a pleckstrin homology (PH) fold but does not bind phospholipids, with conserved surface residues at the canonical ligand-binding site apparently repurposed for protein-protein interactions (PMID:24675080).

1. 2010 High

   Established where ISP1 resides and how it gets there, defining the apical cap as a discrete IMC sub-compartment with a lipid-anchor-dependent targeting signal.

   Evidence Fluorescence microscopy with N-terminal truncation/mutation constructs and live imaging in T. gondii

   PMID:20844581

   Open questions at the time

   • Direct biochemical demonstration of the myristoylation/palmitoylation modifications not shown
   • Enzyme responsible for the lipid modifications not identified in this study
2. 2010 High

   Revealed that ISP1 does more than localize itself — it enforces sub-compartment boundaries by excluding paralogues, defining a hierarchical targeting logic for IMC organization.

   Evidence Gene knockout of ISP1 with fluorescence microscopy of ISP2/ISP3 localization and C-terminal deletion mapping in T. gondii

   PMID:20844581

   Open questions at the time

   • Molecular mechanism of exclusion (steric, binding-partner competition) not resolved
   • Direct interaction partners of the C-terminal region not identified
3. 2012 Medium

   Demonstrated that ISP1 contains a soluble, folded domain amenable to atomic-resolution structural study, enabling later mechanistic interpretation.

   Evidence Recombinant expression in E. coli, crystallization, and X-ray diffraction of N-terminally truncated ISP1

   PMID:22750877

   Open questions at the time

   • No structure solved in this report
   • Fold and functional site not yet assigned
4. 2014 High

   Solved the fold and tested its presumed function, showing ISP1 is a PH-fold protein that has abandoned lipid binding in favor of a protein-interaction surface.

   Evidence X-ray crystallography of ISP1 (2.10 Å) and ISP3 (2.32 Å), phospholipid binding assays, and surface conservation mapping

   PMID:24675080

   Open questions at the time

   • Identity of protein ligands binding the repurposed surface unknown
   • Structural basis of paralogue exclusion not directly demonstrated
5. 2020 Medium

   Identified the enzyme responsible for ISP1 membrane anchoring, linking the predicted palmitoylation to a specific acyltransferase with demonstrated specificity.

   Evidence Conditional knockdown of TgDHHC2 versus TgDHHC14 with microscopy and fractionation to assess ISP1/ISP3 membrane association in T. gondii

   PMID:32329212

   Open questions at the time

   • Direct enzymatic modification of ISP1 by TgDHHC2 (in vitro acylation) not shown
   • Contribution of myristoylation versus palmitoylation to anchoring not disentangled

• The molecular basis by which the ISP1 C-terminal PH-fold surface excludes ISP2/ISP3 and the identity of its direct protein partners remain unresolved.
• No binding partner identified for the repurposed ligand-binding surface
• Mechanism connecting the PH fold to paralogue gate-keeping unknown