PF3D7_1024600

PF3D7_1024600 (RAP-1) is a rhoptry protein of Plasmodium falciparum that functions as the organizing core of low-molecular-weight rhoptry complexes and is required for normal parasitophorous vacuolar membrane (PVM) structure and intra-erythrocytic growth after invasion (PMID:28205319). It is synthesized as an ~84–86 kDa precursor bearing a cleavable N-terminal signal peptide and is routed to the rhoptries through the classical secretory pathway, since brefeldin A, low temperature, and N-ethylmaleimide reversibly or irreversibly block processing of the precursor (PMID:8719244). Within the rhoptries the precursor undergoes two sequential N-terminal proteolytic cleavages to p82 and then p67, and these forms assemble into non-covalent complexes with RAP-2 and RAP-3 (PMID:9574915, PMID:17553576). Contrary to a role in invasion, conditional knockdown demonstrated that the RAP complex is dispensable for rhoptry morphology and erythrocyte invasion but is required post-invasion for normal PVM ultrastructure, growth kinetics, and in vivo parasitaemia (PMID:28205319); RAP-1 also serves as the determinant that tags non-parasitized erythrocytes with rhoptry proteins upon merozoite release (PMID:17624849). In P. berghei, RAP-1 is required to target RAP-2 to the rhoptries and contributes to virulence (PMID:29307564). Monoclonal antibodies against RAP-1 inhibit parasite growth and invasion in vitro, mapping to defined B-cell epitopes near the processing sites (PMID:2200961, PMID:1279419, PMID:9423886).

1. 1990 Medium

   Establishing that RAP-1 is a multi-component, disulfide-linked rhoptry antigen whose components derive from a single precursor set the framework for all subsequent processing and complex studies.

   Evidence Immunoaffinity purification, SDS-PAGE, monoclonal antibody growth-inhibition assay and gene sequencing in P. falciparum

   PMID:2200961

   Open questions at the time

   • Precise identity of each processed form not resolved
   • Molecular basis of growth inhibition by antibodies unknown
2. 1992 Medium

   Mapping inhibitory antibody epitopes near the p80→p65 cleavage site linked the proteolytic processing to merozoite release and defined a functionally important region.

   Evidence Fusion-protein and synthetic-peptide epitope mapping with growth-inhibition assay in P. falciparum

   PMID:1279419

   Open questions at the time

   • Identity of the protease responsible for cleavage unknown
   • Mechanism by which epitope binding inhibits growth unresolved
3. 1992 Low

   Determining that co-purifying metalloproteinases originate from human culture serum rather than the parasite excluded them from RAP-1's intrinsic biology.

   Evidence Co-isolation, proteinase inhibitor and metal-ion assays, immunoprecipitation in P. falciparum

   PMID:1477099

   Open questions at the time

   • Negative result; establishes only what is not part of the complex
   • Does not identify the parasite protease that processes RAP-1
4. 1995 High

   Showing that precursor processing depends on the secretory pathway localized RAP-1 trafficking to the classical ER/Golgi route and defined a cleavable signal peptide.

   Evidence Brefeldin A, low-temperature and NEM blocks with in vitro translation and immunoprecipitation in P. falciparum

   PMID:8719244

   Open questions at the time

   • Nature of the inferred additional post-translational modification undefined
   • Rhoptry-targeting signal not identified
5. 1998 High

   Defining sequential Pr86→p82→p67 processing within the rhoptries and complex formation with RAP-2/RAP-3 established RAP-1's maturation and assembly pathway.

   Evidence Epitope mapping, size fractionation, radiolabeling and IFA on merozoites in P. falciparum

   PMID:9574915

   Open questions at the time

   • Protease responsible for cleavages unidentified
   • Stoichiometry of the complexes unresolved
6. 1998 Medium

   Identification of a second invasion-inhibitory epitope near p67 extended the functional antibody map of RAP-1.

   Evidence Monoclonal antibody 1D6, recombinant epitope mapping, invasion inhibition and owl monkey model in P. falciparum

   PMID:9423886

   Open questions at the time

   • Mechanism of invasion inhibition not defined
   • Whether antibody effect reflects a native RAP-1 invasion function unclear
7. 2007 Medium

   Placing RAP-1 as the core of RAP1/RAP2 and RAP1/RAP3 complexes in detergent-resistant membranes refined the biochemical architecture of the rhoptry complexes.

   Evidence DRM purification, blue native PAGE and mass spectrometry in P. falciparum schizonts

   PMID:17553576

   Open questions at the time

   • Composition of the larger high-molecular-weight species not fully defined
   • Functional consequence of membrane association unknown
8. 2007 Medium

   Gene-disruption plus surface iodination established that RAP-1 is required to tag non-parasitized erythrocytes with rhoptry proteins after merozoite release, revealing a role beyond the invading merozoite.

   Evidence RAP-1 and RAP-3 knockout parasites with surface iodination and antibody binding in P. falciparum

   PMID:17624849

   Open questions at the time

   • Biological purpose of erythrocyte tagging unresolved
   • Receptor on the erythrocyte surface unidentified
9. 2010 Low

   Co-isolation of RAP-1 with MSP-1-associated surface proteins raised the possibility of a broader merozoite-surface complex.

   Evidence Antibody pull-down from merozoite lysate with tandem MS in P. falciparum

   PMID:21175202

   Open questions at the time

   • Single pull-down without reciprocal validation specific to RAP-1
   • Direct versus indirect association not distinguished
10. 2017 High

    Conditional knockdown redefined RAP-1's essential role as post-invasion, showing it is required for normal PVM structure and intra-erythrocytic growth rather than for invasion or rhoptry morphology.

    Evidence Conditional knockdown, PVM electron microscopy, growth assays and mouse infection in P. falciparum

    PMID:28205319

    Open questions at the time

    • Molecular mechanism linking RAP-1 to PVM integrity unknown
    • How the complex functions at the PVM undefined
11. 2018 Medium

    Knockout in P. berghei demonstrated a conserved requirement for RAP-1 in targeting RAP-2 to the rhoptries and a contribution to virulence.

    Evidence Gene knockout in P. berghei ANKA with mouse infection and comparative proteomics

    PMID:29307564

    Open questions at the time

    • Mechanism by which RAP-1 confers virulence unresolved
    • Relationship of the increased early parasitemia to PVM function unclear

• The protease(s) executing RAP-1 processing and the molecular mechanism by which RAP-1 maintains PVM integrity remain unidentified.
• Processing protease unidentified
• Direct molecular partners at the PVM undefined
• Structural basis of complex assembly unknown