Affinage · Plasmodium

GAP45

PF3D7_1222700 · glideosome-associated protein 45

Annotated
2026-09-08
20 papers in source corpus 16 papers cited in narrative 15 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

GAP45 (PF3D7_1222700) is a dually acylated peripheral membrane protein of the Plasmodium falciparum glideosome that is essential for erythrocyte invasion but dispensable for merozoite egress (PMID:29970464). It is N-myristoylated and palmitoylated (PMID:16750579), and these two modifications serve distinct functions: palmitoylation—shown in the P. yoelii ortholog to depend on the IMC palmitoyl-acyl-transferase DHHC2—directs GAP45 to the inner membrane complex (IMC) (PMID:35775739), whereas N-myristoylation is dispensable for localization and glideosome assembly yet is strictly required for a functional invasion step (PMID:34695132). Structurally, GAP45 bridges the plasma membrane (via N-terminal acylation) and the IMC (via its C-terminal extremity), with the C-terminal region recruiting the MyoA/MLC1 motor to the IMC and the N-terminal acylation and coiled-coil domain preserving pellicle integrity, a role conserved enough that P. falciparum GAP45 complements the Toxoplasma ortholog (PMID:20951968). As a core glideosome component it assembles with GAP50, MyoA, and MTIP and is transported to the developing IMC throughout schizogony (PMID:16513191, PMID:17553576, PMID:21239623, PMID:22479457), with GAP40 required to stabilize its steady-state protein level (PMID:37249423). GAP45 is a convergence point for signaling, being phosphorylated at multiple serine/threonine residues by CDPK1, PfPKB, and PKA in a calcium- and cAMP-responsive manner (PMID:18768477, PMID:19549500, PMID:22558243, PMID:23025827); however, these phosphorylation events are not required for its localization or glideosome assembly (PMID:22479457, PMID:22558243).

Mechanistic history

Synthesis pass · year-by-year structured walk · 13 steps
  1. 2006 Medium

    Establishing how GAP45 attaches to membranes was the first mechanistic question, and dual acylation was shown to provide its membrane anchoring.

    Evidence Metabolic labeling with fatty acid analogues and mutagenesis of acylation sites in P. falciparum merozoites

    PMID:16750579

    Open questions at the time
    • Did not establish which acyltransferases add the modifications
    • Did not distinguish the functional roles of myristoylation versus palmitoylation
  2. 2006 Medium

    Whether GAP45 physically belongs to the invasion motor was resolved by demonstrating its assembly into the glideosome with GAP50 and MyoA.

    Evidence Co-immunoprecipitation and immunofluorescence in late schizonts and merozoites of P. falciparum

    PMID:16513191 PMID:17553576

    Open questions at the time
    • Co-IP alone did not define direct versus indirect contacts
    • Did not resolve the domain of GAP45 mediating complex assembly
  3. 2007 Medium

    Linking calcium signaling to motor regulation, PfPKB was found to associate with the motor complex and phosphorylate GAP45.

    Evidence Co-immunoprecipitation and in vitro kinase assay in P. falciparum

    PMID:18165240

    Open questions at the time
    • Functional consequence of PfPKB phosphorylation on invasion not tested
    • Phosphosites not mapped in this study
  4. 2008 High

    Identifying the kinases acting on the motor, GAP45 was established as a CDPK1 substrate with phosphorylation detectable in parasite lysates.

    Evidence In vitro kinase assay with recombinant PfCDPK1 and mass spectrometry of phosphopeptides from parasite lysates

    PMID:18768477

    Open questions at the time
    • Physiological role of CDPK1 phosphorylation of GAP45 not established
    • Only two sites identified at this stage
  5. 2009 High

    The full CDPK1 phosphosite repertoire on GAP45 was mapped and shown to alter its proteolytic and substrate properties, refining the picture of regulated modification.

    Evidence In vitro PfCDPK1 phosphorylation, nano-UPLC-MS/MS with IMAC enrichment, and synthetic phosphopeptide assays

    PMID:19549500

    Open questions at the time
    • In vitro mapping did not confirm all nine sites are used in vivo
    • Downstream effect on motor function untested
  6. 2009 Medium

    Whether glideosome assembly is static or regulated was addressed pharmacologically, showing kinase activity influences GAP45 isoform distribution and complex integrity while cADPR calcium release does not.

    Evidence Pharmacological inhibition (cADPR inhibitors, staurosporine), immunoprecipitation, and isoform analysis in P. falciparum

    PMID:19576251

    Open questions at the time
    • Staurosporine is nonspecific and did not identify the responsible kinase
    • Link between isoform shift and invasion outcome not established
  7. 2010 High

    The domain architecture underlying GAP45's bridging function was defined, assigning C-terminal motor recruitment and N-terminal pellicle-integrity roles, with cross-genera complementation confirming conserved function.

    Evidence Conditional knockdown/deletion, domain mutagenesis, transgenera complementation, and immunofluorescence

    PMID:20951968

    Open questions at the time
    • Egress requirement in P. falciparum not directly tested here
    • Structural basis of C-terminal MLC1-MyoA recruitment not resolved
  8. 2011 Medium

    Complex membership was corroborated from the GAP50 anchor side, confirming GAP45 and MTIP are co-recruited to the IMC.

    Evidence Immunoprecipitation of GFP-tagged GAP50 with photobleaching and immunofluorescence in P. falciparum

    PMID:21239623

    Open questions at the time
    • Did not determine assembly order of components
    • Direct GAP45-GAP50 contact not resolved
  9. 2012 High

    Whether phosphorylation controls GAP45 assembly/localization was tested directly, and phosphosite mutagenesis showed it does not, decoupling modification from trafficking; in parallel PLC/calcium and PKA inputs were mapped onto specific serines.

    Evidence GFP-tagging and live imaging, in vitro kinase assays, site-directed mutagenesis, PLC inhibition, and PKA phosphoproteomics in P. falciparum schizonts

    PMID:22479457 PMID:22558243 PMID:23025827

    Open questions at the time
    • Functional readout of the phosphosite mutants on invasion not resolved
    • How three kinases are coordinated on GAP45 in vivo unknown
    • Whether phosphorylation regulates motor force or dynamics untested
  10. 2018 High

    Conditional deletion settled the physiological requirement, showing GAP45 is essential for invasion but dispensable for egress and thereby that the actinomyosin motor is not needed for egress.

    Evidence DiCre conditional deletion with invasion/egress assays and electron microscopy in P. falciparum asexual blood stages

    PMID:29970464

    Open questions at the time
    • Molecular step of invasion that fails without GAP45 not pinpointed
    • Did not dissect contributions of individual GAP45 modifications
  11. 2021 High

    The specific role of N-myristoylation was isolated, revealing it is required for a functional invasion step independent of membrane targeting or glideosome assembly.

    Evidence NMT inhibition, Gly-to-Ala myristoylation-site mutagenesis, invasion assays, immunofluorescence, and chemical proteomics in P. falciparum

    PMID:34695132

    Open questions at the time
    • The distinct invasion function conferred by myristoylation not mechanistically defined
    • Interplay with palmitoylation not addressed
  12. 2022 Medium

    The enzyme responsible for GAP45 IMC targeting was identified, showing DHHC2-mediated palmitoylation directs its IMC localization.

    Evidence TurboID IMC proteomics, conditional DHHC2 depletion, palmitoylation assay, and immunofluorescence in P. yoelii

    PMID:35775739

    Open questions at the time
    • Shown in P. yoelii, not directly confirmed for the P. falciparum ortholog
    • Whether DHHC2 loss phenocopies GAP45 mislocalization on invasion untested
  13. 2023 Medium

    A determinant of GAP45 steady-state abundance was found, with GAP40 required to stabilize GAP45 protein levels within the glideosome.

    Evidence Conditional knockdown of PfGAP40, co-immunoprecipitation, and western blotting in P. falciparum

    PMID:37249423

    Open questions at the time
    • Mechanism of GAP40-dependent stabilization unknown
    • Whether reduced GAP45 fully accounts for GAP40 knockdown phenotype not resolved

Open questions

Synthesis pass · forward-looking unresolved questions
  • The precise molecular step of invasion that requires GAP45 N-myristoylation, and how multi-kinase phosphorylation is integrated to regulate motor function, remain unresolved.
  • No structural model of the GAP45-anchored motor at the pellicle
  • Functional output of the mapped phosphosites on motor force/dynamics undefined
  • Molecular function distinguishing myristoylation from palmitoylation in invasion unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification
Partners
DHHC2 (PALMITOYL-ACYL-TRANSFERASE, P. YOELII)MTIP/MLC1NMT (N-MYRISTOYLTRANSFERASE)PFCDPK1 (KINASE)PFGAP40PFGAP50PFMYOAPFPKA (KINASE)PFPKB (KINASE)
Complex memberships
Glideosome (GAP45–GAP50–MyoA/PfMyoA–MTIP/MLC1–GAP40)

Evidence

Reading pass · 15 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2006 GAP45 is dually acylated (N-myristoylation and palmitoylation) in P. falciparum merozoites, as demonstrated by metabolic labeling and mutagenesis experiments on the 45 kDa glideosome-associated protein. Metabolic labeling with fatty acid analogues, mutagenesis of acylation sites Molecular and biochemical parasitology Medium 16750579
2006 GAP45 forms a complex with PfGAP50 and PfMyoA (the glideosome) in late schizonts and merozoites of P. falciparum, as shown by co-immunoprecipitation and localization studies consistent with a role in erythrocyte invasion. Co-immunoprecipitation, immunofluorescence Molecular and biochemical parasitology Medium 16513191 17553576
2008 GAP45 is phosphorylated by P. falciparum CDPK1 in vitro at two sites (one of which was also detected in phosphopeptides from parasite lysates), establishing GAP45 as a substrate of CDPK1 in the motor complex. In vitro kinase assay with recombinant PfCDPK1, mass spectrometry of phosphopeptides from parasite lysates The Journal of biological chemistry High 18768477
2007 PfPKB (a Ca2+/calmodulin-activated protein kinase B-like enzyme) associates with the actin-myosin motor complex and phosphorylates GAP45 in P. falciparum, linking calcium signaling to motor complex regulation during erythrocyte invasion. Co-immunoprecipitation, in vitro kinase assay The Journal of biological chemistry Medium 18165240
2009 PfCDPK1 phosphorylates GAP45 in vitro at nine sites (Ser31, Ser89, Ser103, Ser109, Ser121, Ser149, Ser156, Thr158, and Ser173), and phosphorylation at these sites alters proteolytic cleavage properties and kinase substrate properties of GAP45. In vitro phosphorylation by PfCDPK1, nano-UPLC-MS/MS, phosphopeptide enrichment by Ga(III) IMAC, synthetic phosphopeptide substrate assays Analytical biochemistry High 19549500
2009 Inhibition of cADPR-mediated calcium release does not affect GAP45 phosphorylation or glideosome association, but the serine/threonine kinase inhibitor staurosporine affects GAP45 isoform distribution and integrity of the glideosome complex, indicating that assembly of the glideosome is dynamically regulated. Pharmacological inhibition (cADPR pathway inhibitors, staurosporine), immunoprecipitation, isoform analysis by gel electrophoresis Molecular and biochemical parasitology Medium 19576251
2010 GAP45 is anchored to both the plasma membrane (via N-terminal acylation) and the inner membrane complex (via its C-terminal extremity); the C-terminal region recruits MLC1-MyoA to the IMC, while the N-terminal acylation and coiled-coil domain preserve pellicle integrity during invasion. GAP45 is essential for gliding, invasion, and egress in Toxoplasma, and the P. falciparum GAP45 ortholog can functionally complement the Toxoplasma GAP45, shown by transgenera complementation. Conditional knockdown/deletion, domain mutagenesis, transgenera complementation, immunofluorescence Cell host & microbe High 20951968
2012 GAP45 tagged internally with GFP is assembled into the motor complex and transported to the developing IMC in early schizogony in P. falciparum, accumulating there throughout intracellular development until merozoite release; CDPK1 phosphorylates GAP45 in vivo, but substitution of the identified serine residues with alanine or aspartate has no apparent effect on GAP45 assembly into the motor complex or its subcellular localization. GFP-tagging and live imaging, immunoprecipitation, in vitro kinase assay, site-directed mutagenesis PloS one High 22479457
2012 GAP45 is phosphorylated by both PfPKB and PfCDPK1 at S89, S103, and S149 in response to phospholipase C and calcium signaling; the PLC pathway specifically influences phosphorylation at S103 and S149; however, localization of GAP45 and its association with the glideosome appear independent of phosphorylation at these sites. In vitro kinase assay, site-directed mutagenesis, pharmacological inhibition of PLC pathway, co-immunoprecipitation PloS one Medium 22558243
2012 GAP45 was identified as a substrate of cAMP-dependent protein kinase A (PKA) in P. falciparum schizonts, verified by in vitro kinase assay, implicating PKA as an additional regulator of the glideosome motor complex alongside CDPK1. Phosphoproteomic analysis of schizont stage, in vitro PKA kinase assay Journal of proteome research Medium 23025827
2018 Conditional deletion of GAP45 in asexual blood stages of P. falciparum confirms its essential role in erythrocyte invasion but shows it is NOT required for merozoite egress from red blood cells, demonstrating that the actinomyosin motor is dispensable for egress. Conditional genetic deletion (DiCre recombinase system), invasion assays, egress assays, electron microscopy mBio High 29970464
2021 N-myristoylation of GAP45 is essential for erythrocyte invasion in P. falciparum: replacement of the N-terminal glycine with alanine to prevent myristoylation had no impact on GAP45 protein localization or glideosome assembly, but abolished invasion, showing that myristoylation provides a function distinct from membrane targeting per se. NMT inhibitor treatment, site-directed mutagenesis (Gly→Ala), invasion assays, immunofluorescence, chemical proteomics for NMT substrate identification PLoS biology High 34695132
2022 DHHC2, the major palmitoyl-acyl-transferase of the IMC in Plasmodium yoelii, palmitoylates GAP45 for its IMC localization; depletion of DHHC2 disrupts GAP45 IMC targeting, demonstrating that palmitoylation by DHHC2 is required for proper GAP45 subcellular localization during schizont development. TurboID proximity labeling (IMC proteomics), conditional DHHC2 depletion, palmitoylation assay, immunofluorescence eLife Medium 35775739
2023 PfGAP40 co-precipitates with GAP45 and PfGAP50 in P. falciparum, and knockdown of PfGAP40 reduces the expression levels of GAP45 and PfGAP50 in the schizont stage, indicating that GAP40 stabilizes GAP45 protein levels within the glideosome. Conditional knockdown, co-immunoprecipitation, western blotting Microbiology spectrum Medium 37249423
2011 PfGAP50-GFP immunoprecipitation co-precipitates endogenous GAP45 and MTIP, confirming that GAP45 is part of the core glideosome complex anchored to the IMC via GAP50. Immunoprecipitation of GFP-tagged GAP50, fluorescence photobleaching, immunofluorescence Eukaryotic cell Medium 21239623

Source papers

Stage 0 corpus · 20 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2005 A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites. The Journal of biological chemistry 291 16321976
2010 Functional dissection of the apicomplexan glideosome molecular architecture. Cell host & microbe 237 20951968
2008 The motor complex of Plasmodium falciparum: phosphorylation by a calcium-dependent protein kinase. The Journal of biological chemistry 150 18768477
2012 The Plasmodium falciparum schizont phosphoproteome reveals extensive phosphatidylinositol and cAMP-protein kinase A signaling. Journal of proteome research 113 23025827
2006 Dual acylation of the 45 kDa gliding-associated protein (GAP45) in Plasmodium falciparum merozoites. Molecular and biochemical parasitology 90 16750579
2006 Plasmodium falciparum erythrocyte invasion: a conserved myosin associated complex. Molecular and biochemical parasitology 86 16513191
2007 Role of Ca2+/calmodulin-PfPKB signaling pathway in erythrocyte invasion by Plasmodium falciparum. The Journal of biological chemistry 85 18165240
2007 Identification of protein complexes in detergent-resistant membranes of Plasmodium falciparum schizonts. Molecular and biochemical parasitology 73 17553576
2018 The Actinomyosin Motor Drives Malaria Parasite Red Blood Cell Invasion but Not Egress. mBio 56 29970464
2012 Subcellular location, phosphorylation and assembly into the motor complex of GAP45 during Plasmodium falciparum schizont development. PloS one 56 22479457
2011 Tracking Glideosome-associated protein 50 reveals the development and organization of the inner membrane complex of Plasmodium falciparum. Eukaryotic cell 49 21239623
2009 Effects of calcium signaling on Plasmodium falciparum erythrocyte invasion and post-translational modification of gliding-associated protein 45 (PfGAP45). Molecular and biochemical parasitology 44 19576251
2017 Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility. The Journal of biological chemistry 33 28893907
2022 Inner membrane complex proteomics reveals a palmitoylation regulation critical for intraerythrocytic development of malaria parasite. eLife 22 35775739
2009 Protein phosphorylation influences proteolytic cleavage and kinase substrate properties exemplified by analysis of in vitro phosphorylated Plasmodium falciparum glideosome-associated protein 45 by nano-ultra performance liquid chromatography-tandem mass spectrometry. Analytical biochemistry 21 19549500
2012 Regulation of Plasmodium falciparum glideosome associated protein 45 (PfGAP45) phosphorylation. PloS one 20 22558243
2021 Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress and invasion. PLoS biology 18 34695132
2021 Calcium in the Backstage of Malaria Parasite Biology. Frontiers in cellular and infection microbiology 13 34395314
2016 Production of recombinant proteins from Plasmodium falciparum in Escherichia coli. Biomedica : revista del Instituto Nacional de Salud 12 27622630
2023 Plasmodium falciparum GAP40 Plays an Essential Role in Merozoite Invasion and Gametocytogenesis. Microbiology spectrum 7 37249423