GAP50

TgGAP50 is an N-glycosylated integral membrane glycoprotein of the Toxoplasma gondii inner membrane complex (IMC) that serves as the membrane anchor tethering the parasite's actomyosin gliding motor to the IMC, thereby coupling motor force to the cellular functions of gliding motility and invasion (PMID:15123738). Together with the polytopic IMC protein GAP40, it anchors the MyoA-glideosome — comprising the myosin TgMyoA, its light chain TgMLC1, and the peripheral scaffold GAP45 — and the assembled four-protein complex exhibits measurable motor activity in vitro (PMID:20951968, PMID:20084115). The same GAP50/GAP40 integral core also anchors a distinct MyoC-glideosome at the basal polar ring, with the two motors differing in their peripheral scaffolding and localization determinants (GAP45 versus GAP80) (PMID:25393004). All three of GAP50's N-glycosylation sites carry unusual N-glycans that are required for its trafficking from the ER through the Golgi to the IMC and for its assembly with gliding partners and parasite motility (PMID:21610105). Within the IMC, the anchored motor is immobilized in the membrane plane within cholesterol-rich, detergent-resistant membrane domains (PMID:17538016). Beyond its role in motility, GAP50 is essential for IMC biogenesis during intracellular replication, its disruption causing collapse of developing daughter parasites and redistribution of other glideosome components (PMID:26845335).

1. 2004 High

   Establishing how a myosin motor is fixed to the parasite periphery, GAP50 was identified as an integral IMC membrane protein serving as the specific receptor anchoring the myosin XIV motor complex, providing the first molecular description of a membrane anchor for a myosin motor.

   Evidence Co-immunoprecipitation, subcellular fractionation and localization in Toxoplasma gondii

   PMID:15123738

   Open questions at the time

   • Did not define which motor partners contact GAP50 directly versus indirectly
   • No structural basis for the anchoring interaction
2. 2007 Medium

   Addressing how the anchored motor is stabilized to transmit force, the GAP50-anchored motor was shown to be immobilized in the plane of the IMC within cholesterol-dependent detergent-resistant membrane domains.

   Evidence FRAP, detergent-resistant membrane fractionation and cholesterol depletion in Toxoplasma gondii

   PMID:17538016

   Open questions at the time

   • Mechanism by which cholesterol domains organize GAP50 not defined
   • Single-study observation of immobilization
3. 2008 Medium

   Testing whether phosphorylation gates motor assembly, GAP45 Ser163/Ser167 phospho-mimetic mutations were reported to prevent the MyoA-MLC1-GAP45 sub-complex from associating with GAP50.

   Evidence Mass spectrometry phosphosite mapping, Ser→Glu/Ala mutagenesis and Co-IP in Toxoplasma gondii

   PMID:19047362

   Open questions at the time

   • Functional significance later contradicted (see 2014)
   • Effect measured in presence of co-expressed wild-type GAP45
4. 2010 High

   Resolving the composition of the anchoring module, GAP40 was identified as a second integral IMC anchor acting with GAP50, while GAP45 was shown to bridge plasma membrane and IMC and recruit MLC1-MyoA, defining the architecture of glideosome attachment.

   Evidence Co-IP, IFA, transgenera complementation and conditional knockdown in Toxoplasma gondii

   PMID:20951968

   Open questions at the time

   • Stoichiometry of the GAP50/GAP40 core unknown
   • Direct binding interface between GAP50 and partners not mapped
5. 2010 Medium

   Demonstrating that the anchored complex is a functional motor, the intact TgMyoA-MLC1-GAP45-GAP50 complex produced mechanical output in vitro that was inhibitable by post-translational modification of MLC1.

   Evidence In vitro motility assay using motor complexes purified from control and compound-treated Toxoplasma gondii

   PMID:20084115

   Open questions at the time

   • Did not isolate GAP50's specific contribution to mechanical output
   • Single-lab reconstitution
6. 2011 High

   Explaining how GAP50 reaches the IMC and assembles a functional glideosome, all three N-glycosylation sites were shown to be occupied and required for ER-to-Golgi-to-IMC trafficking, partner assembly and motility.

   Evidence Glycomic/proteomic analysis, N-glycosylation site mutagenesis, affinity purification, IFA and motility assays in Toxoplasma gondii

   PMID:21610105

   Open questions at the time

   • Whether glycans act on folding, trafficking receptors, or partner recognition not resolved
   • Identity of trafficking machinery reading the glycans unknown
7. 2014 Medium

   Broadening GAP50's role beyond a single motor, GAP50/GAP40 were shown to also anchor the MyoC-glideosome at the basal polar ring, revealing a shared integral core serving distinct motors defined by their peripheral scaffolds.

   Evidence Co-IP, conditional knockout/knockdown and IFA localization in Toxoplasma gondii

   PMID:25393004

   Open questions at the time

   • How GAP50 discriminates or partitions between MyoA and MyoC glideosomes unknown
   • Localization determinants for each motor not fully mapped
8. 2014 Medium

   Revising the phosphorylation model, clean inducible-knockout complementation showed GAP45 phospho-mutants did not impair GAP50 binding, glideosome assembly, or motility, attributing the earlier phenotype to competition from wild-type GAP45.

   Evidence Phospho-mutant generation, inducible KO complementation, Co-IP and motility assays in Toxoplasma gondii

   PMID:24779470

   Open questions at the time

   • Whether any physiological phospho-regulation of GAP50 assembly exists remains open
   • Directly contradicts 2008 phosphorylation model
9. 2016 Medium

   Uncovering a motility-independent function, conditional deletion of GAP50 caused collapse of developing daughter parasites and glideosome redistribution, establishing an essential structural role in IMC biogenesis during replication.

   Evidence Conditional/inducible gene deletion, IFA, electron microscopy and phenotypic analysis in dividing Toxoplasma gondii

   PMID:26845335

   Open questions at the time

   • Molecular mechanism linking GAP50 to IMC membrane formation unknown
   • Whether the structural and motor roles use the same molecular interactions unclear

• The structural interface by which GAP50 binds its glideosome partners, the mechanism linking it to IMC membrane biogenesis, and how it partitions between the MyoA and MyoC motors remain undefined.
• No high-resolution structure of GAP50 or its complexes
• Molecular basis of the IMC-biogenesis function unresolved
• Determinants of motor-specific anchoring unmapped