GRA44

GRA44 is a Toxoplasma gondii dense granule protein secreted into the parasitophorous vacuole that functions as an essential component of the effector translocation machinery (PMID:32075880, PMID:32075881). It physically associates with the MYR1-containing translocation complex and other secreted vacuolar proteins (PMID:32075880, PMID:32075881), and is required for translocating parasite effectors such as GRA16 across the parasitophorous vacuole membrane into the host cell, enabling downstream host reprogramming including upregulation of c-Myc and cyclin E1 (PMID:32075880, PMID:32075881). Within the vacuole GRA44 is proteolytically processed at an ASP5 motif into two secreted fragments, but cleavage at this site is dispensable for its translocation function (PMID:32075880, PMID:32075881). Beyond these findings, GRA44 carries a computationally predicted acid phosphatase domain whose enzymatic activity has not been demonstrated in the available corpus (PMID:32075881).

1. 2020 High

   Establishing that GRA44 is a functional component of the Toxoplasma effector export system answered whether the MYR1 translocon requires additional partners to move effectors into host cells.

   Evidence Conditional knockdown and genetic disruption of GRA44 with GRA16 translocation and host c-Myc/cyclin E1 readouts, replicated across two labs

   PMID:32075880 PMID:32075881

   Open questions at the time

   • The precise step of translocation GRA44 mediates (cargo recognition, membrane pore formation, or complex stability) is not resolved
   • Whether GRA44 is required for translocation of effectors beyond GRA16 was not tested
2. 2020 High

   Reciprocal co-immunoprecipitation placed GRA44 physically within the MYR1 translocation complex, connecting its functional phenotype to a defined molecular assembly.

   Evidence Affinity purification/Co-IP of MYR1 with mass spectrometry and reciprocal IP of GRA44, in two independent studies

   PMID:32075880 PMID:32075881

   Open questions at the time

   • Direct binding versus indirect association within the complex is not distinguished
   • The stoichiometry and architecture of the GRA44-MYR1 complex are unknown
3. 2020 Medium

   Characterizing GRA44 processing addressed whether maturation by a secretory-pathway protease is required for its function, distinguishing biogenesis from activity.

   Evidence Biochemical fractionation and immunoblotting of cleavage fragments plus ASP5-motif analysis, with genetic testing of processing-site nonessentiality (single study)

   PMID:32075880 PMID:32075881

   Open questions at the time

   • Nonessentiality of the processing site established in a single study
   • Identity of the responsible protease (ASP5 versus another) not definitively assigned
   • Function of the two secreted fragments individually is unknown
4. 2020 Low

   Identification of a putative acid phosphatase domain raised the possibility of an enzymatic role for GRA44 in the vacuole.

   Evidence Sequence/domain prediction; domain described as putative with no enzymatic assay

   PMID:32075881

   Open questions at the time

   • No in vitro or in situ phosphatase activity was demonstrated
   • No substrate identified
   • Whether any predicted enzymatic activity contributes to translocation is untested

• Whether GRA44 possesses catalytic phosphatase activity and how it mechanistically contributes to effector membrane translocation remain unresolved.
• No enzymatic assay confirming or refuting phosphatase activity
• No structural model of GRA44 within the translocation complex
• Mechanism by which the complex moves effectors across the PVM is undefined