ROM5

TgROM5 is an active rhomboid-family intramembrane serine protease of Toxoplasma gondii that acts at the parasite pellicle to cleave the transmembrane domains of surface adhesins during host cell invasion (PMID:15753289, PMID:15913633). It is a bona fide protease, cleaving the transmembrane domain of the heterologous rhomboid substrate Drosophila Spitz (PMID:15913633), and localizes to the pellicle, concentrating at the posterior pole where the translocated MIC2 adhesin accumulates and colocalizes with it during invasion (PMID:15753289, PMID:18346213). Although early cell-based cleavage and co-localization data implicated ROM5 as the principal MIC-adhesin sheddase (PMID:15753289), genetic knockout studies overturned this: single, double, and triple knockouts showed ROM5 plays a negligible role in MIC2/MIC6 shedding and invasion, with ROM4 serving as the dominant adhesin-processing rhomboid (PMID:25336455, PMID:25846828). ROM5's residual physiological role is intramembrane cleavage of AMA1 (and other AMA family members), which becomes detectable only in the absence of ROM4 (PMID:25846828). The sorting determinants directing ROM5 specifically to the pellicle have not been resolved in the available corpus (PMID:18346213).

1. 2005 High

   Establishing whether a Toxoplasma rhomboid could cleave the microneme adhesins that must be shed for invasion identified ROM5 as an active intramembrane protease colocalizing with translocated MIC2 at the posterior pole.

   Evidence Cloning and characterization of T. gondii rhomboids with cell-based cleavage assay and IF co-localization of MIC2 during invasion

   PMID:15753289

   Open questions at the time

   • Cell-based cleavage and co-localization do not establish that ROM5 is the sheddase responsible in vivo
   • No genetic loss-of-function test of ROM5 function
2. 2005 Medium

   Confirming ROM5 was a genuine protease rather than merely a rhomboid homolog, it cleaved the transmembrane domain of the canonical rhomboid substrate Spitz.

   Evidence Cell-based heterologous cleavage assay using Drosophila Spitz

   PMID:15913633

   Open questions at the time

   • Heterologous substrate does not define physiological substrates
   • Single method, single lab
3. 2008 Medium

   Determining where ROM5 acts, it was confirmed to reside at the pellicle, though the trafficking determinants specifying this localization were not resolved for ROM5.

   Evidence Immunofluorescence localization plus domain-exchange chimeras and site-directed mutagenesis of N-terminal cytosolic domains

   PMID:18346213

   Open questions at the time

   • ROM4/ROM5-specific pellicle sorting determinants remain unidentified
   • Localization does not establish substrate access or function
4. 2014 High

   Directly testing ROM5's in vivo requirement via knockouts overturned its assumed role as the primary adhesin sheddase, revealing ROM4 as the dominant adhesin-processing rhomboid and ROM5 as negligible for invasion.

   Evidence Single, double, and triple knockouts with adhesin shedding, gliding, invasion, and moving-junction assays

   PMID:25336455

   Open questions at the time

   • Did not identify a distinct physiological substrate for ROM5
   • Possible functional redundancy masking ROM5 role not fully dissected
5. 2015 High

   Resolving what residual function ROM5 retains, ROM5/ROM4 double-knockout analysis showed ROM5 mediates residual intramembrane cleavage of AMA1 and contributes to invasion only when ROM4 is absent.

   Evidence CRISPR-Cas9 ROM5 knockout and Cre-recombinase ROM4 knockout with MIC2, MIC6, and AMA1 shedding assays and invasion assays

   PMID:25846828

   Open questions at the time

   • Full physiological substrate repertoire of ROM5 not defined
   • Molecular basis of ROM4/ROM5 substrate discrimination unknown

• The determinants that restrict ROM5 to the posterior pole and the basis for its substrate preference for AMA-family adhesins over ROM4-preferred substrates remain unresolved.
• ROM5-specific pellicle/posterior-pole sorting determinants unidentified
• Structural or biochemical basis of ROM5 versus ROM4 substrate selectivity unknown
• No structural model of ROM5