ROM2

TgROM2 is an active rhomboid intramembrane serine protease of Toxoplasma gondii whose expression is restricted to the oocyst stage, distinguishing it from the tachyzoite-expressed rhomboids TgROM1, TgROM4, and TgROM5 (PMID:15913633, PMID:15753289). In cell-based cleavage assays it processes the transmembrane domain of the canonical rhomboid substrate Drosophila Spitz as well as chimeric proteins bearing the transmembrane domains of the microneme proteins TgMIC2 and TgMIC12, establishing its capacity to cleave micronemal TM segments (PMID:15913633). The enzyme localizes to the Golgi apparatus, and this targeting is directed by critical sorting determinants residing in its N-terminal cytosolic domain, as mapped by domain-exchange and site-directed mutagenesis (PMID:18346213). Beyond these findings, the physiological substrates, in vivo function during the oocyst stage, and structural basis of catalysis have not been characterized in the available corpus.

1. 2005 Medium

   Whether TgROM2 is a functional protease and what class of substrates it can act on was unknown; demonstrating cleavage of rhomboid substrates established it as a catalytically active intramembrane serine protease with specificity for microneme protein TM domains.

   Evidence Cell-based cleavage assay with Drosophila Spitz and chimeric TgMIC2/TgMIC12 TM-domain constructs in T. gondii

   PMID:15913633

   Open questions at the time

   • No mutagenesis of the catalytic serine to confirm the active-site mechanism
   • Native full-length microneme substrates not tested, only chimeric TM constructs
   • No structural validation of substrate recognition
2. 2005 Medium

   The life-cycle stage in which TgROM2 operates was undefined; expression profiling placed it exclusively in the oocyst stage, separating its biological role from the tachyzoite rhomboids TgROM1/4/5.

   Evidence Gene cloning and stage-specific expression profiling of the T. gondii rhomboid family

   PMID:15753289 PMID:15913633

   Open questions at the time

   • Functional role within the oocyst stage not established
   • No genetic knockout to test requirement in vivo
3. 2008 High

   Where TgROM2 acts within the cell and how it is targeted there was unknown; localization combined with domain-swap and mutagenesis showed Golgi residence dictated by sorting determinants in the N-terminal cytosolic domain.

   Evidence Subcellular localization, domain-exchange, and site-directed mutagenesis in T. gondii

   PMID:18346213

   Open questions at the time

   • Identity of the trafficking machinery recognizing the N-terminal determinants not defined
   • Whether Golgi localization is required for physiological substrate processing not tested

• The endogenous physiological substrates of TgROM2 and its functional requirement during the oocyst stage remain undefined.
• No in vivo substrate identified
• No loss-of-function phenotype established
• No structural model of the enzyme