Identification of the pro-mature processing site of Toxoplasma ROP1 by mass spectrometry

The rhoptries are specialized secretory organelles that function during host cell invasion in the obligate intracellular parasite Toxoplasma gondii. All T. gondii rhoptry proteins studied to date are synthesized as pro-proteins that are then processed to their mature forms. To understand the role of the pro region in rhoptry protein function, we have precisely defined the processing site of the pro-region of the rhoptry protein ROP1. Efforts to determine such processing sites have been prevented by blocked N-termini of mature proteins isolated from T. gondii. To overcome this problem, we have used an engineered form of ROP1 and mass spectrometry to demonstrate that proROP1 is processed to its mature form between the glutamic acid at position 83 and alanine at position 84. These data also show that mature ROP1 lacks substantial post-translational modifications, a result which has important implications for targeting of rhoptry proteins.