Abstract
The Toxoplasma gondii cytoskeleton contains an intermediate filament network, supporting a quilt of alveolar sheets forming the inner membrane complex (IMC), undergirded by 22 subpellicular microtubules (SPMTs). Embedded within the IMC are the apical annuli: 5-6 ring-shaped pores facilitating dense granule exocytosis. Here we describe a novel apical annuli protein, AAP7. AAP7 depletion causes a severe fitness defect. In stable AAP7-depleted (ATc-resistant) parasites, LMBD3 no longer traffics to the annuli, but accumulates among the secretory organelles. This suggests AAP7 is required to traffic LMDB3 to the plasma membrane through a novel route. Moreover, it indicated that AAP7 connects plasma membrane embedded LMDB3 to the AAP proteins embedded in the IMC sutures. Functionally, AAP7 depletion results in reduced secretion of dense granule proteins. Specifically, parasitophorous vacuole membrane pore forming GRA17 secretion is reduced, causing 'bubble' vacuoles. GRA17 overexpression overcomes AAP7 depletion and reduces bubble vacuoles revealing the critical defect. An additional AAP7 depletion phenotype is the accumulation of polyglutamylated SPMTs at the basal end, indicating slow turnover. Lastly, from a comparative angle, we investigated annuli in Sarcocystis neurona, revealing 6 apical annuli. This is surprising considering S. neurona's 11 alveolar vesicles and expected 11 annuli. Ergo, annuli architecture does not take cues from IMC suture positioning. In summary, our analysis of AAP7 led to equally versatile and novel insights in apical annuli architecture, their assembly (uncovering a potentially novel trafficking process), how they interface with the IMC and impact the SPMTs, and their critical function in facilitating GRA17 secretion required for the pore across the parasitophorous vacuole membrane.