Cell cycle regulation of organelle biogenesis and apicoplast protein trafficking in Toxoplasma gondii

Apicomplexan parasites employ an unusual mechanism for cell replication, assembling daughters within the mother. Because rapid multiplication correlates with pathogenesis, it is important to understand the coordination of parasite assembly. Organellar segregation is critical in these organisms harboring single copy organelles, and their highly polarized subcellular organization is essential for host cell invasion. In order to understand how these unicellular eukaryotes efficiently package a complete set of organelles, we have used time-lapse microscopy of fluorescent markers targeted to various subcellular structures, and constructed a complete timetable for organellokinesis in Toxoplasma gondii. Golgi division and elongation of the apicoplast (a secondary endosymbiotic plastid) are among the first morphologically observable events, associated with centriole migration and preceding the formation of the daughter cytoskeletal scaffolds. Cytoskeletal growth proceeds from the apical end of the parasite, first encapsulating the ER/Golgi from which the apical secretory organelles are produced de novo. Further growth of the cytoskeletal scaffold results in the partition of the apicoplast, nucleus, and ultimately the mitochondrion, which enters the developing daughters rapidly and late during mitosis. Defining an accurate timetable for organellokinesis permitted a detailed analysis of nuclear-encoded protein trafficking to the apicoplast. The targeting of nuclear-encoded apicoplast lumenal proteins is dependent on a bipartite N-terminal extension consisting of a signal sequence followed by a transit peptide; but the targeting of an apicoplast membrane protein (a phosphate translocator) is not. Analysis using fluorescence recovery after photobleaching shows that proteins target to the apicoplast via vesicles only when the organelle is elongating prior to daughter cell formation. Electron microscopy reveals that these vesicles are morphologically distinct from other known ER or Golgi-derived vesicles. Treatment with Brefeldin A or overexpression of dominant negative mutation of ADP-ribosylation factor 1 causes apicoplast lumenal or membrane proteins to accumulate in the ER or the tubulo-vesicular Golgi region respectively, only during the apicoplast elongation stage. In sum, these results indicate a tight correlation between organelle biogenesis, protein trafficking and cell division in T. gondii