Identification of Fis1 interactors in Toxoplasma gondii reveals a novel protein required for peripheral distribution of the mitochondrion.

Toxoplasma gondii9s singular mitochondrion is very dynamic and undergoes morphological changes throughout the parasite9s life cycle. During parasite division, the mitochondrion elongates, enters the daughter cells just prior to cytokinesis and undergoes fission. Extensive morphological changes also occur as the parasite transitions from the intracellular to the extracellular environment. We show that treatment with the ionophore monensin causes reversible constriction of the mitochondrial outer membrane, and that this effect depends on the function of the fission related protein Fis1. We also observed that mislocalization of the endogenous Fis1 causes a dominant negative effect that affects the morphology of the mitochondrion. As this suggests Fis1 interacts with proteins critical for maintenance of mitochondrial structure, we performed various protein interaction trap screens. In this manner we identified a novel outer mitochondrial membrane protein, LMF1, which is essential for positioning of the mitochondrion in intracellular parasites. Normally, while inside a host cell, the parasite mitochondrion is maintained in a lasso shape that stretches around the parasite periphery where it has regions of coupling with the parasite pellicle, suggesting the presence of membrane contact sites. In intracellular parasites lacking LMF1 the mitochondrion is retracted away from the pellicle and instead is collapsed, as only normally seen in extracellular parasites. We show that this phenotype is associated with defects in parasite fitness and mitochondrial segregation. Thus, LMF1 is necessary for mitochondrial association with the parasite pellicle during intracellular growth and proper mitochondrial morphology is a prerequisite for mitochondrial division.