IMC10 and LMF1 mediate membrane contact between the mitochondrion and the inner membrane complex in Toxoplasma gondii

Abstract

The single mitochondrion of Toxoplasma gondii is highly dynamic, being predominantly in a peripherally distributed lasso-shape in intracellular parasites and collapsed in extracellular ones. The peripheral positioning of the mitochondrion is associated with apparent contacts between the mitochondrion membrane and the parasite pellicle. The outer mitochondrial membrane-associated protein LMF1 is critical for the correct positioning of the mitochondrion, and in its absence, intracellular parasites fail to form the lasso-shaped mitochondrion. To identify other proteins that participate in tethering the parasites mitochondrion to the pellicle, we performed a yeast two-hybrid screen for LMF1 interactors. We identified 70 putative interactors, six of which are known to localize to the apical end of the parasite, two to the mitochondrial membrane, and three localize to the inner membrane complex (IMC), a component of the parasite pellicle. Using reciprocal immunoprecipitation and proximity ligation assays, we confirmed the interaction of LMF1 with the pellicle protein IMC10, with a hypothetical protein known to be part of the conoid, and with an ATPase-Guanylyl Cyclase. Conditional knockdown of IMC10 does not affect parasite viability but severely affects mitochondrial morphology in intracellular parasites and mitochondrial distribution to the daughter cells during division. In effect, IMC10 knockdown phenocopies disruption of LMF1, suggesting that these two proteins define a novel membrane tether between Toxoplasmas mitochondrion and the inner membrane complex. IMPORTANCE Toxoplasma gondii is an opportunistic parasite that can cause life-threatening disease in immunocompromised patients and those infected congenitally. As current therapies against this parasite can be poorly tolerated and are not effective against the latent stage of the parasite, there is an urgent need to identify new drug targets. The single mitochondrion of this parasite is a validated drug target, but little is known about the machinery that controls its division and structure, information that would be critical for a thorough exploration of the mitochondrion as a drug target. We have identified parasite-specific proteins that are essential to maintain the normal structure of the mitochondrion. We have discovered a complex of two proteins that tether the mitochondrion to the periphery of the parasite. Loss of this connection results in changes in mitochondrial morphology and cell division defects. Our results provide important insight into the molecular mechanisms regulating Toxoplasma mitochondrial morphology.