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 parasite's 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 Toxoplasma's mitochondrion and the inner membrane complex.

Myosin A and F-Actin play a critical role in mitochondrial dynamics and inheritance inToxoplasma gondii

“…Thus, it appears that the LMF1/IMC10 tether might be important not only for maintaining the lasso shape in non-dividing parasites but also for the distribution of the mitochondrion among the daughter parasites. Accordingly, we observe that IMC10 and LMF1 form a tether immediately upon mitochondrion entrance into the daughter cells (6). It is plausible that the formation of the tether is critical for positioning the mitochondrion for entry into the daughter cell using the IMC as a scaffold or anchor for that process.…”

“…2). Based on the previous description of the mitochondrion; we classified the morphology as either lasso, sperm-like or collapsed (3, 5, 27, 28) (Fig. 2A).…”

“…3700S) all from Cell Signaling Technology. Anti-TgATOM40 rabbit G579 (1:5000), anti-TgACP rabbit (1:5000), anti-Tg-beta-tubulin (1:5000) (all from Dr. Michael Reese, UT Southwestern), Anti-IMC10 rat 1:5,000 (6), anti-IMC6 rabbit 1:5,000 (from Dr. Peter Bradley, UCLA), anti-eIF2α rabbit 1:10,000 (from Dr. William J. Sullivan, Indiana University School of Medicine), and anti-GFP tag mouse (168AT1211 cat. no.…”

“…Previous work used a Yeast Two-Hybrid (Y2H) interaction screen to identify LMF1 interactors (6) and obtained a list of 15 proteins, which were prioritized based on cellular localization. Among these proteins, three proteins were located within the pellicle, Myosin-A (MyoA) among them (Fig.…”

“…To mediate the contact site, LMF1 interacts with IMC10, a protein located in the parasite's IMC. Similarly to LMF1, loss of IMC10 causes mitochondrion morphological defects, in addition to a few division-related defects (6). Interestingly, loss of either LMF1 or IMC10 results in parasites that lack mitochondrion and in mitochondrial material outside of the parasites.…”