Mitochondria shed their outer membrane in response to infection-induced stress

Description Mitochondria shed their SPOTs Outer mitochondrial membrane (OMM) function is essential for cellular health. How mitochondria respond to naturally occurring OMM stress is unknown. Li et al. show that, upon infection with the human parasite Toxoplasma gondii, mitochondria shed large structures positive for OMM (SPOTs). SPOT formation required the parasite effector TgMAF1 and its interaction with the host mitochondrial receptor TOM70 and translocase SAM50. TOM70-dependent SPOT formation mediated a depletion of mitochondrial proteins and optimal parasite growth. SPOT-like structures also formed after OMM perturbations independently of infection. Thus, membrane remodeling is a feature of cellular responses to OMM stress that Toxoplasma hijacks during infection. —SMH Infection-induced stress remodels mitochondria by inducing the release of structures from the outer mitochondrial membrane. INTRODUCTION Mitochondria are dynamic organelles that coordinate many cellular functions that are essential for life, including diverse metabolic processes, cell division and differentiation, and immune signaling. To carry out these diverse functions, mitochondria must communicate with the cytosol, a task mediated by the outer mitochondrial membrane (OMM), the gateway between mitochondria and the rest of the cell. Thus, preserving the integrity of the OMM is essential for cellular homeostasis. Although responses to stress that is artificially induced by small molecules have been described, little is known of the mechanisms by which mammalian cells respond to naturally occurring stresses of the OMM. RATIONALE Several intracellular microbes come in contact with the host OMM or release proteins that target the OMM. We reasoned that microbial infection would serve as a model with which to study cellular responses to natural OMM stress. To this end, we chose the human parasite Toxoplasma gondii because it tethers host mitochondria to its parasite vacuole; in an infected cell, areas of close membrane apposition form between the host OMM and the parasite vacuole membrane. To address how Toxoplasma affected the OMM, we performed live-cell imaging of infected mammalian cells stably expressing OMM-targeted green fluorescent protein (GFP). We found that mitochondria in contact with the Toxoplasma vacuole released large structures that were positive for OMM, which we termed “SPOTs. ” Analysis of SPOTs in fixed and live cells revealed that SPOTs did not contain proteins of the mitochondrial matrix and inner mitochondrial membrane (IMM). RESULTS Having identified Toxoplasma infection as a natural stress that induced OMM remodeling and the shedding of SPOTs, we next dissected how these structures are formed. We found that the secreted effector protein TgMAF1 (Toxoplasma gondii mitochondrial association factor 1), which tethers the host OMM to the parasite vacuole membrane, was required for SPOT formation. TgMAF1 led to a decrease in the amount of mitochondrial proteins during infection. In particular, the OMM proteins mitofusin 1 and mitofusin 2 were degraded during infection. These proteins, which mediate a nutritional defense against Toxoplasma by promoting mitochondrial uptake of fatty acids, localized to SPOTs. The ability of TgMAF1 to induce SPOTs depended on its binding to the host OMM import receptor TOM70 (translocase of the outer membrane 70), whose import function TgMAF1 impaired. TOM70 was required for optimal parasite growth and enabled an interaction between TgMAF1 and the OMM translocase SAM50 (sorting assembly machinery 50 kDa subunit), which is a key component of the OMM-IMM mitochondrial intermembrane space bridging (MIB) complex. The genetic ablation of SAM50 and the overexpression of an OMM-targeted protein induced the formation of SPOT-like structures independently of infection. CONCLUSION Because SAM50 is the only component of mitochondrial import machinery that bridges the OMM and IMM, it is in a position to translate OMM stress into the removal of compromised OMM. TgMAF1 behaves as a mitochondrial preprotein that uses the host receptor TOM70 to interact with SAM50. This enables Toxoplasma to hijack a cellular response to OMM stress—the formation of SPOTs—and drive the constitutive shedding of the OMM. Consequently, levels of mitochondrial proteins that restrict parasite growth are depleted, and import machinery that is required for mitochondrial biogenesis is sequestered on SPOTs. In an infection-independent context, however, we propose that SPOT-like structures could mitigate OMM stress by enabling the excision of dysfunctional OMM machinery, such as import receptors or translocases during defective import. Our finding that OMM remodeling occurs during infection and infection-independent scenarios sheds light on potential cellular mechanisms that safeguard OMM function. Mitochondria shed SPOTs during Toxoplasma infection. (Left) Mitochondria surrounding the parasite Toxoplasma (red) shed SPOTs, large structures positive for OMM (yellow) that lack mitochondrial matrix (cyan). (Right) Cartoon depiction of image at left as infection progresses (clock). OMM proteins—including the import translocase SAM50, and MFN1 and MFN2, which are required for mitochondrial fusion—are sequestered on SPOTs. The outer mitochondrial membrane (OMM) is essential for cellular homeostasis. Yet little is known of the mechanisms that remodel it during natural stresses. We found that large “SPOTs” (structures positive for OMM) emerge during Toxoplasma gondii infection in mammalian cells. SPOTs mediated the depletion of the OMM proteins mitofusin 1 and 2, which restrict parasite growth. The formation of SPOTs depended on the parasite effector TgMAF1 and the host mitochondrial import receptor TOM70, which is required for optimal parasite proliferation. TOM70 enabled TgMAF1 to interact with the host OMM translocase SAM50. The ablation of SAM50 or the overexpression of an OMM-targeted protein promoted OMM remodeling independently of infection. Thus, Toxoplasma hijacks the formation of SPOTs, a cellular response to OMM stress, to promote its growth.