Proteomic Analysis of Mitochondrial-Associated ER Membranes (MAM) during RNA Virus Infection Reveals Dynamic Changes in Protein and Organelle Trafficking

Abstract

RIG-I pathway signaling of innate immunity against RNA virus infection is organized between the ER and mitochondria on a subdomain of the ER called the mitochondrial-associated ER membrane (MAM). The RIG-I adaptor protein MAVS transmits downstream signaling of antiviral immunity, with signaling complexes assembling on the MAM in association with mitochondria and peroxisomes. To identify components that regulate MAVS signalosome assembly on the MAM, we characterized the proteome of MAM, ER, and cytosol from cells infected with either chronic (hepatitis C) or acute (Sendai) RNA virus infections, as well as mock-infected cells. Comparative analysis of protein trafficking dynamics during both chronic and acute viral infection reveals differential protein profiles in the MAM during RIG-I pathway activation. We identified proteins and biochemical pathways recruited into and out of the MAM in both chronic and acute RNA viral infections, representing proteins that drive immunity and/or regulate viral replication. In addition, by using this comparative proteomics approach, we identified 3 new MAVS-interacting proteins, RAB1B, VTN, and LONP1, and defined LONP1 as a positive regulator of the RIG-I pathway. Our proteomic analysis also reveals a dynamic cross-talk between subcellular compartments during both acute and chronic RNA virus infection, and demonstrates the importance of the MAM as a central platform that coordinates innate immune signaling to initiate immunity against RNA virus infection.

The coming of age of the mitochondria–ER contact: a matter of thickness

“…MAMs purification yields the ensemble of membranes that are bound to the ER, including the lipid-MERCs, the ion-MERCs, the phago-MERCs, the ribo-MERCs and the sites where ER tubules contact mitochondria to mediate the Drp-1-dependent constriction that guides mitochondria division, 81,82 the fission-MERC. As such, the current MAM proteomes, 83,84 are representative of the protein collections of all types of MERCs in the tissue and cell type from which the MAMs were isolated. Similarly, localization of major protein complexes like the γ-secretase 30,57 and mTORC2 85 at the MAMs will need to be investigated by immunogold analysis, to associate their localization to a specific MERC thickness and, therefore, function.…”

Molecular Mechanisms and Physiological Significance of Organelle Interactions and Cooperation

“…It could regulate the interaction between positive and negative regulators distributed on different organelles in order to fine-tune the RIG-1 induced innate immune response (Horner et al, 2011) (Figure 1). Proteomic analysis of MAM during RNA virus infection revealed an increased presence of peroxisomal proteins if compared to control cells, supporting physical interactions between peroxisomes and mitochondria (or MAM) during antiviral response (Horner et al, 2015).…”

“…This organelle connecting assembly was suggested to act as signaling hub for the regulation of mitochondrial and peroxisomal innate immune responses after viral infection (Horner et al, 2011). In a subsequent publication the authors further reported that the MAM proteome dynamically changes after virus infection in particular increasing the amounts of individual MAVS interacting proteins (Horner et al, 2015). Evaluating their findings, the authors speculated that the MAM may be used to coordinate mitochondrial and peroxisomal metabolism according to the requirements during virus infection.…”

“…To date only one protein has been described as specific to the MAM-the phosphatidylethanolamine-Nmethyltransferase-2 (Cui et al, 1993), which appears to be only expressed in liver (Cui et al, 1995). Proteomic approaches to define the MAM in different tissues led to the identification of approximately 1000 proteins each (Poston et al, 2013;Horner et al, 2015). However, the overlap between proteins identified in different tissues using different approaches is far lower.…”

“…Peroxisomes and mitochondria cooperate in cellular lipid metabolism, in particular the breakdown of fatty acids via their organellespecific β-oxidation pathways and can both act as subcellular source, sink or target of ROS Wanders and Waterham, 2006;Ivashchenko et al, 2011;. Although peroxisomes and mitochondria can be observed in close proximity, e.g., in ultrastructural studies in mammalian cells and can also be co-purified at distinct buoyant densities (Hicks and Fahimi, 1977;Islinger et al, 2006), studies on the molecular background of physical interactions and their physiological importance are scarce (Horner et al, 2011(Horner et al, , 2015Van Bergeijk et al, 2015). Recent studies in yeast localized peroxisomes to specific mitochondrial subdomains such as mitochondria-ER junctions and sites of acetyl-CoA synthesis (Cohen et al, 2014).…”