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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.

The AAA+ ATPase VCP regulates the extraction of SUMO and ubiquitin-modified DNA replication factors from chromatin. We have previously described that active DNA synthesis is associated with a SUMO-high/ubiquitin-low environment governed by the deubiquitylase USP7. Here, we unveil a functional cooperation between USP7 and VCP in DNA replication, which is conserved from Caenorhabditis elegans to mammals. The role of VCP in chromatin is defined by its cofactor FAF1, which facilitates the extraction of SUMOylated and ubiquitylated proteins that accumulate after the block of DNA replication in the absence of USP7. The inactivation of USP7 and FAF1 is synthetically lethal both in C. elegans and mammalian cells. In addition, USP7 and VCP inhibitors display synergistic toxicity supporting a functional link between deubiquitylation and extraction of chromatin-bound proteins. Our results suggest that USP7 and VCPFAF1 facilitate DNA replication by controlling the balance of SUMO/Ubiquitin-modified DNA replication factors on chromatin

Eukaryotic cells are subdivided into membrane-bound compartments specialized in different cellular functions and requiring dedicated sets of proteins. Although cells developed compartment-specific mechanisms for protein quality control, chaperones and ubiquitin are generally required for maintaining cellular proteostasis. Proteotoxic stress is signalled from one compartment into another to adjust the cellular stress response. Moreover, transport of misfolded proteins between different compartments can buffer local defects in protein quality control. Mitochondria are special organelles in that they possess an own expression, folding and proteolytic machinery, of bacterial origin, which do not have ubiquitin. Nevertheless, the importance of extensive crosstalk between mitochondria and other subcellular compartments is increasingly clear. Here, we will present local quality control mechanisms and discuss how cellular proteostasis is affected by the interplay between mitochondria and the ubiquitin proteasome system.

Mitochondria are essential organelles important for energy production, proliferation, and cell death. Biogenesis, homeostasis, and degradation of this organelle are tightly controlled to match cellular needs and counteract chronic stress conditions. Despite providing their own DNA, the vast majority of mitochondrial proteins are encoded in the nucleus, synthesized by cytosolic ribosomes, and subsequently imported into different mitochondrial compartments. The integrity of the mitochondrial proteome is permanently challenged by defects in folding, transport, and turnover of mitochondrial proteins. Therefore, damaged proteins are constantly sequestered from the outer mitochondrial membrane and targeted for proteasomal degradation in the cytosol via mitochondrial-associated degradation (MAD). Recent studies identified specialized quality control mechanisms important to decrease mislocalized proteins, which affect the mitochondrial import machinery. Interestingly, central factors of these ubiquitin-dependent pathways are shared with the ER-associated degradation (ERAD) machinery, indicating close collaboration between both tubular organelles. Here, we summarize recently described cellular stress response mechanisms, which are triggered by defects in mitochondrial protein import and quality control. Moreover, we discuss how ubiquitin-dependent degradation is integrated with cytosolic stress responses, particularly focused on the crosstalk between MAD and ERAD.

Cdc48/p97, a ubiquitin-selective chaperone, orchestrates the function of E3 ligases and deubiquitylases (DUBs). Here, we identify a new function of Cdc48 in ubiquitin-dependent regulation of mitochondrial dynamics. The DUBs Ubp12 and Ubp2 exert opposing effects on mitochondrial fusion and cleave different ubiquitin chains on the mitofusin Fzo1. We demonstrate that Cdc48 integrates the activities of these two DUBs, which are themselves ubiquitylated. First, Cdc48 promotes proteolysis of Ubp12, stabilizing pro-fusion ubiquitylation on Fzo1. Second, loss of Ubp12 stabilizes Ubp2 and thereby facilitates removal of ubiquitin chains on Fzo1 inhibiting fusion. Thus, Cdc48 synergistically regulates the ubiquitylation status of Fzo1, allowing to control the balance between activation or repression of mitochondrial fusion. In conclusion, we unravel a new cascade of ubiquitylation events, comprising Cdc48 and two DUBs, fine-tuning the fusogenic activity of Fzo1.

In this protocol we describe the separation and visualization of ubiquitylated forms of the yeast mitofusin Fzo1 by Western blot. To this aim, we express HA-tagged Fzo1 in Saccharomyces cerevisiae, break the cells to extract a membrane-enriched fraction, solubilize the membranes using detergent and then specifically immunoprecipitate the tagged protein using anti-HA affinity beads. Subsequently, we separate the higher molecular weight (ubiquitylated) forms of Fzo1 via SDS-PAGE. Finally, immunoblotting and immunodecoration are used to detect the protein and its ubiquitylated forms using an HA-specific antibody. By using this protocol, it is possible to separate and visualize higher molecular weight forms of low abundant proteins such as Fzo1 and detect sharp and distinct bands above the unmodified protein by Western blot.