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Mitochondria carry out essential functions for the cell, including energy production, various biosynthesis pathways, formation of co-factors and cellular signalling in apoptosis and inflammation. The functionality of mitochondria requires the import of about 900-1300 proteins from the cytosol in baker's yeast Saccharomyces cerevisiae and human cells, respectively. The vast majority of these proteins pass the outer membrane in a largely unfolded state through the translocase of the outer mitochondrial membrane (TOM) complex. Subsequently, specific protein translocases sort the precursor proteins into the outer and inner membranes, the intermembrane space and matrix. Premature folding of mitochondrial precursor proteins, defects in the mitochondrial protein translocases or a reduction of the membrane potential across the inner mitochondrial membrane can cause stalling of precursors at the protein import apparatus. Consequently, the translocon is clogged and non-imported precursor proteins accumulate in the cell, which in turn leads to proteotoxic stress and eventually cell death. To prevent such stress situations, quality control mechanisms remove non-imported precursor proteins from the TOM channel. The highly conserved ubiquitin-proteasome system of the cytosol plays a critical role in this process. Thus, the surveillance of protein import via the TOM complex involves the coordinated activity of mitochondria-localized and cytosolic proteins to prevent proteotoxic stress in the cell., (© 2024 The Author(s). Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

The functionality of mitochondria depends on the import of proteins synthesized on cytosolic ribosomes. Impaired import into mitochondria results in mitochondrial dysfunction and proteotoxic accumulation of precursor proteins in the cytosol. All proteins sorted to inner mitochondrial compartments are imported via the translocase of the outer membrane (TOM) complex. Premature protein folding, a reduction of the mitochondrial membrane potential or defects in translocases can result in precursor arrest during translocation, thereby clogging the TOM channel and blocking protein import. In recent years, different pathways have been identified, which employ the cytosolic ubiquitin-proteasome system in the extraction and turnover of precursor proteins from the TOM complex. Central events in this process are the modification of arrested precursor proteins with ubiquitin, their extraction by AAA-ATPases and subsequent degradation by the 26 S proteasome. Analysis of these processes is largely facilitated by the expression of model proteins that function as efficient "cloggers" of the import machinery. Here we describe the use of such clogger proteins and how their handling by the protein quality control machinery can be monitored. We provide protocols to study the extent of clogging, the ubiquitin-modification of arrested precursor proteins and their turnover by the 26 S proteasome., (Copyright © 2024. Published by Elsevier Inc.)

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.

Mitochondria import about 1000 proteins that are produced as precursors on cytosolic ribosomes. Defects in mitochondrial protein import result in the accumulation of non-imported precursor proteins and proteotoxic stress. The cell is equipped with different quality control mechanisms to monitor protein transport into mitochondria. First, molecular chaperones guide unfolded proteins to mitochondria and deliver non-imported proteins to proteasomal degradation. Second, quality control factors remove translocation stalled precursor proteins from protein translocases. Third, protein translocases monitor protein sorting to mitochondrial subcompartments. Fourth, AAA proteases of the mitochondrial subcompartments remove mislocalized or unassembled proteins. Finally, impaired efficiency of protein transport is an important sensor for mitochondrial dysfunction and causes the induction of cellular stress responses, which could eventually result in the removal of the defective mitochondria by mitophagy. In this review, we summarize our current understanding of quality control mechanisms that govern mitochondrial protein transport., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The article presents a study which examines the effect of Toxoplasma on outer mitochondrial membrane (OMM) through the use of live-cell imaging of infected mammalian cells expressing OMM-targeted green fluorescent protein. Topics discussed include the functions of mitochondria, and a dissection of how structures released by Toxoplasma vacuole are formed.