The C-terminus of the prototypical M2 muscarinic receptor localizes to the mitochondria and regulates cell respiration under stress conditions.

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.

The mitochondrial protein Sideroflexin 3 (SFXN3) influences neurodegeneration pathways in vivo.

Synapses are a primary pathological target in neurodegenerative diseases. Identifying therapeutic targets at the synapse could delay progression of numerous conditions. The mitochondrial protein SFXN3 is a neuronally enriched protein expressed in synaptic terminals and regulated by key synaptic proteins, including α-synuclein. We first show that SFXN3 uses the carrier import pathway to insert into the inner mitochondrial membrane. Using high-resolution proteomics on Sfxn3-KO mice synapses, we then demonstrate that SFXN3 influences proteins and pathways associated with neurodegeneration and cell death (including CSPα and Caspase-3), as well as neurological conditions (including Parkinson's disease and Alzheimer's disease). Overexpression of SFXN3 orthologues in Drosophila models of Parkinson's disease significantly reduced dopaminergic neuron loss. In contrast, the loss of SFXN3 was insufficient to trigger neurodegeneration in mice, indicating an anti- rather than pro-neurodegeneration role for SFXN3. Taken together, these results suggest a potential role for SFXN3 in the regulation of neurodegeneration pathways.

The mitochondrial intermembrane space: the most constricted mitochondrial sub-compartment with the largest variety of protein import pathways.

The mitochondrial intermembrane space (IMS) is the most constricted sub-mitochondrial compartment, housing only about 5% of the mitochondrial proteome, and yet is endowed with the largest variability of protein import mechanisms. In this review, we summarize our current knowledge of the major IMS import pathway based on the oxidative protein folding pathway and discuss the stunning variability of other IMS protein import pathways. As IMS-localized proteins only have to cross the outer mitochondrial membrane, they do not require energy sources like ATP hydrolysis in the mitochondrial matrix or the inner membrane electrochemical potential which are critical for import into the matrix or insertion into the inner membrane. We also explore several atypical IMS import pathways that are still not very well understood and are guided by poorly defined or completely unknown targeting peptides. Importantly, many of the IMS proteins are linked to several human diseases, and it is therefore crucial to understand how they reach their normal site of function in the IMS. In the final part of this review, we discuss current understanding of how such IMS protein underpin a large spectrum of human disorders.

The biogenesis of mitochondrial intermembrane space proteins.

The mitochondrial intermembrane space (IMS) houses a large spectrum of proteins with distinct and critical functions. Protein import into this mitochondrial sub-compartment is underpinned by an intriguing variety of pathways, many of which are still poorly understood. The constricted volume of the IMS and the topological segregation by the inner membrane cristae into a bulk area surrounded by the boundary inner membrane and the lumen within the cristae is an important factor that adds to the complexity of the protein import, folding and assembly processes. We discuss the main import pathways into the IMS, but also how IMS proteins are degraded or even retro-translocated to the cytosol in an integrated network of interactions that is necessary to maintain a healthy balance of IMS proteins under physiological and cellular stress conditions. We conclude this review by highlighting new and exciting perspectives in this area with a view to develop a better understanding of yet unknown, likely unconventional import pathways, how presequence-less proteins can be targeted and the basis for dual localisation in the IMS and the cytosol. Such knowledge is critical to understanding the dynamic changes of the IMS proteome in response to stress, and particularly important for maintaining optimal mitochondrial fitness.

AIF meets the CHCHD4/Mia40-dependent mitochondrial import pathway.

In the mitochondria of healthy cells, Apoptosis-Inducing factor (AIF) is required for the optimal functioning of the respiratory chain machinery, mitochondrial integrity, cell survival, and proliferation. In all analysed species, it was revealed that the downregulation or depletion of AIF provokes mainly the post-transcriptional loss of respiratory chain Complex I protein subunits. Recent progress in the field has revealed that AIF fulfils its mitochondrial pro-survival function by interacting physically and functionally with CHCHD4, the evolutionarily-conserved human homolog of yeast Mia40. The redox-regulated CHCHD4/Mia40-dependent import machinery operates in the intermembrane space of the mitochondrion and controls the import of a set of nuclear-encoded cysteine-motif carrying protein substrates. In addition to their participation in the biogenesis of specific respiratory chain protein subunits, CHCHD4/Mia40 substrates are also implicated in the control of redox regulation, antioxidant response, translation, lipid homeostasis and mitochondrial ultrastructure and dynamics. Here, we discuss recent insights on the AIF/CHCHD4-dependent protein import pathway and review current data concerning the CHCHD4/Mia40 protein substrates in metazoan. Recent findings and the identification of disease-associated mutations in AIF or in specific CHCHD4/Mia40 substrates have highlighted these proteins as potential therapeutic targets in a variety of human disorders.

MiR-195 regulates mitochondrial function by targeting mitofusin-2 in breast cancer cells.

Mitochondrial dynamics is a highly dysregulated process in cancer. Apoptosis and mitochondrial fission are two concurrent events wherein increased mitochondrial fragmentation serves as a hallmark of apoptosis. We have shown earlier that miR-195 exerts pro-apoptotic effects in breast cancer cells. Herein, we have demonstrated miR-195 as a modulator of mitochondrial dynamics and function. Imaging experiments upon miR-195 treatment have shown that mitochondria undergo extensive fission. We validated mitofusin2 as a potential target of miR-195. This may provide a molecular explanation for the respiratory defects induced by miR-195 over-expression in breast cancer cells. Active, but not total, mitochondrial mass, was reduced with increasing levels of miR-195. We have further shown that miR-195 enhances mitochondrial SOD-2 expression but does not affect PINK1 levels in breast cancer cells. Collectively, we have revealed that miR-195 is a modulator of mitochondrial dynamics by targeting MFN2 thereby impairing mitochondrial function. Concomitantly, it enhances the scavenger of reactive oxygen species (SOD-2) to maintain moderate levels of oxidative stress. Our findings suggest a therapeutic potential of miR-195 in both ER-positive as well as ER-negative breast cancer cells.

The Yeast Voltage-Dependent Anion Channel Porin: More IMPORTant than Just Metabolite Transport.

Porin is crucial for metabolite flux in mitochondria. In this issue of Molecular Cell, Sakaue et al. (2019) and Ellenrieder et al. (2019) describe an unexpected role for Porin in mitochondrial protein import by regulating the oligomeric state of the major protein import gate, the TOM complex, and the inner membrane insertion of metabolite carriers.