Familial Parkinson disease is associated with mutations in ␣-synuclein (␣-syn), a presynaptic protein that has been localized not only to the cytosol, but also to mitochondria. We report here that wild-type ␣-syn from cell lines, and brain tissue from humans and mice, is present not in mitochondria but rather in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM), a structurally and functionally distinct subdomain of the ER. Remarkably, we found that pathogenic point mutations in human ␣-syn result in its reduced association with MAM, coincident with a lower degree of apposition of ER with mitochondria, a decrease in MAM function, and an increase in mitochondrial fragmentation compared with wild-type. Although overexpression of wild-type ␣-syn in mutant ␣-synexpressing cells reverted the fragmentation phenotype, neither overexpression of the mitochondrial fusion/MAM-tethering protein MFN2 nor inhibition/ablation of the mitochondrial fission protein DRP1 was able to do so, implying that ␣-syn operates downstream of the mitochondrial fusion/fission machinery. These novel results indicate that wild-type ␣-syn localizes to the MAM and modulates mitochondrial morphology, and that these behaviors are impaired by pathogenic mutations in ␣-syn. We believe that our results have far-reaching implications for both our understanding of ␣-syn biology and the treatment of synucleinopathies.

PINK1 is a mitochondrial kinase proposed to have a role in the pathogenesis of Parkinson's disease through the regulation of mitophagy. Here, we show that the PINK1 main cleavage product, PINK1 52, after being generated inside mitochondria, can exit these organelles and localize to the cytosol, where it is not only destined for degradation by the proteasome but binds to Parkin. The interaction of cytosolic PINK1 with Parkin represses Parkin translocation to the mitochondria and subsequent mitophagy. Our work therefore highlights the existence of two cellular pools of PINK1 that have different effects on Parkin translocation and mitophagy.

Aggregation-prone amyloid β-peptides occurring in Alzheimer’s disease (AD) inhibit the import of nuclear-encoded mitochondrial precursor proteins. The observation of insoluble coaggregates between preproteins and Aβ peptides provides a biochemical explanation for mitochondrial dysfunction typically observed in AD-affected cells.

Mitochondrial dysfunction represents a prominent pathological feature in many neurodegenerative diseases, particularly in Parkinson's disease (PD). Mutations in the genes encoding the proteins Pink1 and Parkin have been identified as genetic risk factors in familiar cases of PD. Research during the last decade has identified both proteins as crucial components of an organellar quality control system that contributes to the maintenance of mitochondrial function in healthy cells. The Pink1/Parkin system acts as a sensor for mitochondrial quality and is activated, in particular, after the loss of the electric potential across the inner mitochondrial membrane. Pink1 molecules accumulate at the surface of damaged mitochondria to recruit and activate Parkin, which, in turn, elicits a signaling pathway eventually leading to the autophagic removal of the damaged organelles. This review summarizes recent advances in our knowledge of the functional role of the Pink1/Parkin system in preventing the accumulation of damaged mitochondria by mitophagy.

Proteins in mammalian cells exhibit optimal stability at physiological temperatures, and even small temperature variations may cause unfolding and nonspecific aggregation. Because this process leads to a loss of function of the affected polypeptides and to cytotoxic stress, formation of protein aggregates has been recognized as a major pathogenic factor in human diseases. In this study, we determined the impact of physiological heat stress on mitochondria isolated from HeLa cells. We found that the heat-stressed mitochondria had lower membrane potential and ATP level and exhibited a decreased production of reactive oxygen species. An analysis of the mitochondrial proteome by 2D PAGE showed that the overall solubility of endogenous proteins was only marginally affected by elevated temperatures. However, a small subset of polypeptides exhibited an high sensitivity to heat stress. The mitochondrial translation elongation factor Tu (Tufm), a protein essential for organellar protein biosynthesis, was highly aggregation-prone and lost its solubility already under mild heat-stress conditions. Moreover, mitochondrial translation and the import of cytosolic proteins were defective in the heat-stressed mitochondria. Both types of nascent polypeptides, produced by translation or imported into the mitochondria, exhibited a strong tendency to aggregate in the heat-exposed mitochondria. We propose that a fast and specific inactivation of elongation factors may prevent the accumulation of misfolded nascent polypeptides and may thereby attenuate proteotoxicity under heat stress.

Isolated cytochrome c oxidase (complex IV) deficiency is one of the most frequent respiratory chain defects in humans and is usually caused by mutations in proteins required for assembly of the complex. Mutations in nuclear-encoded structural subunits are very rare. In a patient with Leigh-like syndrome presenting with leukodystrophy and severe epilepsy, we identified a homozygous splice site mutation in COX8A, which codes for the ubiquitously expressed isoform of subunit VIII, the smallest nuclear-encoded subunit of complex IV. The mutation, affecting the last nucleotide of intron 1, leads to aberrant splicing, a frame-shift in the highly conserved exon 2, and decreased amount of the COX8A transcript. The loss of the wild-type COX8A protein severely impairs the stability of the entire cytochrome c oxidase enzyme complex and manifests in isolated complex IV deficiency in skeletal muscle and fibroblasts, similar to the frequent c.845_846delCT mutation in the assembly factor SURF1 gene. Stability and activity of complex IV could be rescued in the patient's fibroblasts by lentiviral expression of wild-type COX8A. Our findings demonstrate that COX8A is indispensable for function of human complex IV and its mutation causes human disease.

The alternative oxidase (AOX) of Neurospora crassa transfers electrons from ubiquinol to oxygen. The enzyme is not expressed under normal conditions. However, when the function of the standard electron transport chain is compromised, AOX is induced, providing cells with a means to continue respiration and growth. Induction of the enzyme represents a form of retrograde regulation because AOX is encoded by a nuclear gene that responds to signals produced from inefficiently functioning mitochondria. To identify genes required for AOX expression, we have screened the N. crassa gene knockout library for strains that are unable to grow in the presence of antimycin A, an inhibitor of complex III of the standard electron transport chain. From the 7800 strains containing knockouts of different genes, we identified 62 strains that have reduced levels of AOX when grown under conditions known to induce the enzyme. Some strains have virtually no AOX, whereas others have only a slight reduction of the protein. A broad range of seemingly unrelated functions are represented in the knockouts. For example, we identified transcription factors, kinases, the mitochondrial import receptor Tom70, three subunits of the COP9 signalosome, a monothiol glutaredoxin, and several hypothetical proteins as being required for wild-type levels of AOX production. Our results suggest that defects in many signaling or metabolic pathways have a negative effect on AOX expression and imply that complex systems control production of the enzyme.

Aggregated polypeptides accumulating inside mitochondria are sequestered in a single cellular quality compartment, called IMiQ. Its formation retains proteotoxic aggregates in a distinct cellular localization, increasing mitochondrial fitness by relieving the protein quality control system of misfolded polypeptides.