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Mitochondrial RTG (an acronym for ReTroGrade) signaling plays a cytoprotective role under various intracellular or environmental stresses. We have previously shown its contribution to osmoadaptation and capacity to sustain mitochondrial respiration in yeast. Here, we studied the interplay between RTG2, the main positive regulator of the RTG pathway, and HAP4, encoding the catalytic subunit of the Hap2-5 complex required for the expression of many mitochondrial proteins that function in the tricarboxylic acid (TCA) cycle and electron transport, upon osmotic stress. Cell growth features, mitochondrial respiratory competence, retrograde signaling activation, and TCA cycle gene expression were comparatively evaluated in wild type and mutant cells in the presence and in the absence of salt stress. We showed that the inactivation of HAP4 improved the kinetics of osmoadaptation by eliciting both the activation of retrograde signaling and the upregulation of three TCA cycle genes: citrate synthase 1 (CIT1), aconitase 1 (ACO1), and isocitrate dehydrogenase 1 (IDH1). Interestingly, their increased expression was mostly dependent on RTG2. Impaired respiratory competence in the HAP4 mutant does not affect its faster adaptive response to stress. These findings indicate that the involvement of the RTG pathway in osmostress is fostered in a cellular context of constitutively reduced respiratory capacity. Moreover, it is evident that the RTG pathway mediates peroxisomes–mitochondria communication by modulating the metabolic function of mitochondria in osmoadaptation.

Mitochondrial carriers, which transport metabolites, nucleotides, and cofactors across the mitochondrial inner membrane, have six transmembrane α-helices enclosing a translocation pore with a central substrate binding site whose access is controlled by a cytoplasmic and a matrix gate (M-gate). The salt bridges formed by the three PX[DE]XX[RK] motifs located on the odd-numbered transmembrane α-helices greatly contribute to closing the M-gate. We have measured the transport rates of cysteine mutants of the charged residue positions in the PX[DE]XX[RK] motifs of the bovine oxoglutarate carrier, the yeast GTP/GDP carrier, and the yeast NAD+ transporter, which all lack one of these charged residues. Most single substitutions, including those of the non-charged and unpaired charged residues, completely inactivated transport. Double mutations of charged pairs showed that all three carriers contain salt bridges non-essential for activity. Two double substitutions of these non-essential charge pairs exhibited higher transport rates than their corresponding single mutants, whereas swapping the charged residues in these positions did not increase activity. The results demonstrate that some of the residues in the charged residue positions of the PX[DE]XX[KR] motifs are important for reasons other than forming salt bridges, probably for playing specific roles related to the substrate interaction-mediated conformational changes leading to the M-gate opening/closing.

Uncoupling proteins (UCPs) form a distinct subfamily of the mitochondrial carrier family (MCF) SLC25. Four UCPs, DmUCP4A-C and DmUCP5, have been identified in Drosophila melanogaster on the basis of their sequence homology with mammalian UCP4 and UCP5. In a Parkinson’s disease model, DmUCP4A showed a protective role against mitochondrial dysfunction, by increasing mitochondrial membrane potential and ATP synthesis. To date, DmUCP4A is still an orphan of a biochemical function, although its possible involvement in mitochondrial uncoupling has been ruled out. Here, we show that DmUCP4A expressed in bacteria and reconstituted in phospholipid vesicles catalyzes a unidirectional transport of aspartate, which is saturable and inhibited by mercurials and other mitochondrial carrier inhibitors to various degrees. Swelling experiments carried out in yeast mitochondria have demonstrated that the unidirectional transport of aspartate catalyzed by DmUCP4 is not proton-coupled. The biochemical function of DmUCP4A has been further confirmed in a yeast cell model, in which growth has required an efflux of aspartate from mitochondria. Notably, DmUCP4A is the first UCP4 homolog from any species to be biochemically characterized. In Drosophila melanogaster, DmUCP4A could be involved in the transport of aspartate from mitochondria to the cytosol, in which it could be used for protein and nucleotide synthesis, as well as in the biosynthesis of ß-alanine and N-acetylaspartate, which play key roles in signal transmission in the central nervous system.

Mitochondrial diseases result from inherited or spontaneous mutations in mitochondrial or nuclear DNA, leading to an impairment of the oxidative phosphorylation responsible for the synthesis of ATP. To date, there are no effective pharmacological therapies for these pathologies. We performed a yeast-based screening to search for therapeutic drugs to be used for treating mitochondrial diseases associated with dominant mutations in the nuclear ANT1 gene, which encodes for the mitochondrial ADP/ATP carrier. Dominant ANT1 mutations are involved in several degenerative mitochondrial pathologies characterized by the presence of multiple deletions or depletion of mitochondrial DNA in tissues of affected patients. Thanks to the presence in yeast of the AAC2 gene, orthologue of human ANT1, a yeast mutant strain carrying the M114P substitution equivalent to adPEO-associated L98P mutation was created. Five molecules were identified for their ability to suppress the defective respiratory growth phenotype of the haploid aac2M114P. Furthermore, these molecules rescued the mtDNA mutability in the heteroallelic AAC2/aac2M114P strain, which mimics the human heterozygous condition of adPEO patients. The drugs were effective in reducing mtDNA instability also in the heteroallelic strain carrying the R96H mutation equivalent to the more severe de novo dominant missense mutation R80H, suggesting a general therapeutic effect on diseases associated with dominant ANT1 mutations.

Oxidative stress is considered to be a key factor of the pathogenesis of Parkinson’s disease, a multifactorial neurodegenerative disorder characterized by reduced dopaminergic neurons in the substantia nigra pars compacta and accumulated protein aggregates. Rotenone is a worldwide-used pesticide that induces the most common features of Parkinson’s by direct inhibition of the mitochondrial complex I. Rotenone-induced Parkinson’s models, as well as brain tissues from Parkinson’s patients, are characterized by the presence of both lipid peroxidation and protein oxidation markers resulting from the increased level of free radical species. Oxidation introduces several modifications in protein structure, including carbonylation and nitrotyrosine formation, which severely compromise cell function. Due to the link existing between oxidative stress and Parkinson’s disease, antioxidant molecules could represent possible therapeutic tools for this disease. In this study, we evaluated the effect of curcumin, a natural compound known for its antioxidant properties, in dopaminergic PC12 cells treated with rotenone, a cell model of Parkinsonism. Our results demonstrate that the treatment of PC12 cells with rotenone causes severe protein damage, with formation of both carbonylated and nitrotyrosine-derived proteins, whereas curcumin (10 µM) co-exposure exerts protective effects by reducing the levels of oxidized proteins. Curcumin also promotes proteasome activation, abolishing the inhibitory effect exerted by rotenone on this degradative system.