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Although structurally related, mitochondrial carrier family (MCF) proteins catalyze the specific transport of a range of diverse substrates including nucleotides, amino acids, dicarboxylates, tricarboxylates, cofactors, vitamins, phosphate and H+. Despite their name, they do not, however, always localize to the mitochondria, with plasma membrane, peroxisomal, chloroplast and thylakoid and endoplasmic reticulum localizations also being reported. The existence of plastid-specific MCF proteins is suggestive that the evolution of these proteins occurred after the separation of the green lineage. That said, plant-specific MCF proteins are not all plastid-localized, with members also situated at the endoplasmic reticulum and plasma membrane. While by no means yet comprehensive, the in vivo function of a wide range of these transporters is carried out here, and we discuss the employment of genetic variants of the MCF as a means to provide insight into their in vivo function complementary to that obtained from studies following their reconstitution into liposomes.

The mitochondrial ATP-Mg/Pi carrier is responsible for the calcium-dependent regulation of adenosine nucleotide concentrations in the mitochondrial matrix, which allows mitochondria to respond to changing energy requirements of the cell. The carrier is expressed in mitochondria of fungi, plants and animals and belongs to the family of mitochondrial carriers. The carrier is unusual as it consists of three separate domains: (i) an N-terminal regulatory domain with four calcium-binding EF-hands similar to calmodulin, (ii) a loop domain containing an amphipathic α-helix and (iii) a mitochondrial carrier domain related to the mitochondrial ADP/ATP carrier. This striking example of three domains coming together from different origins to provide new functions represents an interesting quirk of evolution. In this review, we outline how the carrier was identified and how its physiological role was established with a focus on human isoforms. We exploit the sequence and structural information of the domains to explore the similarities and differences to their closest counterparts; mitochondrial ADP/ATP carriers and proteins with four EF-hands. We discuss how their combined function has led to a mechanism for calcium-regulated transport of adenosine nucleotides. Finally, we compare the ATP-Mg/Pi carrier with the mitochondrial aspartate/glutamate carrier, the only other mitochondrial carrier regulated by calcium, and we will argue that they have arisen by convergent rather than divergent evolution. © 2018 The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(12):1222-1232, 2018.

Metformin, a commonly used agent in the treatment of type 2 diabetes, is also associated with reduced risk of cancer development and improvement in cancer survival. Although much is known about metformin, the mechanisms behind its anti-cancer properties are not fully understood. In this study we addressed the role of a mitochondrial transporter commonly upregulated in cancer cells, SLC25A10, for cell survival and metabolism in the presence of metformin. SLC25A10 is a carrier in the mitochondrial inner membrane that transports malate and succinate out of the mitochondria, in exchange of phosphate and sulfate. We show that metformin treatment results in decreased gene expression of the SLC25A10 carrier both in lung cancer A549 mock cells and A549 SLC25A10 knockdown (siSLC25A10) cells. The decrease was even more pronounced when cells were grown at low glucose concentrations. The expression levels of key enzymes in glucose metabolism showed slightly altered mean values for all genes tested in both control cells and siSLC25A10 cells upon metformin treatment. The gene expression of the metabolic regulator glutamic-oxaloacetic transaminase 1 decreased in wild type cells upon metformin treatment whereas there was a trend of increased expression in the siSLC25A10 cells upon metformin treatment. In addition, the gene expression of the cyclin-dependent kinase inhibitor 1A was markedly increased in the siSLC25A10 compared to control A549 cells, and with even larger increases in the presence of metformin and at low glucose concentration. Our data show that in siSLC25A10 cell lines, metformin significantly alters the SLC25A10 carrier at both mRNA and protein levels and can thereby affect the supply of nutrients and the metabolic state of cancer cells.

Phosphate transporters play an important role in promoting the uptake and transport of phosphate in plants. In this study, the McPht gene from the Mesembryanthemum crystallinum, a mitochondrial phosphate transporter, was isolated and constructed onto a constitutive expression vector carrying 35S::GFP, and the recombinant constructs were transferred into Oryza sativa japonica L. cv. Kitaake to investigate the regulatory role of the McPht gene under phosphorus deficiency. The McPht gene encodes a protein of 357 amino acids with six transmembrane domains and is located to the mitochondria, and the mRNA transcripts of the McPht gene are highly accumulated in the shoots of M. crystallinum in response to phosphorus deficiency. However, more mRNA transcripts of the McPht gene were accumulated in the roots of the transgenic rice under phosphorus deficiency. Measurements showed that the transgenic rice demonstrated an enhanced promotion in the root development, the root activities, and phosphate uptake under phosphorus deficiency. Transcriptome sequencing showed that the transgenic rice exhibited total of 198 differentially expressed genes. Of these, total of 154 differentially expressed genes were up-regulated and total 44 genes were down-regulated comparing to the wild type in response to phosphorus deficiency. The selective six genes of the up-regulated differentially expressed genes showed an enhanced increase in mRNA transcripts in response to phosphorus deficiency, however, the transcripts of the mitochondrial carrier protein transporter in rice, a homologous gene of the McPht, in both the transgenic line and the wild type had no obvious differences. Functional enrichment analyses revealed that the most of the up-regulated genes are involved in the cytoplasmic membrane-bounded vesicle, and most of the down-regulated genes are involved in the mitochondrion and cytoplasmic membrane-bounded vesicle. The differentially expressed genes were highly enriched in plant secondary metabolisms and plant-pathogen interaction. These results indicated that the overexpression of the McPht gene might participate in the physiological adaptive modulation of the transgenic rice to phosphorus deficiency by up- or down-regulating the differentially expressed genes.

Conserved amongst all eukaryotes is a family of mitochondrial carrier proteins (SLC25A) responsible for the import of various solutes across the inner mitochondrial membrane. We previously reported that the human parasite Trypanosoma brucei possesses 26 SLC25A proteins (TbMCPs) amongst which two, TbMCP11 and TbMCP8, were predicted to function as phosphate importers. The transport of inorganic phosphate into the mitochondrion is a prerequisite to drive ATP synthesis by substrate level and oxidative phosphorylation and thus crucial for cell viability. In this paper we describe the functional characterization of TbMCP11. In procyclic form T. brucei, the RNAi of TbMCP11 blocked ATP synthesis on mitochondrial substrates, caused a drop of the mitochondrial oxygen consumption and drastically reduced cell viability. The functional complementation in yeast and mitochondrial swelling experiments suggested a role for TbMCP11 as inorganic phosphate carrier. Interestingly, procyclic form T. brucei cells in which TbMCP11 was depleted displayed an inability to either replicate or divide the kinetoplast DNA, which resulted in a severe cytokinesis defect.

Inhibition of mTOR signaling using rapamycin has been shown to increase lifespan and healthspan in multiple model organisms; however, the precise mechanisms for the beneficial effects of rapamycin remain uncertain. We have previously reported that rapamycin delays senescence in human cells and that enhanced mitochondrial biogenesis and protection from mitochondrial stress is one component of the benefit provided by rapamycin treatment. Here, using two models of senescence, replicative senescence and senescence induced by the presence of the Hutchinson-Gilford progeria lamin A mutation, we report that senescence is accompanied by elevated glycolysis and increased oxidative phosphorylation, which are both reduced by rapamycin. Measurements of mitochondrial function indicate that direct mitochondria targets of rapamycin are succinate dehydrogenase and matrix alanine aminotransferase. Elevated activity of these enzymes could be part of complex mechanisms that enable mitochondria to resume their optimal oxidative phosphorylation and resist senescence. This interpretation is supported by the fact that rapamycin-treated cultures do not undergo a premature senescence in response to the replacement of glucose with galactose in the culture medium, which forces a greater reliance on oxidative phosphorylation. Additionally, long-term treatment with rapamycin increases expression of the mitochondrial carrier protein UCP2, which facilitates the movement of metabolic intermediates across the mitochondrial membrane. The results suggest that rapamycin impacts mitochondrial function both through direct interaction with the mitochondria and through altered gene expression of mitochondrial carrier proteins.

In vertebrates the coding sequences of the mitochondrial carrier (MC) genes are highly conserved; in these genes, relatively few intronic sequences are well conserved in chicken, mouse and human, and many more in mouse and human. In the chicken/mouse/human MC intronic alignments encompassing about 13,700 nucleotides, 77.8% of the homotopic nucleotides are identical in all three species. The non-identical nucleotides form three distinct subpopulations of nucleotides: chicken-specific, mouse-specific and human-specific. The nucleotide compositions of the chicken-specific and human-specific subpopulations do not differ significantly from each other and from the identical-nucleotide subpopulation. In contrast, the mouse-specific subpopulation is remarkably different from the chicken-specific and human-specific ones, being richer in C and G and poorer in A and T. A similar analysis was carried out for mouse/human MC intronic alignments encompassing about 56,800 nucleotides, and 74.9% of the homotopic nucleotides were found to be identical in the two species. Also in this case the human-specific subpopulation appears to be markedly similar to the mouse/human identical-nucleotide subpopulation, whereas the mouse-specific subpopulation is richer in C and G and poorer in A and T. These results can be interpreted as indicating that the human and chicken intronic sequences of the MC genes investigated largely retain the characteristics of the bird/mammal ancestor sequence, while the mouse intronic sequences diverge from those of the rodent/primate ancestor much more than the human sequences do. Remarkably, the bird/mammal and rodent/primate ancestors probably shared a significant amount of homotopic nucleotides of the investigated intronic sequences.

The procyclic form of the human parasite Trypanosoma brucei harbors one single, large mitochondrion containing all tricarboxylic acid (TCA) cycle enzymes and respiratory chain complexes present also in higher eukaryotes. Metabolite exchange among subcellular compartments such as the cytoplasm, the mitochondrion, and the peroxisomes is crucial for redox homeostasis and for metabolic pathways whose enzymes are dispersed among different organelles. In higher eukaryotes, mitochondrial carrier family (MCF) proteins transport TCA-cycle intermediates across the inner mitochondrial membrane. Previously, we identified several MCF members that are essential for T. brucei survival. Among these, only one MCF protein, TbMCP12, potentially could transport dicarboxylates and tricarboxylates. Here, we conducted phylogenetic and sequence analyses and functionally characterised TbMCP12 in vivo. Our results suggested that similarly to its homologues in plants, TbMCP12 transports both dicarboxylates and tricarboxylates across the mitochondrial inner membrane. Deleting this carrier in T. brucei was not lethal, while its overexpression was deleterious. Our results suggest that the intracellular abundance of TbMCP12 is an important regulatory element for the NADPH balance and mitochondrial ATP-production.

In Saccharomyces cerevisiae the export of 2-oxoglutarate from the mitochondria, catalyzed by Yhm2p, Odc1p and Odc2p or by at least one of these transporters, has recently been shown to be essential for glutamate biosynthesis in glucose-supplemented minimal synthetic (SM) medium without glutamate, because the triple mutant yhm2∆odc1∆odc2∆ displays a growth defect under these conditions. Surprisingly, in this study it was found that yhm2∆odc1∆odc2∆ cells grow like wild-type (WT) cells in the same medium supplemented with non-fermentable carbon sources. Direct transport assays of 2-oxoglutarate/2-oxoglutarate homoexchange activity in mitochondria from WT and yhm2∆odc1∆odc2∆ cells (solubilized and reconstituted into liposomes) showed that the mitochondrial extract from yhm2∆odc1∆odc2∆ was completely inactive at variance with that from WT cells, showing that S. cerevisiae mitochondria do not contain additional proteins capable of catalyzing 2-oxoglutarate transport efficiently besides Yhm2p, Odc1p and Odc2p. Furthermore, quantitative real-time PCR experiments showed that in both WT and yhm2∆odc1∆odc2∆ cells the expression of GDH1 is low on lactate and high on glucose and, vice versa, the expression of GDH3 is high on lactate and low on glucose. These results may be interpreted to indicate that in S. cerevisiae, grown in glucose-supplemented SM medium, glutamate is synthesized by Gdh1p in the cytosol, whereas in lactate-supplemented SM medium glutamate is synthesized by Gdh3p in the mitochondria; therefore, the pathway of ammonia assimilation under fermentative conditions requires export of 2-oxoglutarate from the mitochondria, whereas the alternative pathway under respiratory conditions does not.

Iron is universally important to cellular metabolism, and mitoferrin-1 and -2 have been proposed to be the iron importers of mitochondria, the cell's assembly plant of heme and iron–sulfur clusters. These iron-containing prosthetic groups are critical for a host of physiological processes ranging from oxygen transport and energy consumption to maintaining protein structural integrity. Mitoferrin-1 (Mfrn1) belongs to the mitochondrial carrier (MC) family and is atypical given its putative metallic cargo; most MCs transport nucleotides, amino acids, or other small- to medium-size metabolites. Despite the clear importance of Mfrn1 in iron utilization, its transport activity has not been demonstrated unambiguously. To bridge this knowledge gap, we have purified recombinant Mfrn1 under non-denaturing conditions and probed its metal ion–binding and transport functions. Isothermal titration calorimetry indicates that Mfrn1 has micromolar affinity for Fe(II), Mn(II), Co(II), and Ni(II). Mfrn1 was incorporated into defined liposomes, and iron transport was reconstituted in vitro, demonstrating that Mfrn1 can transport iron. Mfrn1 can also transport manganese, cobalt, copper, and zinc but discriminates against nickel. Experiments with candidate ligands for cellular labile iron reveal that Mfrn1 transports free iron and not a chelated iron complex and selects against alkali divalent ions. Extensive mutagenesis identified multiple residues that are crucial for metal binding, transport activity, or both. There is a clear abundance of residues with side chains that can coordinate first-row transition metal ions, suggesting that these could form primary or auxiliary metal-binding sites during the transport process.

The DNA sequence corresponding to the second exon of the SLC25A3 gene is duplicated in vertebrates. The second exon codes for the first transmembrane segment and parts of the immediately adjoining intermembrane and mitochondrial matrix segments. The two genomic exon 2 sequences are 84% similar in zebrafish (slc25a3b gene), 70% in chicken, 66% in mouse and 67% in human. The amino acid identity is 86% in zebrafish, 77% in chicken and 70% in mouse and human. The two copies of exon 2 are separated by an intronic interval. Translation of both exon 2 sequences would alter the reading frame of the downstream sequence, generating a modified aa sequence which would soon be truncated by a stop codon. As a matter of fact the splicing machinery is tuned in such a way that in some species only one of the two copies is expressed and the other is spliced out, while in other species both copies are expressed but only one at a time, generating two alternative protein products.

Copper is required for the activity of cytochrome c oxidase (COX), the terminal electron-accepting complex of the mitochondrial respiratory chain. The likely source of copper used for COX biogenesis is a labile pool found in the mitochondrial matrix. In mammals, the proteins that transport copper across the inner mitochondrial membrane remain unknown. We previously reported that the mitochondrial carrier family protein Pic2 in budding yeast is a copper importer. The closest Pic2 ortholog in mammalian cells is the mitochondrial phosphate carrier SLC25A3. Here, to investigate whether SLC25A3 also transports copper, we manipulated its expression in several murine and human cell lines. SLC25A3 knockdown or deletion consistently resulted in an isolated COX deficiency in these cells, and copper addition to the culture medium suppressed these biochemical defects. Consistent with a conserved role for SLC25A3 in copper transport, its heterologous expression in yeast complemented copper-specific defects observed upon deletion of PIC2. Additionally, assays in Lactococcus lactis and in reconstituted liposomes directly demonstrated that SLC25A3 functions as a copper transporter. Taken together, these data indicate that SLC25A3 can transport copper both in vitro and in vivo.

International audience; The Arabidopsis thaliana genome contains 58 members of the solute carrier family SLC25, also called the mitochondrial carrier family, many of which have been shown to transport specific metabolites, nucleotides, and cofactors across the mitochondrial membrane. Here, two Arabidopsis members of this family, AtUCP1 and AtUCP2, which were previously thought to be uncoupling proteins and hence named UCP1/PUMP1 and UCP2/PUMP2, respectively, are assigned with a novel function. They were expressed in bacteria, purified, and reconstituted in phospholipid vesicles. Their transport properties demonstrate that they transport amino acids (aspartate, glutamate, cysteine sulfinate, and cysteate), dicarboxylates (malate, oxaloacetate, and 2-oxoglutarate), phosphate, sulfate, and thiosulfate. Transport was saturable and inhibited by mercurials and other mitochondrial carrier inhibitors to various degrees. AtUCP1 and AtUCP2 catalyzed a fast counterexchange transport as well as a low uniport of substrates, with transport rates of AtUCP1 being much higher than those of AtUCP2 in both cases. The aspartate/glutamate heteroexchange mediated by AtUCP1 and AtUCP2 is electroneutral, in contrast to that mediated by the mammalian mitochondrial aspartate glutamate carrier. Furthermore, both carriers were found to be targeted to mitochondria. Metabolite profiling of single and double knockouts shows changes in organic acid and amino acid levels. Notably, AtUCP1 and AtUCP2 are the first reported mitochondrial carriers in Arabidopsis to transport aspartate and glutamate. It is proposed that the primary function of AtUCP1 and AtUCP2 is to catalyze an aspartate(out)/glutamate(in) exchange across the mitochondrial membrane and thereby contribute to the export of reducing equivalents from the mitochondria in photorespiration.

Membrane organelles comprise both proteins and lipids. Remodeling of these membrane structures is controlled by interactions between specific proteins and lipids. Mitochondrial structure and function depend on regulated fusion and the division of both the outer and inner membranes. Here we discuss recent advances in the regulation of mitochondrial dynamics by two critical phospholipids, phosphatidic acid (PA) and cardiolipin (CL). These two lipids interact with the core components of mitochondrial fusion and division (Opa1, mitofusin, and Drp1) to activate and inhibit these dynamin-related GTPases. Moreover, lipid-modifying enzymes such as phospholipases and lipid phosphatases may organize local lipid composition to spatially and temporarily coordinate a balance between fusion and division to establish mitochondrial morphology.

Background Combined oxidative phosphorylation deficiency 28 (COXPD28) is associated with mitochondrial dysfunction caused by mutations in SLC25A26, the gene which encodes the mitochondrial S-adenosylmethionine carrier (SAMC) that responsible for the transport of S-adenosylmethionine (SAM) into the mitochondria. Objective To identify and characterize pathogenic variants of SLC25A26 in a Chinese pedigree, provide a basis for clinical diagnosis and genetic counseling. Methods We conducted a systematic analysis of the clinical characteristics of a female with COXPD28. Whole-exome and mitochondrial genome sequencing was applied for the genetic analysis, together with bioinformatic analysis of predicted consequences of the identified variant. A homotrimer model was built to visualize the affected region and predict possible outcomes of this mutation. Then a literature review was performed by online searching all cases reported with COXPD28. Results The novel compound heterozygous SLC25A26 variants (c.34G > C, p.A12P; c.197C > A; p.A66E) were identified in a Chinese patient with COXPD28. These two variants are located in the transmembrane region 1 and transmembrane region 2, respectively. As a member of the mitochondrial carrier family, the transmembrane region of SAMC is highly conserved. The variants were predicted to be pathogenic by in silico analysis and lead to a change in the protein structure of SAMC. And the change of the SAMC structure may lead to insufficient methylation and cause disease by affecting the SAM transport. Conclusions The variants in this region probably resulted in a variable loss of mitochondrial SAMC transport function and cause the COXPD28. This study that further refine genotype-phenotype associations can provide disease prognosis with a basis and families with reproductive planning options.

Protein complexes involved in respiration, ATP synthesis, and protein import reside in the mitochondrial inner membrane; thus, proper regulation of these proteins is essential for cell viability. The m-AAA protease, a conserved hetero-hexameric AAA (ATPase associated with diverse cellular activities) protease, composed of the Yta10 and Yta12 proteins, regulates mitochondrial proteostasis by mediating protein maturation and degradation. It also recognizes and mediates the dislocation of membrane-embedded substrates, including foreign transmembrane (TM) segments, but the molecular mechanism involved in these processes remains elusive. This study investigated the role of the TM domains in the m-AAA protease by systematic replacement of one TM domain at a time in yeast. Our data indicated that replacement of the Yta10 TM2 domain abolishes membrane dislocation for only a subset of substrates, whereas replacement of the Yta12 TM2 domain impairs membrane dislocation for all tested substrates, suggesting different roles of the TM domains in each m-AAA protease subunit. Furthermore, m-AAA protease–mediated membrane dislocation was impaired in the presence of a large downstream hydrophilic moiety in a membrane substrate. This finding suggested that the m-AAA protease cannot dislocate large hydrophilic domains across the membrane, indicating that the membrane dislocation probably occurs in a lipid environment. In summary, this study highlights previously underappreciated biological roles of TM domains of the m-AAA proteases in mediating the recognition and dislocation of membrane-embedded substrates.

Mitochondrial structure and function are influenced by the unique phospholipid composition of its membranes. While mitochondria contain all the major classes of phospholipids, recent studies have highlighted specific roles of the non-bilayer forming phospholipids phosphatidylethanolamine (PE) and cardiolipin (CL) in the assembly and activity of mitochondrial respiratory chain (MRC) complexes. The non-bilayer phospholipids are cone-shaped molecules that introduce curvature stress in the bilayer membrane and have been shown to impact mitochondrial fusion and fission. In addition to their overlapping roles in these mitochondrial processes, each non-bilayer phospholipid also plays a unique role in mitochondrial function; for example, CL is specifically required for MRC supercomplex formation. Recent discoveries of mitochondrial PE and CL trafficking proteins and prior knowledge of their biosynthetic pathways have provided targets for precisely manipulating non-bilayer phospholipid levels in the mitochondrial membranes in vivo. Thus, the genetic mutants of these pathways could be valuable tools in illuminating molecular functions and biophysical properties of non-bilayer phospholipids in driving mitochondrial bioenergetics and dynamics.

Differentiation from proplastids into chloroplasts is a light- and energy-dependent process. How this process is regulated is still poorly understood at the molecular level. We herein report a new putative plastidial adenine nucleotide transporter, BRITTLE1-3 (referred to as OsBT1-3), encoded by the rice (Oryza sativa) White Stripe Leaf 2 (WSL2) gene. Loss of OsBT1-3 function results in defective chloroplast biogenesis, severely reduced photosynthetic efficiency, and finally a white stripe leaf phenotype in the first four leaves. The expression levels of genes related to chlorophyll biosynthesis and photosynthesis are drastically reduced, accompanied with over accumulation of reactive oxygen species (ROS) in the wsl2 mutant. OsBT1-3 is targeted to the chloroplasts and it expresses in almost all tissues in plants, especially in young leaves. OsBT1-3 consists of 419 amino acids and exhibits features of all mitochondrial carrier proteins, including a typical transmembrane-spanning domain and a highly conserved sequence motif designated as the ‘mitochondrial energy transfer signatures’. Phylogenetic analysis shows that OsBT1-3 is a putative plastidial adenine nucleotide transporter and is most closely related to ZmBT1-2. Together, these observations suggest that the new putative adenine nucleotide transporter, OsBT1-3, plays an essential role in regulating chloroplast biogenesis and maintenance of ROS homeostasis during rice seedling de-etiolation.

Yeast Prx1 is a mitochondrial 1-Cys peroxiredoxin that catalyzes the reduction of endogenously generated H2O2. Prx1 is synthesized on cytosolic ribosomes as a preprotein with a cleavable N-terminal presequence that is the mitochondrial targeting signal, but the mechanisms underlying Prx1 distribution to distinct mitochondrial subcompartments are unknown. Here, we provide direct evidence of the following dual mitochondrial localization of Prx1: a soluble form in the intermembrane space and a form in the matrix weakly associated with the inner mitochondrial membrane. We show that Prx1 sorting into the intermembrane space likely involves the release of the protein precursor within the lipid bilayer of the inner membrane, followed by cleavage by the inner membrane peptidase. We also found that during its import into the matrix compartment, Prx1 is sequentially cleaved by mitochondrial processing peptidase and then by octapeptidyl aminopeptidase 1 (Oct1). Oct1 cleaved eight amino acid residues from the N-terminal region of Prx1 inside the matrix, without interfering with its peroxidase activity in vitro. Remarkably, the processing of peroxiredoxin (Prx) proteins by Oct1 appears to be an evolutionarily conserved process because yeast Oct1 could cleave the human mitochondrial peroxiredoxin Prx3 when expressed in Saccharomyces cerevisiae. Altogether, the processing of peroxiredoxins by Imp2 or Oct1 likely represents systems that control the localization of Prxs into distinct compartments and thereby contribute to various mitochondrial redox processes.

The mitochondrial carrier system (MCS) transports small molecules between mitochondria and the cytoplasm. It is integral to the core mitochondrial function to regulate cellular chemistry by metabolism. The mammalian MCS comprises the transporters of the 53 member canonical SLC25A family and a lesser number of identified non-canonical transporters. The recent discovery and investigations of the mitochondrial pyruvate carrier (MPC) illustrate the diverse effects a single mitochondrial carrier may exert on cellular function. However, the transport selectivities of many carriers remain unknown, and most have not been functionally investigated in mammalian cells. The mechanisms coordinating their function as a unified system remain undefined. Increased accessibility to molecular-genetic and metabolomic technologies now greatly enables investigation of the MCS. Continued investigation of the MCS may reveal how mitochondria encode complex regulatory information within chemical thermodynamic gradients. This understanding may enable precision modulation of cellular chemistry to counteract the dysmetabolism inherent in disease.

Mammalian mitochondrial genome encodes a small set of tRNAs, rRNAs, and mRNAs. The RNA synthesis process has been well characterized. How the RNAs are degraded, however, is poorly understood. It was long assumed that the degradation happens in the matrix where transcription and translation machineries reside. Here we show that contrary to the assumption, mammalian mitochondrial RNA degradation occurs in the mitochondrial intermembrane space (IMS) and the IMS-localized RNASET2 is the enzyme that degrades the RNAs. This provides a new paradigm for understanding mitochondrial RNA metabolism and transport. Electronic supplementary material The online version of this article (doi:10.1007/s13238-017-0448-9) contains supplementary material, which is available to authorized users.

The ATP-Mg/phosphate carriers (APCs) modulate the intramitochondrial adenine nucleotide pool size. In this study the concentration-dependent effects of Mg2+ and other divalent cations (Me2+) on the transport of [3H]ATP in liposomes reconstituted with purified human and Arabidopsis APCs (hAPCs and AtAPCs, respectively, including some lacking their N-terminal domains) have been investigated. The transport of Me2+ mediated by these proteins was also measured. In the presence of a low external concentration of [3H]ATP (12 μM) and increasing concentrations of Me2+, Mg2+ stimulated the activity (measured as initial transport rate of [3H]ATP) of hAPCs and decreased that of AtAPCs; Fe2+ and Zn2+ stimulated markedly hAPCs and moderately AtAPCs; Ca2+ and Mn2+ markedly AtAPCs and moderately hAPCs; and Cu2+ decreased the activity of both hAPCs and AtAPCs. All the Me2+-dependent effects correlated well with the amount of ATP-Me complex present. The transport of [14C]AMP, which has a much lower ability of complexation than ATP, was not affected by the presence of the Me2+ tested, except Cu2+. Furthermore, the transport of [3H]ATP catalyzed by the ATP/ADP carrier, which is known to transport only free ATP and ADP, was inhibited by all the Me2+ tested in an inverse relationship with the formation of the ATP-Me complex. Finally, direct measurements of Mg2+, Mn2+, Fe2+, Zn2+ and Cu2+ showed that they are cotransported with ATP by both hAPCs and AtAPCs. It is likely that in vivo APCs transport free ATP and ATP-Mg complex to different degrees, and probably trace amounts of other Me2+ in complex with ATP.

Cancer cells down-regulate different genes to give them a selective advantage in invasiveness and/or metastasis. The SLC25A26 gene encodes the mitochondrial carrier that catalyzes the import of S-adenosylmethionine (SAM) into the mitochondrial matrix, required for mitochondrial methylation processes, and is down-regulated in cervical cancer cells. In this study we show that SLC25A26 is down-regulated due to gene promoter hypermethylation, as a mechanism to promote cell survival and proliferation. Furthermore, overexpression of SLC25A26 in CaSki cells increases mitochondrial SAM availability and promotes hypermethylation of mitochondrial DNA, leading to decreased expression of key respiratory complex subunits, reduction of mitochondrial ATP and release of cytochrome c. In addition, increased SAM transport into mitochondria leads to impairment of the methionine cycle with accumulation of homocysteine at the expense of glutathione, which is strongly reduced. All these events concur to arrest the cell cycle in the S phase, induce apoptosis and enhance chemosensitivity of SAM carrier-overexpressing CaSki cells to cisplatin.

Mitochondria have many different functions the most important one of which is oxidative phosphorylation. They originated from an endosymbiotic event between a bacterium and an archaeal host cell. It was the evolution of a protein import system that marks the boundary between the endosymbiotic ancestor of the mitochondrion and a true organelle that is under the control of the nucleus. In present day mitochondria more than 95% of all proteins are imported from the cytosol, in a process mediated by hetero-oligomeric protein complexes in the outer and inner mitochondrial membranes. In this review we compare mitochondrial protein import in the best studied model system yeast and the parasitic protozoan Trypanosoma brucei. The two organisms are phylogenetically only remotely related. Despite the fact that mitochondrial protein import has the same function in both species, only very few subunits of their import machineries are conserved. Moreover, while yeast has two inner membrane protein translocases, one specialized for presequence-containing and one for mitochondrial carrier proteins, T. brucei has a single inner membrane translocase only, that mediates import of both types of substrates. The evolutionary implications of these findings are discussed.

Channels in the mitochondrial outer membrane exchange metabolites, ions, and proteins with the rest of the cell. Kruger et al. identify several new types of channel and suggest that the outer mitochondrial membrane is a more selective molecular sieve with a greater variety of channel-forming proteins than previously appreciated., The mitochondrial outer membrane is essential for communication between mitochondria and the rest of the cell and facilitates the transport of metabolites, ions, and proteins. All mitochondrial outer membrane channels known to date are β-barrel membrane proteins, including the abundant voltage-dependent anion channel and the cation-preferring protein-conducting channels Tom40, Sam50, and Mdm10. We analyzed outer membrane fractions of yeast mitochondria and identified four new channel activities: two anion-preferring channels and two cation-preferring channels. We characterized the cation-preferring channels at the molecular level. The mitochondrial import component Mim1 forms a channel that is predicted to have an α-helical structure for protein import. The short-chain dehydrogenase-related protein Ayr1 forms an NADPH-regulated channel. We conclude that the mitochondrial outer membrane contains a considerably larger variety of channel-forming proteins than assumed thus far. These findings challenge the traditional view of the outer membrane as an unspecific molecular sieve and indicate a higher degree of selectivity and regulation of metabolite fluxes at the mitochondrial boundary.

All animal subcellular membranes require cholesterol, which influences membrane fluidity and permeability, fission and fusion processes, and membrane protein function. The distribution of cholesterol among subcellular membranes is highly heterogeneous and the cholesterol content of each membrane must be carefully regulated. Compared to other subcellular membranes, mitochondrial membranes are cholesterol-poor, particularly the inner mitochondrial membrane (IMM). As a result, steroidogenesis can be controlled through the delivery of cholesterol to the IMM, where it is converted to pregnenolone. The low basal levels of cholesterol also make mitochondria sensitive to changes in cholesterol content, which can have a relatively large impact on the biophysical and functional characteristics of mitochondrial membranes. Increased mitochondrial cholesterol levels have been observed in diverse pathological conditions including cancer, steatohepatitis, Alzheimer disease and Niemann-Pick Type C1-deficiency, and are associated with increased oxidative stress, impaired oxidative phosphorylation, and changes in the susceptibility to apoptosis, among other alterations in mitochondrial function. Mitochondria are not included in the vesicular trafficking network; therefore, cholesterol transport to mitochondria is mostly achieved through the activity of lipid transfer proteins at membrane contact sites or by cytosolic, diffusible lipid transfer proteins. Here we will give an overview of the main mechanisms involved in mitochondrial cholesterol import, focusing on the steroidogenic acute regulatory protein StAR/STARD1 and other members of the StAR-related lipid transfer (START) domain protein family, and we will discuss how changes in mitochondrial cholesterol levels can arise and affect mitochondrial function. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.

Mitochondria are double-membrane organelles with varying shapes influenced by metabolic conditions, developmental stage, and environmental stimuli1–4. Their dynamic morphology is realized through regulated and balanced fusion and fission processes5, 6. Fusion is crucial for the health and physiological functions of mitochondria, including complementation of damaged mitochondrial DNAs and maintenance of membrane potential6–8. Mitofusins (Mfns) are dynamin-related GTPases essential for mitochondrial fusion9, 10. They are embedded in the mitochondrial outer membrane and thought to fuse adjacent mitochondria via concerted oligomerization and GTP hydrolysis11–13. However, the molecular mechanisms behind this process remains elusive. Here we present crystal structures of engineered human Mfn1 containing the GTPase domain and a helical domain in different stages of GTP hydrolysis. The helical domain is composed of elements from widely dispersed sequence regions of Mfn1 and resembles the Neck of the bacterial dynamin-like protein. The structures reveal unique features of its catalytic machinery and explain how GTP binding induces conformational changes to promote G domain dimerization in the transition state. Disruption of G domain dimerization abolishes the fusogenic activity of Mfn1. Moreover, a conserved aspartate trigger was found in Mfn1 to affect mitochondrial elongation, likely through a GTP-loading-dependent domain rearrangement. Based on these results, we propose a mechanistic model for Mfn1-mediated mitochondrial tethering. Our study provides important insights in the molecular basis of mitochondrial fusion and mitofusin-related human neuromuscular disorders14.

The processes of mitochondrial restitution are controlled by nuclear genes that encode proteins synthesized in ER and cytosol and delivered as organelle- and membrane-specific transport vesicles. The analysis of the transporters recovered from inner mitochondrial space (Mitosol) revealed that the ER-synthesized mitochondria-specific transport vesicles consist of two carriers, one remaining in outer mitochondrial membrane (OMM), and the other that transfers specific membrane segments to the inner mitochondrial membrane (IMM). The ER-assembled and IMM-committed membrane segments, while first integrated into OMM, undergo intra-mitochondrial lipid modification reflected in the synthesis of cardiolipin (CL) and inversion into Mitosol with load of IMM associated cytosolic proteins. Then, the CL-bedecked vesicles are released from OMM to Mitosol and upon contact with IMM fuse with the membrane, and the release of cytosolic cargo ensues. While ER-assembled mitochondria-specific transport vesicles fuse with OMM with the aid of the cytosolic, phosphatidylglycerol (PG)-specific phospholipase A2 (PLA2), the Mitosol-contained CL-specific PLA guides vesicles fusion with IMM. The described path of translocation of the membrane segments and the cytosol synthesized proteins into the designated mitochondrial compartments sustains growth and identity of OMM, IMM, maintains protein delivery for intra-mitochondrial lipid and protein modification in Mitosol, and ensures conformity of the cytosolic proteins cargo delivered to matrix.