Metal Uptake by Mitochondrial Carrier Family Proteins Using Lactococcus lactis.

Metal ion homeostasis in mitochondria is essential to maintaining proper cellular physiology. However, the ability of metals to bind off target or form complexes with multiple metabolites presents major challenges to understanding the mechanisms that govern this homeostasis. Adding further to the complexity, some of the major mitochondrial transporters have shown substrate promiscuity. In many cases, mitochondrial metals are found in the matrix compartment that is surrounded by the impermeable inner membrane. Four major classes of transporters facilitate the movement of solute across the inner membrane. These are mitochondrial carrier family, ATP-binding cassette transporters, mitochondrial pyruvate carriers, and sideroflexins. For iron, the matrix is the site of iron-sulfur clusters and heme synthesis and therefore transport must occur in a coordinated fashion with the cellular needs for these critical cofactors. Iron could be transported in numerous forms as it has been shown to form complexes with abundant metabolites such as citrate, nucleotides, or glutathione. Here, we describe assays to study iron (or any metal) transport by mitochondrial carrier family proteins expressed in Lactococcus lactis using a nisin-controlled expression system.

MTCH2 in Metabolic Diseases, Neurodegenerative Diseases, Cancers, Embryonic Development and Reproduction.

Mitochondrial carrier homolog 2 (MTCH2) is a member of the solute carrier 25 family, located on the outer mitochondrial membrane. MTCH2 was first identified in 2000. The development in MTCH2 research is rapidly increasing. The most well-known role of MTCH2 is linking to the pro-apoptosis BID to facilitate mitochondrial apoptosis. Genetic variants in MTCH2 have been investigated for their association with metabolic and neurodegenerative diseases, however, no intervention or therapeutic suggestions were provided. Recent studies revealed the physiological and pathological function of MTCH2 in metabolic diseases, neurodegenerative diseases, cancers, embryonic development and reproduction via regulating mitochondrial apoptosis, metabolic shift between glycolysis and oxidative phosphorylation, mitochondrial fusion/fission, epithelial-mesenchymal transition, etc. This review endeavors to assess a total of 131 published articles to summarise the structure and physiological/pathological role of MTCH2, which has not previously been conducted. This review concludes that MTCH2 plays a crucial role in metabolic diseases, neurodegenerative diseases, cancers, embryonic development and reproduction, and the predominant molecular mechanism is regulation of mitochondrial function. This review gives a comprehensive state of current knowledgement on MTCH2, which will promote the therapeutic research of MTCH2.

Human mitochondrial carriers of the SLC25 family function as monomers exchanging substrates with a ping-pong kinetic mechanism.

Members of the SLC25 mitochondrial carrier family link cytosolic and mitochondrial metabolism and support cellular maintenance and growth by transporting compounds across the mitochondrial inner membrane. Their monomeric or dimeric state and kinetic mechanism have been a matter of long-standing debate. It is believed by some that they exist as homodimers and transport substrates with a sequential kinetic mechanism, forming a ternary complex where both exchanged substrates are bound simultaneously. Some studies, in contrast, have provided evidence indicating that the mitochondrial ADP/ATP carrier (SLC25A4) functions as a monomer, has a single substrate binding site, and operates with a ping-pong kinetic mechanism, whereby ADP is imported before ATP is exported. Here we reanalyze the oligomeric state and kinetic properties of the human mitochondrial citrate carrier (SLC25A1), dicarboxylate carrier (SLC25A10), oxoglutarate carrier (SLC25A11), and aspartate/glutamate carrier (SLC25A13), all previously reported to be dimers with a sequential kinetic mechanism. We demonstrate that they are monomers, except for dimeric SLC25A13, and operate with a ping-pong kinetic mechanism in which the substrate import and export steps occur consecutively. These observations are consistent with a common transport mechanism, based on a functional monomer, in which a single central substrate-binding site is alternately accessible.

The Hansenula polymorpha mitochondrial carrier family protein Mir1 is dually localized at peroxisomes and mitochondria.

Peroxisomes are ubiquitous cell organelles involved in various metabolic pathways. In order to properly function, several cofactors, substrates and products of peroxisomal enzymes need to pass the organellar membrane. So far only a few transporter proteins have been identified. We analysed peroxisomal membrane fractions purified from the yeast Hansenula polymorpha by untargeted label-free quantitation mass spectrometry. As expected, several known peroxisome-associated proteins were enriched in the peroxisomal membrane fraction. In addition, several other proteins were enriched, including mitochondrial transport proteins. Localization studies revealed that one of them, the mitochondrial phosphate carrier Mir1, has a dual localization on mitochondria and peroxisomes. To better understand the molecular mechanisms of dual sorting, we localized Mir1 in cells lacking Pex3 or Pex19, two peroxins that play a role in targeting of peroxisomal membrane proteins. In these cells Mir1 only localized to mitochondria, indicating that Pex3 and Pex19 are required to sort Mir1 to peroxisomes. Analysis of the localization of truncated versions of Mir1 in wild-type H. polymorpha cells revealed that most of them localized to mitochondria, but only one, consisting of the transmembrane domains 3-6, was peroxisomal. Peroxisomal localization of this construct was lost in a MIR1 deletion strain, indicating that full-length Mir1 was required for the localization of the truncated protein to peroxisomes. Our data suggest that only full-length Mir1 sorts to peroxisomes, while Mir1 contains multiple regions with mitochondrial sorting information. Data are available via ProteomeXchange with identifier PXD050324.

A mitochondrial carrier transports glycolytic intermediates to link cytosolic and mitochondrial glycolysis in the human gut parasite Blastocystis.

Stramenopiles form a clade of diverse eukaryotic organisms, including multicellular algae, the fish and plant pathogenic oomycetes, such as the potato blight Phytophthora, and the human intestinal protozoan Blastocystis. In most eukaryotes, glycolysis is a strictly cytosolic metabolic pathway that converts glucose to pyruvate, resulting in the production of NADH and ATP (Adenosine triphosphate). In contrast, stramenopiles have a branched glycolysis in which the enzymes of the pay-off phase are located in both the cytosol and the mitochondrial matrix. Here, we identify a mitochondrial carrier in Blastocystis that can transport glycolytic intermediates, such as dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, across the mitochondrial inner membrane, linking the cytosolic and mitochondrial branches of glycolysis. Comparative analyses with the phylogenetically related human mitochondrial oxoglutarate carrier (SLC25A11) and dicarboxylate carrier (SLC25A10) show that the glycolytic intermediate carrier has lost its ability to transport the canonical substrates malate and oxoglutarate. Blastocystis lacks several key components of oxidative phosphorylation required for the generation of mitochondrial ATP, such as complexes III and IV, ATP synthase, and ADP/ATP carriers. The presence of the glycolytic pay-off phase in the mitochondrial matrix generates ATP, which powers energy-requiring processes, such as macromolecular synthesis, as well as NADH, used by mitochondrial complex I to generate a proton motive force to drive the import of proteins and molecules. Given its unique substrate specificity and central role in carbon and energy metabolism, the carrier for glycolytic intermediates identified here represents a specific drug and pesticide target against stramenopile pathogens, which are of great economic importance.

All living organisms breakdown food molecules to generate energy for processes, such as growing, reproducing and movement. The series of chemical reactions that breakdown sugars into smaller molecules ­ known as glycolysis ­ is so important that it occurs in all life forms, from bacteria to humans. In higher organisms, such as fungi and animals, these reactions take place in the cytosol, the space surrounding the cell's various compartments. A transport protein then shuttles the end-product of glycolysis ­ pyruvate ­ into specialised compartments, known as the mitochondria, where most energy is produced. However, recently it was discovered that a group of living organisms, called the stramenopiles, have a branched glycolysis in which the enzymes involved in the second half of this process are located in both the cytosol and mitochondrial matrix. But it was not known how the intermediate molecules produced after the first half of glycolysis enter the mitochondria. To answer this question, Pyrihová et al. searched for transport protein(s) that could link the two halves of the glycolysis pathway. Computational analyses, comparing the genetic sequences of many transport proteins from several different species, revealed a new group found only in stramenopiles. Pyrihová et al. then used microscopy to visualise these new transport proteins ­ called GIC-1 and GIC-2 ­ in the parasite Blastocystis, which infects the human gut, and observed that they localise to mitochondria. Further biochemical experiments showed that GIC-1 and GIC-2 can physically bind these intermediate molecules, but only GIC-2 can transport them across membranes. Taken together, these observations suggest that GIC-2 links the two halves of glycolysis in Blastocystis. Further analyses could reveal corresponding transport proteins in other stramenopiles, many of which have devastating effects on agriculture, such as Phytophthora, which causes potato blight, or Saprolegnia, which causes skin infections in farmed salmon. Since human cells do not have equivalent transporters, they could be new drug targets not only for Blastocystis, but for these harmful pathogens as well.

Mitochondrial carrier homolog 2 increases malignant phenotype of human gastric epithelial cells and promotes proliferation, invasion, and migration of gastric cancer cells.

BACKGROUND: The precise role of mitochondrial carrier homolog 2 (MTCH2) in promoting malignancy in gastric mucosal cells and its involvement in gastric cancer cell metastasis have not been fully elucidated. AIM: To determine the role of MTCH2 in gastric cancer. METHODS: We collected 65 samples of poorly differentiated gastric cancer tissue and adjacent tissues, constructed MTCH2-overexpressing and MTCH2-knockdown cell models, and evaluated the proliferation, migration, and invasion of human gastric epithelial cells (GES-1) and human gastric cancer cells (AGS) cells. The mitochondrial membrane potential (MMP), mitochondrial permeability transformation pore (mPTP) and ATP fluorescence probe were used to detect mitochondrial function. Mitochondrial function and ATP synthase protein levels were detected via Western blotting. RESULTS: The expression of MTCH2 and ATP2A2 in gastric cancer tissues was significantly greater than that in adjacent tissues. Overexpression of MTCH2 promoted colony formation, invasion, migration, MMP expression and ATP production in GES-1 and AGS cells while upregulating ATP2A2 expression and inhibiting cell apoptosis; knockdown of MTCH2 had the opposite effect, promoting overactivation of the mPTP and promoting apoptosis. CONCLUSION: MTCH2 can increase the malignant phenotype of GES-1 cells and promote the proliferation, invasion, and migration of gastric cancer cells by regulating mitochondrial function, providing a basis for targeted therapy for gastric cancer cells.

Structural mechanisms of mitochondrial uncoupling protein 1 regulation in thermogenesis.

In mitochondria, the oxidation of nutrients is coupled to ATP synthesis by the generation of a protonmotive force across the mitochondrial inner membrane. In mammalian brown adipose tissue (BAT), uncoupling protein 1 (UCP1, SLC25A7), a member of the SLC25 mitochondrial carrier family, dissipates the protonmotive force by facilitating the return of protons to the mitochondrial matrix. This process short-circuits the mitochondrion, generating heat for non-shivering thermogenesis. Recent cryo-electron microscopy (cryo-EM) structures of human UCP1 have provided new molecular insights into the inhibition and activation of thermogenesis. Here, we discuss these structures, describing how purine nucleotides lock UCP1 in a proton-impermeable conformation and rationalizing potential conformational changes of this carrier in response to fatty acid activators that enable proton leak for thermogenesis.

Layered mechanisms regulating the human mitochondrial NAD+ transporter SLC25A51.

SLC25A51 is the primary mitochondrial NAD+ transporter in humans and controls many local reactions by mediating the influx of oxidized NAD+. Intriguingly, SLC25A51 lacks several key features compared with other members in the mitochondrial carrier family, thus its molecular mechanism has been unclear. A deeper understanding would shed light on the control of cellular respiration, the citric acid cycle, and free NAD+ concentrations in mammalian mitochondria. This review discusses recent insights into the transport mechanism of SLC25A51, and in the process highlights a multitiered regulation that governs NAD+ transport. The aspects regulating SLC25A51 import activity can be categorized as contributions from (1) structural characteristics of the transporter itself, (2) its microenvironment, and (3) distinctive properties of the transported ligand. These unique mechanisms further evoke compelling new ideas for modulating the activity of this transporter, as well as new mechanistic models for the mitochondrial carrier family.