Mitochondrial Carrier Protein Family Research Articles

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.

Expression, purification and folding of native like mitochondrial carrier proteins in lipid membranes

Mitochondrial Carrier Family proteins (MCFs) are located in the mitochondrial inner membrane and play essential roles in various cellular processes. Due to the relatively low abundance of many members of the family, in vitro structure and function determination of most MCFs require over-expression and purification of recombinant versions of these proteins. In this study, we report on a new method for overexpression of MCFs in Escherichia coli (E. coli) membranes, efficient purification of native-like proteins, and their reconstitution in mitochondrial inner membrane lipid mimics. cDNAs of Uncoupling Protein 4 (UCP4), Adenine Nucleotide Translocase (ANT) and Phosphate Translocase (PiT) were subcloned into the pET26b (+) expression vector such that fusion proteins with a short N-terminal pelB leader sequence and a six-histidine tag were produced to target the proteins toward the inner membrane of E. coli and facilitate affinity purification, respectively. Utilizing a modified autoinduction method, these proteins were overexpressed and extracted from the membrane of E. coli BL21 (DE3) and two modified strains, E. coli BL21 C43 (DE3) and E. coli BL21 Lobstr (DE3), in high yields. The proteins were then purified by immobilized metal affinity chromatography as monomers. Purity, identity, and concentration of the eluted monomers were determined by semi-native SDS-PAGE, Western blotting and mass spectrometry, and a modified Lowry assay, respectively. Cleavage of the pelB leader sequence from proteins was verified by mass spectrometric analysis. The purified proteins, surrounded by a shell of bacterial membrane lipids, were then reconstituted from the mild non-denaturing octyl glucoside (OG) detergent into phospholipid liposomes. Monomeric UCP4 spontaneously self-associated to form stable tetramers in lipid membranes, which is consistent with our previous studies. However, PiT and ANT remained dominantly monomeric in both detergent and liposome milieus, as detected by a combination of spectroscopic and electrophoretic methods. Native-like helical conformations of proteins were then confirmed by circular dichroism spectroscopy. Overall, this study demonstrates that targeting mitochondrial carrier family proteins to E. coli membranes provides an effective expression system for producing this family of proteins for biophysical studies.

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.

Adenine nucleotide carrier protein dysfunction in human disease.

Adenine nucleotide translocase (ANT) is the prototypical member of the mitochondrial carrier protein family, primarily involved in ADP/ATP exchange across the inner mitochondrial membrane. Several carrier proteins evolutionarily related to ANT, including SLC25A24 and SLC25A25, are believed to promote the exchange of cytosolic ATP-Mg2+ with phosphate in the mitochondrial matrix. They allow a net accumulation of adenine nucleotides inside mitochondria, which is essential for mitochondrial biogenesis and cell growth. In the last two decades, mutations in the heart/muscle isoform 1 of ANT (ANT1) and the ATP-Mg2+ transporters have been found to cause a wide spectrum of human diseases by a recessive or dominant mechanism. Although loss-of-function recessive mutations cause a defect in oxidative phosphorylation and an increase in oxidative stress which drives the pathology, it is unclear how the dominant missense mutations in these proteins cause human diseases. In this review, we focus on how yeast was productively used as a model system for the understanding of these dominant diseases. We also describe the relationship between the structure and function of ANT and how this may relate to various pathologies. Particularly, mutations in Aac2, the yeast homolog of ANT, were recently found to clog the mitochondrial protein import pathway. This leads to mitochondrial precursor overaccumulation stress (mPOS), characterized by the toxic accumulation of unimported mitochondrial proteins in the cytosol. We anticipate that in coming years, yeast will continue to serve as a useful model system for the mechanistic understanding of mitochondrial protein import clogging and related pathologies in humans.

Abstract 6121: UCP2 inhibitor eradicates cancer stem-like population in trastuzumab-resistant HER2-positive breast cancer

Abstract Purpose: Uncoupling protein 2 (UCP2) is a member of the mitochondrial anion carrier protein family that plays an important role in stabilizing the inner mitochondrial membrane potential (MMP, ΔΨm) and controlling reactive oxygen species (ROS) production. A selective UCP2 inhibitor, genipin is known to elicit cytotoxicity in several cancers, however, its effects on cancer stem cells (CSCs)-like properties and trastuzumab resistance in HER2-positive breast cancer cells have not been fully elucidated. In the present study, we sought to investigate the mechanism of action of genipin responsible for the induction of apoptosis and its effects on CSC-like features, expression of HER family member and trastuzumab resistance in HER2-positive breast cancer cells in vitro and in vivo. Experimental Designs: The effects of genipin on trastuzuamb-sensitive [BT474 and SKBR3] and trastuzumab-resistant [JIMT-1 and MDA-MB-453] HER2-positive breast cancer cell lines in vitro were evaluated for cell viability, Sub-G1, ROS, MMP, ALDH1 activity, CD44+/CD24- subpopulation and mammosphere formation. To confirm the physiological relevance of our in vitro observations, we explored the impact of genipin on tumor growth and angiogenesis and expression of p95HER2 and ALDH1A1 in trastuzumab-resistant xenograft model in vivo. Results: HER2-positive breast cancer cells harbored a higher level of UCP2, when compared to their counterparts. Genipin significantly downregulated UCP2 and mitochondrial dysfunction coinciding with increased ROS generation and disruption of MMP. These phenomena were accompanied with upregulation of the Bax/Bcl-2 ratio and activation of caspase-3 and caspase-7. Genipin treatment led to significant reduction in levels of truncated p95HER2, p-HER2, p-HER3 and p-Akt levels in both trastuzumab-sensitive and -resistant lines. Marked decline of CD44 and ALDH1A1 expression by genipin treatment was associated with attenuation of mammosphere-forming ability. UCP2 level is predominantly upregulated in CSC-enriched populations, while its knockdown significantly suppressed CSC-like characteristics concomitant with decreased ALDH1A1 and CD44 expression as well as impairment of ALDH1 activity. Genipin administration significantly retarded tumor growth and angiogenesis in trastuzumab-resistant JIMT-1 xenograft tumors. The antitumor effect occurred concomitantly with a decrease in Ki-67 proliferating index and enhancement of apoptosis. Furthermore, individuals receiving genipin exhibited markedly lower levels of p95HER2, full-length p185HER2, CD44 and ALDH1A1 expression compared to their control counterparts. Conclusion: To our knowledge, our findings are the first reported instance of genipin-induced suppression of CSC-like properties and HER2/HER3/Akt axis, implying that genipin treatment may have application in addressing trastuzumab resistance. Citation Format: Minsu Park, Soeun Park, Juyeon Seo, Dongmi Ko, Seongjae Kim, Yong Koo Kang, Kee Dal Nam, So Ra Seuk, Tae-Min Cho, Eunsun Jung, Yoon-Jae Kim, Ji Young Kim, Jae Hong Seo. UCP2 inhibitor eradicates cancer stem-like population in trastuzumab-resistant HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6121.

Investigation of the structural and dynamical properties of human uncoupling protein 2 through molecular dynamics simulations

Uncoupling protein 2 (UCP2) is an integral membrane protein that belongs to the family of mitochondrial anion carrier proteins. The absence of human UCP2 structure, lack of understanding of Cl− ion transport mechanism in the UCP2 and the associated biological functions motivated us to model the protein and investigate its structural and dynamical properties in a realistic mitochondrial lipid membrane system. The lipid-protein and protein-protein interactions were probed since they were found to be responsible for the conformational changes of the transmembrane (TM) helices which are involved in facilitating Cl− ion transport. Here, we employed multiscale molecular dynamics simulations including unbiased and biased MD for the investigation of the transport pathway in hUCP2 and interactions of the ion with TM helices within a membrane environment. We initially validated the hUCP2 model in the lipid membrane and then explored the transport pathway of Cl− ion and its interaction with positive residues of TM2 helix that have been reported to play a major role in the Cl− ion transport along with other TM helices of the protein. The simulation results suggest that the TM2 helix plays an important role in the formation of a stable ion channel due to the presence of arginine residues, in particular Arg88 which was found to be a key residue to maintain the channel pore through which the movement of Cl− ions occurs. Based on the results, it can be said that the study provides an atomic-level description of the Cl− ion transport mechanism in hUCP2 embedded in the mitochondrial lipid membrane.

Impacts of Mn, Fe, and Oxidative Stressors on MnSOD Activation by AtMTM1 and AtMTM2 in Arabidopsis.

It has been reported that the mitochondrial carrier family proteins of AtMTM1 and AtMTM2 are necessary for manganese superoxide dismutase (MnSOD) activation in Arabidopsis, and are responsive to methyl viologen (MV)-induced oxidative stress. In this study, we showed that MnSOD activity was enhanced specifically by Mn treatments. By using AtMnSOD-overexpressing and AtMnSOD-knockdown mutant plants treated with the widely used oxidative stressors including MV, NaCl, H2O2, and tert-butyl hydroperoxide (t-BH), we revealed that Arabidopsis MnSOD was crucial for root-growth control and superoxide scavenging ability. In addition, it has been reported that E. coli MnSOD activity is inhibited by Fe and that MTM1-mutated yeast cells exhibit elevated Fe content and decreased MnSOD activity, which can be restored by the Fe2+-specific chelator, bathophenanthroline disulfonate (BPS). However, we showed that BPS inhibited MnSOD activity in AtMTM1 and AtMTM2 single- and double-mutant protoplasts, implying that altered Fe homeostasis affected MnSOD activation through AtMTM1 and AtMTM2. Notably, we used inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis to reveal an abnormal Fe/Mn ratio in the roots and shoots of AtMTM1 and AtMTM2 mutants under MV stress, indicating the importance of AtMTM1 in roots and AtMTM2 in shoots for maintaining Fe/Mn balance.

Citrin, a mitochondrial transporter, interacts with NLRP3 and regulates inflammasome activity

Abstract The NLRP3 protein is a key initiator of inflammation in humans. NLRP3 becomes activated by a multitude of danger signals, including microbial infection, metabolic dysfunction, and cell-internalized particulates. Upon activation, NLRP3 nucleates formation of a multiprotein complex called the inflammasome, in which caspase-1 activity mediates processing of the pro-inflammatory cytokines IL-1β and IL-18 and induces pyroptosis, a pro-inflammatory form of cell death. While multiple regulators of the NLRP3 inflammasome have been described, specific ligands of NLRP3 and its mechanism of activation remain largely unknown. We performed a proteomics screen using co-immunoprecipitation (IP) and mass spectrometry to identify cellular proteins that bind NLRP3. Using this screen, we identified multiple NLRP3 interactors, including a family of mitochondrial solute carrier proteins. Co-IP experiments verified that four of these carriers, namely CTP, ANT3, OGCP, and Citrin, specifically interact with NLRP3. Because of its linkage with known metabolic disease, we further assessed the role of Citrin, a calcium-binding aspartate/glutamate carrier, in inflammasome function. When ectopically expressed, Citrin induced IL-1β release in cells, while CRISPR/Cas9 knockout of Citrin from THP-1 macrophages significantly abrogated NLRP3-dependent pyroptosis, IL-1β release, and ASC speck formation. This regulatory activity of Citrin was dependent on its calcium-sensing N-terminal domain. Importantly, this regulation was specific to NLRP3 and did not affect the AIM2 inflammasome or other cell death pathways. Altogether, our studies reveal a novel role for Citrin in regulation of the NLRP3 inflammasome and inflammatory cytokine release.

Mitochondrial copper and phosphate transporter specificity was defined early in the evolution of eukaryotes.

The mitochondrial carrier family protein SLC25A3 transports both copper and phosphate in mammals, yet in Saccharomyces cerevisiae the transport of these substrates is partitioned across two paralogs: PIC2 and MIR1. To understand the ancestral state of copper and phosphate transport in mitochondria, we explored the evolutionary relationships of PIC2 and MIR1 orthologs across the eukaryotic tree of life. Phylogenetic analyses revealed that PIC2-like and MIR1-like orthologs are present in all major eukaryotic supergroups, indicating an ancient gene duplication created these paralogs. To link this phylogenetic signal to protein function, we used structural modeling and site-directed mutagenesis to identify residues involved in copper and phosphate transport. Based on these analyses, we generated an L175A variant of mouse SLC25A3 that retains the ability to transport copper but not phosphate. This work highlights the utility of using an evolutionary framework to uncover amino acids involved in substrate recognition by mitochondrial carrier family proteins.

Characterization of In Vivo Function(s) of Members of the Plant Mitochondrial Carrier Family

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 phosphate carrier TbMCP11 is essential for mitochondrial function in the procyclic form of Trypanosoma brucei

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.

Mitochondrial Carriers for Aspartate, Glutamate and Other Amino Acids: A Review

Members of the mitochondrial carrier (MC) protein family transport various molecules across the mitochondrial inner membrane to interlink steps of metabolic pathways and biochemical processes that take place in different compartments; i.e., are localized partly inside and outside the mitochondrial matrix. MC substrates consist of metabolites, inorganic anions (such as phosphate and sulfate), nucleotides, cofactors and amino acids. These compounds have been identified by in vitro transport assays based on the uptake of radioactively labeled substrates into liposomes reconstituted with recombinant purified MCs. By using this approach, 18 human, plant and yeast MCs for amino acids have been characterized and shown to transport aspartate, glutamate, ornithine, arginine, lysine, histidine, citrulline and glycine with varying substrate specificities, kinetics, influences of the pH gradient, and capacities for the antiport and uniport mode of transport. Aside from providing amino acids for mitochondrial translation, the transport reactions catalyzed by these MCs are crucial in energy, nitrogen, nucleotide and amino acid metabolism. In this review we dissect the transport properties, phylogeny, regulation and expression levels in different tissues of MCs for amino acids, and summarize the main structural aspects known until now about MCs. The effects of their disease-causing mutations and manipulation of their expression levels in cells are also considered as clues for understanding their physiological functions.

Evolutionary Genomics of Metchnikovella incurvata (Metchnikovellidae): An Early Branching Microsporidium.

Metchnikovellids are highly specialized hyperparasites, which infect and reproduce inside gregarines (Apicomplexa) inhabiting marine invertebrates. Their phylogenetic affiliation was under constant discussion until recently, when analysis of the first near-complete metchnikovellid genome, that of Amphiamblys sp., placed it in a basal position with respect to most Microsporidia. Microsporidia are a highly diversified lineage of extremely reduced parasites related to Rozellida (Rozellosporidia = Rozellomycota = Cryptomycota) within the Holomycota clade of Opisthokonta. By sequencing DNA from a single-isolated infected gregarine cell we obtained an almost complete genome of a second metchnikovellid species, and the first one of a taxonomically described and well-documented species, Metchnikovella incurvata. Our phylogenomic analyses show that, despite being considerably divergent from each other, M. incurvata forms a monophyletic group with Amphiamplys sp., and confirm that metchnikovellids are one of the deep branches of Microsporidia. Comparative genomic analysis demonstrates that, like most Microsporidia, metchnikovellids lack mitochondrial genes involved in energy transduction and are thus incapable of synthesizing their own ATP via mitochondrial oxidative phosphorylation. They also lack the horizontally acquired ATP transporters widespread in most Microsporidia. We hypothesize that a family of mitochondrial carrier proteins evolved to transport ATP from the host into the metchnikovellid cell. We observe the progressive reduction of genes involved in DNA repair pathways along the evolutionary path of Microsporidia, which might explain, at least partly, the extremely high evolutionary rate of the most derived species. Our data also suggest that genome reduction and acquisition of novel genes co-occurred during the adaptation of Microsporidia to their hosts.

Uncoupling Protein 2 in Cardiovascular Health and Disease.

Uncoupling protein 2 (UCP2) belongs to the family of mitochondrial anion carrier proteins. It uncouples oxygen consumption from ATP synthesis. UCP2 is ubiquitously expressed in most cell types to reduce oxidative stress. It is tightly regulated at the transcriptional, translational, and post-translational levels. UCP2 in the cardiovascular system is being increasingly recognized as an important molecule to defend against various stress signals such as oxidative stress in the pathology of vascular dysfunction, atherosclerosis, hypertension, and cardiac injuries. UCP2 protects against cellular dysfunction through reducing mitochondrial oxidative stress and modulation of mitochondrial function. In view of the different functions of UCP2 in various cell types that contribute to whole body homeostasis, cell type-specific modification of UCP2 expression may offer a better approach to help understanding how UCP2 governs mitochondrial function, reactive oxygen species production and transmembrane proton leak and how dysfunction of UCP2 participates in the development of cardiovascular diseases. This review article provided an update on the physiological regulation of UCP2 in the cardiovascular system, and also discussed the involvement of UCP2 deficiency and associated oxidative stress in the pathogenesis of several common cardiovascular diseases. Drugs targeting UCP2 expression and activity might serve another effective strategy to ameliorate cardiovascular dysfunction. However, more detailed mechanistic study will be needed to dissect the role of UCP2, the regulation of UCP2 expression, and the cellular responses to the changes of UCP2 expression in normal and stressed situations at different stages of cardiovascular diseases.

A plant-like mitochondrial carrier family protein facilitates mitochondrial transport of di- and tricarboxylates in Trypanosoma brucei

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.

The mammalian phosphate carrier SLC25A3 is a mitochondrial copper transporter required for cytochrome c oxidase biogenesis

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.

Amino acid composition analysis of human secondary transport proteins and implications for reliable membrane topology prediction

Secondary transporters in humans are a large group of proteins that transport a wide range of ions, metals, organic and inorganic solutes involved in energy transduction, control of membrane potential and osmotic balance, metabolic processes and in the absorption or efflux of drugs and xenobiotics. They are also emerging as important targets for development of new drugs and as target sites for drug delivery to specific organs or tissues. We have performed amino acid composition (AAC) and phylogenetic analyses and membrane topology predictions for 336 human secondary transport proteins and used the results to confirm protein classification and to look for trends and correlations with structural domains and specific substrates and/or function. Some proteins showed statistically high contents of individual amino acids or of groups of amino acids with similar physicochemical properties. One recurring trend was a correlation between high contents of charged and/or polar residues with misleading results in predictions of membrane topology, which was especially prevalent in Mitochondrial Carrier family proteins. We demonstrate how charged or polar residues located in the middle of transmembrane helices can interfere with their identification by membrane topology tools resulting in missed helices in the prediction. Comparison of AAC in the human proteins with that in 235 secondary transport proteins from Escherichia coli revealed similar overall trends along with differences in average contents for some individual amino acids and groups of similar amino acids that are presumed to result from a greater number of functions and complexity in the higher organism.

Overlap of copper and iron uptake systems in mitochondria in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the mitochondrial carrier family protein Pic2 imports copper into the matrix. Deletion of PIC2 causes defects in mitochondrial copper uptake and copper-dependent growth phenotypes owing to decreased cytochrome c oxidase activity. However, copper import is not completely eliminated in this mutant, so alternative transport systems must exist. Deletion of MRS3, a component of the iron import machinery, also causes a copper-dependent growth defect on non-fermentable carbon. Deletion of both PIC2 and MRS3 led to a more severe respiratory growth defect than either individual mutant. In addition, MRS3 expressed from a high copy number vector was able to suppress the oxygen consumption and copper uptake defects of a strain lacking PIC2. When expressed in Lactococcus lactis, Mrs3 mediated copper and iron import. Finally, a PIC2 and MRS3 double mutant prevented the copper-dependent activation of a heterologously expressed copper sensor in the mitochondrial intermembrane space. Taken together, these data support a role for the iron transporter Mrs3 in copper import into the mitochondrial matrix.

An Atypical Mitochondrial Carrier That Mediates Drug Action in Trypanosoma brucei.

Elucidating the mechanism of action of trypanocidal compounds is an important step in the development of more efficient drugs against Trypanosoma brucei. In a screening approach using an RNAi library in T. brucei bloodstream forms, we identified a member of the mitochondrial carrier family, TbMCP14, as a prime candidate mediating the action of a group of anti-parasitic choline analogs. Depletion of TbMCP14 by inducible RNAi in both bloodstream and procyclic forms increased resistance of parasites towards the compounds by 7-fold and 3-fold, respectively, compared to uninduced cells. In addition, down-regulation of TbMCP14 protected bloodstream form mitochondria from a drug-induced decrease in mitochondrial membrane potential. Conversely, over-expression of the carrier in procyclic forms increased parasite susceptibility more than 13-fold. Metabolomic analyses of parasites over-expressing TbMCP14 showed increased levels of the proline metabolite, pyrroline-5-carboxylate, suggesting a possible involvement of TbMCP14 in energy production. The generation of TbMCP14 knock-out parasites showed that the carrier is not essential for survival of T. brucei bloodstream forms, but reduced parasite proliferation under standard culture conditions. In contrast, depletion of TbMCP14 in procyclic forms resulted in growth arrest, followed by parasite death. The time point at which parasite proliferation stopped was dependent on the major energy source, i.e. glucose versus proline, in the culture medium. Together with our findings that proline-dependent ATP production in crude mitochondria from TbMCP14-depleted trypanosomes was reduced compared to control mitochondria, the study demonstrates that TbMCP14 is involved in energy production in T. brucei. Since TbMCP14 belongs to a trypanosomatid-specific clade of mitochondrial carrier family proteins showing very poor similarity to mitochondrial carriers of mammals, it may represent an interesting target for drug action or targeting.

The genetic association study between polymorphisms in uncoupling protein 2 and uncoupling protein 3 and metabolic data in dogs.

BackgroundThe uncoupling proteins (UCPs) in the mitochondrial inner membrane are members of the mitochondrial anion carrier protein family that play an important role in energy homeostasis. Genetic association studies have shown that human UCP2 and UCP3 variants (SNPs and indels) are associated with obesity, insulin resistance, type 2 diabetes mellitus, and metabolic syndrome. The aim of this study was to examine the genetic association between polymorphisms in UCP2 and UCP3 and metabolic data in dogs.ResultsWe identified 10 SNPs (9 intronic and 1 exonic) and 4 indels (intronic) in UCP2, and 13 SNPs (11 intronic and 2 exonic) and one indel (exonic) in UCP3, by DNA sequence analysis of 11 different dog breeds (n = 119). An association study between these UCP2 and UCP3 variants and the biochemical parameters of glucose, total cholesterol, lactate dehydrogenase and triglyceride in Labrador Retrievers (n = 50) showed that none of the UCP2 polymorphisms were significantly associated with the levels of these parameters. However, four UCP3 SNPs (intron 1) were significantly associated with total cholesterol levels. In addition, the allele frequencies of two of the four SNPs associated with higher total cholesterol levels in a breed that is susceptible to hypercholesterolemia (Shetland Sheepdogs, n = 30), compared with the control breed (Shiba, n = 30).ConclusionThe results obtained from a limited number of individuals suggest that the UCP3 gene in dogs may be associated with total cholesterol levels. The examination of larger sample sizes and further analysis will lead to increased precision of these results.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-904) contains supplementary material, which is available to authorized users.