Mitochondrial Carrier Protein Family Research Articles

Developmental expression of sideroflexin family genes in Xenopus embryos

Emerging evidence indicates that mitochondrial carriers are not only crucial for metabolism, but also important for embryonic development. Sideroflexin is a novel family of mitochondrial tricarboxylate carrier proteins, of which the in vivo function is largely unknown. Here, we report on the expression patterns of five sideroflexin genes in Xenopus embryos. Whole-mount in situ hybridization analysis reveals that while sideroflexin2 is expressed in the pancreas, sideroflexin1 and 3 display a complex expression in the central nervous system, somites, pronephros, liver, and pancreas. In contrast, only a weak expression of sideroflexin4 and 5 was detected in embryonic brain. Taken together, the five sideroflexin genes show both overlapping and nonoverlapping expression during Xenopus embryogenesis. As the primary structures of the five sideroflexin proteins are also quite similar, their functional redundancy should be taken into consideration for gene targeting studies.

Mutations in the hyperosmotic stress-responsive mitochondrial BASIC AMINO ACID CARRIER2 enhance proline accumulation in Arabidopsis.

Mitochondrial carrier family proteins are diverse in their substrate specificity, organellar location, and gene expression. In Arabidopsis (Arabidopsis thaliana), 58 genes encode these six-transmembrane-domain proteins. We investigated the biological role of the basic amino acid carrier Basic Amino Acid Carrier2 (BAC2) from Arabidopsis that is structurally and functionally similar to ARG11, a yeast ornithine and arginine carrier, and to Arabidopsis BAC1. By studying the expression of BAC2 and the consequences of its mutation in Arabidopsis, we showed that BAC2 is a genuine mitochondrial protein and that Arabidopsis requires expression of the BAC2 gene in order to use arginine. The BAC2 gene is induced by hyperosmotic stress (with either 0.2 m NaCl or 0.4 m mannitol) and dark-induced senescence. The BAC2 promoter contains numerous stress-related cis-regulatory elements, and the transcriptional activity of BAC2:beta-glucuronidase is up-regulated by stress and senescence. Under hyperosmotic stress, bac2 mutants express the P5CS1 proline biosynthetic gene more strongly than the wild type, and this correlates with a greater accumulation of Pro. Our data suggest that BAC2 is a hyperosmotic stress-inducible transporter of basic amino acids that contributes to proline accumulation in response to hyperosmotic stress in Arabidopsis.

Mitochondrial Uncoupler Proteins

Mitochondrial uncoupling proteins (UCPs), members of a family of mitochondrial anion carrier proteins (MACP), are nuclear-encoded transmembrane transporter proteins located in the mitochondrial inner membrane. UCP1, mainly expressed in brown adipose tissue (BAT), was the first to be discovered and is responsible for animal thermogenesis; UCP2, originally thought to play a role in nonshivering thermogenesis, obesity and diabetes, its main function appears to be in the control of mitochondria-derived reactive oxygen species (ROS). Another uncoupling protein homologue, the UCP3 is mainly expressed in skeletal muscle and brown adipose tissue and its gene is transcribed from tissue-specific promoters in humans but not in rodents. All the members of this protein family possess a common feature of shunting protons across the mitochondrial inner membrane and reduce ATP synthesis; however, this common mechanism of action is used to carry out different functions by the different UCPs. The distribution and abundance of UCPs are tissue specific, which is also reflected into the processes that these proteins are thought to be participating. UCPs other than UCP1 are involved in several biological processes such as fatty acid (FA) metabolism, insulin secretion, oxidative stress (OS), heart pathophysiology and macrophage activation. New discoveries are advancing our understanding of UCPs roles in cardiovascular physiology.

Not all mitochondrial carrier proteins support permeability transition pore formation: no involvement of uncoupling protein 1

The mPTP (mitochondrial permeability transition pore) is a non-specific channel that is formed in the mitochondrial inner membrane in response to several stimuli, including elevated levels of matrix calcium. The pore is proposed to be composed of the ANT (adenine nucleotide translocase), voltage-dependent anion channel and cyclophilin D. Knockout studies, however, have demonstrated that ANT is not essential for permeability transition, which has led to the proposal that other members of the mitochondrial carrier protein family may be able to play a similar function to ANT in pore formation. To investigate this possibility, we have studied the permeability transition properties of BAT (brown adipose tissue) mitochondria in which levels of the mitochondrial carrier protein, UCP1 (uncoupling protein 1), can exceed those of ANT. Using an improved spectroscopic assay, we have quantified mPTP formation in de-energized mitochondria from wild-type and Ucp1KO (Ucp1-knockout) mice and assessed the dependence of pore formation on UCP1. When correctly normalized for differences in mitochondrial morphology, we find that calcium-induced mPTP activity is the same in both types of mitochondria, with similar sensitivity to GDP (~50% inhibited), although the portion sensitive to cyclosporin A is higher in mitochondria lacking UCP1 (~80% inhibited, compared with ~60% in mitochondria containing UCP1). We conclude that UCP1 is not a component of the cyclosporin A-sensitive mPTP in BAT and that playing a role in mPTP formation is not a general characteristic of the mitochondrial carrier protein family but is, more likely, restricted to specific members including ANT.

Nutritional and hormonal regulation of uncoupling protein 2

Uncoupling proteins (UCPs) belong to a family of mitochondrial carrier proteins that are present in the mitochondrial inner membrane. Genetic and experimental studies have shown that UCP dysfunction can be involved in metabolic disorders and in obesity. Uncoupling protein-1 (UCP1; also known as thermogenin) was identified in 1988 and found to be highly expressed in brown adipose tissue. UCP1 allows the leak of protons in respiring mitochondria, dissipating the energy as heat; the enzyme has an important role in nonshivering heat production induced by cold exposure or food intake. In 1997, two homologs of UCP1 were identified and named UCP2 and UCP3. These novel proteins also lower mitochondrial membrane potential, but whether they can dissipate metabolic energy as heat as efficiently as UCP1 is open to dispute. Even after a decade of study, the physiological roles of these novel proteins have still not been completely elucidated. This review aims to shed light on the nutritional and hormonal regulation of UCP2 and on its physiological roles.

Defining the Mitochondrial Stress Response in Arabidopsis thaliana

To obtain a global overview of how mitochondria respond to stress, we aimed to define the plant mitochondrial stress response (MSR). By combining a set of 1196 Arabidopsis thaliana genes that putatively encode mitochondrial proteins with 16 microarray experiments on stress-related conditions, 45 nuclear encoded genes were defined as widely stress-responsive. Using green fluorescent protein (GFP) fusion assays, the mitochondrial targeting of a large number of these proteins was tested, confirming in total 26 proteins as mitochondrially targeted. Several of these proteins were observed to be dual targeted to mitochondria and plastids, including the small heat shock proteins sHSP23.5 and sHSP23.6. In addition to the well defined stress components of mitochondria, such as alternative oxidases, nicotinamide adenine dinucleotide (NAD(P)H) dehydrogenases, and heat shock proteins, a variety of other proteins, many with unknown function, were identified. The mitochondrial carrier protein family was over-represented in the stress-responsive genes, suggesting that stress induces altered needs for metabolite transport across the mitochondrial inner membrane. Although the genes encoding many of these proteins contain common cis-acting regulatory elements, it was apparent that a number of distinct regulatory processes or signals likely triggered the MSR. Therefore, these genes provide new model systems to study mitochondrial retrograde regulation, in addition to the widely used alternative oxidase model. Additionally, as changes in proteins responsive to stress did not correlate well with changes at a transcript level, it suggests that post-transcriptional mechanisms also play an important role in defining the MSR.

Mutations in mitochondrial carrier family gene SLC25A38 cause nonsyndromic autosomal recessive congenital sideroblastic anemia

The sideroblastic anemias are a heterogeneous group of congenital and acquired hematological disorders whose morphological hallmark is the presence of ringed sideroblasts--bone marrow erythroid precursors containing pathologic iron deposits within mitochondria. Here, by positional cloning, we define a previously unknown form of autosomal recessive nonsyndromic congenital sideroblastic anemia, associated with mutations in the gene encoding the erythroid specific mitochondrial carrier family protein SLC25A38, and demonstrate that SLC25A38 is important for the biosynthesis of heme in eukaryotes.

A novel route for ATP acquisition by the remnant mitochondria of Encephalitozoon cuniculi

Mitochondria use transport proteins of the eukaryotic mitochondrial carrier family (MCF) to mediate the exchange of diverse substrates, including ATP, with the host cell cytosol. According to classical endosymbiosis theory, insertion of a host-nuclear-encoded MCF transporter into the protomitochondrion was the key step that allowed the host cell to harvest ATP from the enslaved endosymbiont. Notably the genome of the microsporidian Encephalitozoon cuniculi has lost all of its genes for MCF proteins. This raises the question of how the recently discovered microsporidian remnant mitochondrion, called a mitosome, acquires ATP to support protein import and other predicted ATP-dependent activities. The E. cuniculi genome does contain four genes for an unrelated type of nucleotide transporter used by plastids and bacterial intracellular parasites, such as Rickettsia and Chlamydia, to import ATP from the cytosol of their eukaryotic host cells. The inference is that E. cuniculi also uses these proteins to steal ATP from its eukaryotic host to sustain its lifestyle as an obligate intracellular parasite. Here we show that, consistent with this hypothesis, all four E. cuniculi transporters can transport ATP, and three of them are expressed on the surface of the parasite when it is living inside host cells. The fourth transporter co-locates with mitochondrial Hsp70 to the E. cuniculi mitosome. Thus, uniquely among eukaryotes, the traditional relationship between mitochondrion and host has been subverted in E. cuniculi, by reductive evolution and analogous gene replacement. Instead of the mitosome providing the parasite cytosol with ATP, the parasite cytosol now seems to provide ATP for the organelle.

Molecular identification of three Arabidopsis thaliana mitochondrial dicarboxylate carrier isoforms: organ distribution, bacterial expression, reconstitution into liposomes and functional characterization

Screening of the Arabidopsis thaliana genome revealed three potential homologues of mammalian and yeast mitochondrial DICs (dicarboxylate carriers) designated as DIC1, DIC2 and DIC3, each belonging to the mitochondrial carrier protein family. DIC1 and DIC2 are broadly expressed at comparable levels in all the tissues investigated. DIC1-DIC3 have been reported previously as uncoupling proteins, but direct transport assays with recombinant and reconstituted DIC proteins clearly demonstrate that their substrate specificity is unique to plants, showing the combined characteristics of the DIC and oxaloacetate carrier in yeast. Indeed, the Arabidopsis DICs transported a wide range of dicarboxylic acids including malate, oxaloacetate and succinate as well as phosphate, sulfate and thiosulfate at high rates, whereas 2-oxoglutarate was revealed to be a very poor substrate. The role of these plant mitochondrial DICs is discussed with respect to other known mitochondrial carrier family members including uncoupling proteins. It is proposed that plant DICs constitute the membrane component of several metabolic processes including the malate-oxaloacetate shuttle, the most important redox connection between the mitochondria and the cytosol.

The diverse members of the mitochondrial carrier family in plants

Sequencing of plant genomes allowed the identification of various members of the mitochondrial carrier family (MCF). In plants, these structurally related proteins are involved in the transport of solutes like nucleotides, phosphate, di- and tricarboxylates across the mitochondrial membrane and therefore exhibit physiological functions similar to known isoforms from animal or yeast mitochondria. Interestingly, various studies led to the recognition of MCF proteins which mediate the transport of different substrates like folates, S-adenosylmethionine, ADPglucose or ATP, ADP and AMP in plastids.

Molecular Identification of an ArabidopsisS-Adenosylmethionine Transporter. Analysis of Organ Distribution, Bacterial Expression, Reconstitution into Liposomes, and Functional Characterization

Despite much study of the role of S-adenosylmethionine (SAM) in the methylation of DNA, RNA, and proteins, and as a cofactor for a wide range of biosynthetic processes, little is known concerning the intracellular transport of this essential metabolite. Screening of the Arabidopsis (Arabidopsis thaliana) genome yielded two potential homologs of yeast (Saccharomyces cerevisiae) and human SAM transporters, designated as SAMC1 and SAMC2, both of which belong to the mitochondrial carrier protein family. The SAMC1 gene is broadly expressed at the organ level, although only in specialized tissues of roots with high rates of cell division, and appears to be up-regulated in response to wounding stress, whereas the SAMC2 gene is very poorly expressed in all organs/tissues analyzed. Direct transport assays with the recombinant and reconstituted SAMC1 were utilized to demonstrate that this protein displays a very narrow substrate specificity confined to SAM and its closest analogs. Further experiments revealed that SAMC1 was able to function in uniport and exchange reactions and characterized the transporter as highly active, but sensitive to physiologically relevant concentrations of S-adenosylhomocysteine, S-adenosylcysteine, and adenosylornithine. Green fluorescent protein-based cell biological analysis demonstrated targeting of SAMC1 to mitochondria. Previous proteomic analyses identified this protein also in the chloroplast inner envelope. In keeping with these results, bioinformatics predicted dual localization for SAMC1. These findings suggest that the provision of cytosolically synthesized SAM to mitochondria and possibly also to plastids is mediated by SAMC1 according to the relative demands for this metabolite in the organelles.

Characterization and Developmentally Regulated Localization of the Mitochondrial Carrier Protein Homologue MCP6 from Trypanosoma brucei

Proteins of the mitochondrial carrier family (MCF) are located mainly in the inner mitochondrial membrane and mediate the transport of a large range of metabolic intermediates. The genome of Trypanosoma brucei harbors 29 genes encoding different MCF proteins. We describe here the characterization of MCP6, a novel T. brucei MCF protein. Sequence comparison and phylogenetic reconstruction revealed that MCP6 is closely related to different mitochondrial ADP/ATP and calcium-dependent solute carriers, including the ATP-Mg/Pi carrier of Homo sapiens. However, MCP6 lacks essential amino acids and sequence motifs conserved in these metabolite transporters, and functional reconstitution and transport assays with E. coli suggested that this protein indeed does not function as an ADP/ATP or ATP-Mg/Pi carrier. The subcellular localization of MCP6 is developmentally regulated: in bloodstream-form trypanosomes, the protein is predominantly glycosomal, whereas in the procyclic form, it is found mainly in the mitochondria. Depletion of MCP6 in procyclic trypanosomes resulted in growth inhibition, an increased cell size, aberrant numbers of nuclei and kinetoplasts, and abnormal kinetoplast morphology, suggesting that depletion of MCP6 inhibits division of the kinetoplast.

Identification of a mitochondrial transporter for pyrimidine nucleotides in Saccharomyces cerevisiae: bacterial expression, reconstitution and functional characterization

Pyrimidine (deoxy)nucleoside triphosphates are required in mitochondria for the synthesis of DNA and the various types of RNA present in these organelles. In Saccharomyces cerevisiae, these nucleotides are synthesized outside the mitochondrial matrix and must therefore be transported across the permeability barrier of the mitochondrial inner membrane. However, no protein has ever been found to be associated with this transport activity. In the present study, Rim2p has been identified as a yeast mitochondrial pyrimidine nucleotide transporter. Rim2p (replication in mitochondria 2p) is a member of the mitochondrial carrier protein family having some special features. The RIM2 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes and its transport properties and kinetic parameters were characterized. It transported the pyrimidine (deoxy)nucleoside tri- and di-phosphates and, to a lesser extent, pyrimidine (deoxy)nucleoside monophosphates, by a counter-exchange mechanism. Transport was saturable, with an apparent K(m) of 207 microM for TTP, 404 microM for UTP and 435 microM for CTP. Rim2p was strongly inhibited by mercurials, bathophenanthroline, tannic acid and Bromocresol Purple, and partially inhibited by bongkrekic acid. Furthermore, the Rim2p-mediated heteroexchanges, TTP/TMP and TTP/TDP, are electroneutral and probably H+-compensated. The main physiological role of Rim2p is proposed to be to transport (deoxy)pyrimidine nucleoside triphosphates into mitochondria in exchange for intramitochondrially generated (deoxy)pyrimidine nucleoside monophosphates.

Uncoupling Proteins 2 and 3 Function in Concert to Augment Tolerance to Cardiac Ischemia

Transient cardiac ischemia activates cell survival signaling, conferring subsequent ischemia tolerance to the heart. This biological phenomenon, termed ischemic preconditioning, results in improved clinical outcome and attenuated infarct size following myocardial infarction. To explore genomic modifications underpinning this ischemia tolerance, we delineated the regulation and function of the cardiac enriched mitochondrial uncoupling proteins 2 and 3 during delayed ischemic preconditioning in the rat. Cardiac transcripts of genes encoding uncoupling proteins 2 and 3 are up-regulated in parallel with infarct size reduction in preconditioned hearts. Mitochondria isolated from preconditioned hearts exhibit an augmented inducible proton leak. In parallel, following anoxia-reoxygenation these mitochondria generate less hydrogen peroxide compared with non-preconditioned mitochondria. Preconditioning in rat cardiac derived myoblasts is abolished following uncoupling protein-2 depletion by RNA-interference. RNAi of uncoupling protein-3 partially attenuates the capacity to precondition these cells. Functional characterization of anoxia and reoxygenation tolerance following uncoupling protein 2 or 3 and combined 2 and 3 RNAi shows the largest reduction in viability follows depletion of both homologues. Uncoupling protein-2 depletion alone significantly attenuates anoxia-reoxygenation tolerance but uncoupling protein-3 depletion does not reduce anoxia tolerance. In parallel combined uncoupling protein depletion and isolated uncoupling protein-2 depletion augments ROS production in viable cardiomyocytes following anoxia-reoxygenation. Concurrent anti-oxidant administration ameliorates the uncoupling protein-depleted anoxia-susceptible phenotype. In conclusion, mitochondrial uncoupling proteins are necessary components of ischemia tolerance and function as components of the cellular antioxidant defense program. In the cytoprotective hierarchy, uncoupling protein-2 appears to play a greater role than uncoupling protein-3 in modulating ischemia/anoxia tolerance in heart-derived cells.

Mitochondrial Carrier Homolog 2: A Clue to Cracking the BCL-2 Family Riddle?

BCL-2 family members are pivotal regulators of the apoptotic process. Mitochondria are a major site-of-action for these proteins. Several prominent alterations occur to mitochondria during apoptosis that seem to be part of the "mitochondrial apoptotic program." The BCL-2 family members are believed to be the major regulators of this program, however their exact mechanism of action still remains a mystery. BID, a pro-apoptotic BCL-2 family member plays an essential role in initiating this program. Recently, we have revealed that in apoptotic cells the activated/truncated form of BID, tBID, interacts with a novel, uncharacterized protein named mitochondrial carrier homolog 2 (Mtch2). Mtch2 is a conserved protein that is similar to members of the mitochondrial carrier protein (MCP) family. This review summarizes the current knowledge regarding BCL-2 family members and the mitochondrial apoptotic program and examines the possible involvement of Mtch2 in this program.

Mitochondrial carrier homolog 2 is a target of tBID in cells signaled to die by tumor necrosis factor alpha.

BID, a proapoptotic BCL-2 family member, plays an essential role in the tumor necrosis factor alpha (TNF-alpha)/Fas death receptor pathway in vivo. Activation of the TNF-R1 receptor results in the cleavage of BID into truncated BID (tBID), which translocates to the mitochondria and induces the activation of BAX or BAK. In TNF-alpha-activated FL5.12 cells, tBID becomes part of a 45-kDa cross-linkable mitochondrial complex. Here we describe the biochemical purification of this complex and the identification of mitochondrial carrier homolog 2 (Mtch2) as part of this complex. Mtch2 is a conserved protein that is similar to members of the mitochondrial carrier protein family. Our studies with mouse liver mitochondria indicate that Mtch2 is an integral membrane protein exposed on the surface of mitochondria. Using blue-native gel electrophoresis we revealed that in viable FL5.12 cells Mtch2 resides in a protein complex of ca. 185 kDa and that the addition of TNF-alpha to these cells leads to the recruitment of tBID and BAX to this complex. Importantly, this recruitment was partially inhibited in FL5.12 cells stably expressing BCL-X(L). These results implicate Mtch2 as a mitochondrial target of tBID and raise the possibility that the Mtch2-resident complex participates in the mitochondrial apoptotic program.

Cloning, ontogenesis, and localization of an atypical uncoupling protein 4 inXenopus laevis

Uncoupling protein 1 (UCP1) is the first UCP described. It belongs to the family of mitochondrial carrier proteins and is expressed mainly in brown adipose tissue. Recently, the family of the UCPs has rapidly been growing due to the successive cloning of UCP2, UCP3, UCP4, and UCP5, also called brain mitochondrial carrier protein 1. Phylogenetic studies suggest that UCP1/UCP2/UCP3 on one hand and UCP4/UCP5 on the other hand belong to separate subfamilies. In this study, we report the cloning from a frog Xenopus laevis (Xl) oocyte cDNA library of a novel UCP that was shown, by sequence homology, to belong to the family of ancestral UCP4. This cloning provides a milestone in the gap between Drosophila melanogaster or Caenorhabditis elegans on one hand and mammalian UCP4 on the other. Xl UCP4 is already expressed in the oocyte, being the first UCP described in germ cell lineage. During development, it segregates in the neural cord, and, in the adult, in situ hybridization shows its expression in the neurons and also in the choroid plexus of the brain. By RT-PCR analysis, it was found that Xl UCP4 is present in all the subdivisions of the brain and also that it differs from mammalian UCP4 by a very high relative level of expression in peripheral tissues such as the liver and kidney. The peripheral tissue distribution of Xl UCP4 reinforces the hypothesis that UCP4 might be the ancestral UCP from which other UCPs diverged from.

A Novel ADP/ATP Transporter in the Mitosome of the Microaerophilic Human Parasite Entamoeba histolytica

Recent data suggest that microaerophilic and parasitic protozoa, which lack oxidative phosphorylation, nevertheless contain mitochondrial homologs [1-6], organelles that share common ancestry with mitochondria. Such widespread retention suggests there may be a common function for mitochondrial homologs that makes them essential for eukaryotic cells. We determined the mitochondrial carrier family (MCF) complement of the Entamoeba histolytica mitochondrial homolog, also known as a crypton [5] or more commonly as a mitosome [3]. MCF proteins support mitochondrial metabolic energy generation, DNA replication, and amino-acid metabolism by linking biochemical pathways in the mitochondrial matrix with those in the cytosol [7]. MCF diversity thus closely mirrors important facets of mitochondrial metabolic diversity. The Entamoeba histolytica mitosome has lost all but a single type of MCF protein, which transports ATP and ADP via a novel mechanism that is not reliant on a membrane potential. Phylogenetic analyses confirm that the Entamoeba ADP/ATP carrier is distinct from archetypal mitochondrial ADP/ATP carriers, an observation that is supported by its different substrate and inhibitor specificity. Because many functions of yeast and human mitochondria rely on solutes transported by specialized members of this family, the Entamoeba mitosome must contain only a small subset of these processes requiring adenine nucleotide exchange.

The plant uncoupling protein homologues: a new family of energy-dissipating proteins in plant mitochondria

Uncoupling proteins (UCPs) form a subfamily within the mitochondrial carrier protein family, which catalyze a free fatty acid-mediated proton recycling and can modulate the tightness of coupling between mitochondrial respiration and ATP synthesis. As in mammalian tissues, UCPs are rather ubiquitous in the plant kingdom and widespread in plant tissues in which they could have various physiological roles, such as heat production or protection against free oxygen radicals. The simultaneous occurrence in plant mitochondria of two putative energy-dissipating systems, namely UCP which dissipates the proton motive force, and alternative oxidase (AOX) which dissipates the redox potential, raises the question of their functional interactions.

Uncoupling protein-3 sensitizes cells to mitochondrial-dependent stimulus of apoptosis.

The mitochondrial uncoupling protein-3 is a member of the mitochondrial carrier protein family. As a homologue of the thermogenic brown fat uncoupling protein-1, it possesses a mitochondrial uncoupling activity and thus can influence cell energy metabolism but its exact biological function remains unclear. In the present study, uncoupling protein-3 was expressed in 293 cells using the tetracycline-inducible system and its impact on cell bioenergetics and responsiveness to the apoptotic stimulus was determined. The induction of uncoupling protein-3 expression in mitochondria did not lead to uncontrolled respiratory uncoupling in intact cells. However, it caused a GDP-inhibition of state 4 respiration and a GDP-induced re-polarization of the inner mitochondrial membrane in the presence of fatty acids, in agreement with its expected physiological behavior as an uncoupling protein (UCP). Uncoupling protein-3 expression did not cause apoptosis per se but increased the responsiveness of the cells to a mitochondrial apoptotic stimulus (i.e., addition of staurosporine in the culture medium). It enhanced caspase 3 and caspase 9 activation and favored cytochrome c release. Moreover, cells in which uncoupling protein-3 expression had been induced showed a higher mitochondrial Bax/Bcl-2 ratio essentially due to enhanced translocation of Bax from cytosol to mitochondria. Finally, the induction of uncoupling protein-3 also increased the sensitivity of mitochondria to open the permeability transition pore in response to calcium. It is concluded that the presence of uncoupling protein-3 in mitochondria sensitizes cells to apoptotic stimuli involving mitochondrial pathways.