**6.1. Mitochondrial nucleotide transporter**

Several proteins have been identified as carriers for purine nucleotides, their analogues, as well as pyrimidine nucleotides. The ANT was identified by Kramer and Klingenberg [98]. Other specific proteins as GTP/GDP carriers, peroxisomal adenine nucleotide transporter, CoA and S-adenosylmethionine transporters belong in [99–102].

#### *6.1.1. ADP/ATP translocase*

Deoxynucleotide carrier ((DNC) SLC25A19) transports all dNDPs in exchange for ATP or ADP [103]. The protein was later identified as thiamine pyrophosphate carrier (TPC) [104], transporting thiamine pyrophosphate, thiamine monophosphate, and deoxynucleotides in descending order of potency dNDP > dNTP > dNMP. The protein is also capable of nucleotide transport, though less efficiently. The protein in ubiquitously expressed within tissues, with the highest levels in the kidney and lung [103].

The mutation in gene-encoded DNC (chromosomal localization 17q25.1) is known to be associated with Amish microcephaly. Amish microcephaly has only been observed in Old Order Amish community in Pennsylvania, U.S.A, with a high prevalence of about 1:500. The disease is characterized by severe congenital microcephaly, elevated levels of α-ketoglutarate in urine, and premature death. The only non-CNS physical anomaly is moderate micrognathia. Patients manifest no orientation to sight or sound and no fine or gross motor development and have metabolic acidosis enhanced by episodic viral illnesses, and in some cases patients have mild hepatomegaly and difficulty maintaining normal body temperature and develop increasing irritability [97]. Study on SLC25A19 knock-out mice has shown that metabolic abnormalities in humans are due to absent TPC activity [104].

#### *6.1.2. Deoxynucleotide carrier*

via MIM would lead to a reduction in the membrane potential and ATP formation [1]. The relative impermeability of the MIM is the basis of chemiosmotic hypothesis proposed by Mitchell. As discussed in O'Rourke [90], Mitchell recognized three modes of ion transport. Symporters cotransport multiple ions (or an ion and a metabolite) in the same direction across the membrane often utilizing the asymmetric electrochemical ion gradient to drive the trans-

Antiporters exchange ions on different sides of the membrane. Antiporters can be electro-

For electrogenic transporters, ion flux is driven by both the electrochemical gradients of the transported ions and the membrane potential. For uniporters, the transport rates are in the

the rate of decay of the pH gradient induced by a pulse of oxygen. This suggested the presence of anion transport systems coupled to proton movement, leading to the identification of the anion/metabolite-coupled cotransporter family. Inner membrane anion uniporters have been less well studied, but in the 1980s, an inner membrane anion channel was postulated to account for anion-selective mitochondrial swelling responses [92]. Moreover, some mitochondrial membrane proteins (e.g., mitochondrial uncoupling protein) were identified to display anion channel activity [90]. Based on the research, seven metabolite-specific mitochondrial transporters or carriers were proposed. Studies of amino acid sequence composition showed that the carriers form a well-defined family (in humans known as the solute carrier 25 family (SLC25)), with the one defining feature, a tripartite structure of three homologous sequence repeats of about 100 amino acid residues each, which was first noted in the published sequence of the bovine ADP/ATP carrier [93]. A signature motif containing P-X-[D/E]-X-X-[R/K] sequence is conserved in all members and in all three sequence repeats [94]. According to typical sequence repeats and signature motif, eukaryotic mitochondria were found to contain 35–55 different carriers when compared to genomic DNA database [95]. The human genome encodes 48 members of the SLC25 family, of which 30 are identified [96]. The isoforms of carrier members are encoded by different genes, and only the phosphate carrier has two alternatively spliced isoforms [97].

Several proteins have been identified as carriers for purine nucleotides, their analogues, as well as pyrimidine nucleotides. The ANT was identified by Kramer and Klingenberg [98]. Other specific proteins as GTP/GDP carriers, peroxisomal adenine nucleotide transporter,

Deoxynucleotide carrier ((DNC) SLC25A19) transports all dNDPs in exchange for ATP or ADP [103]. The protein was later identified as thiamine pyrophosphate carrier (TPC) [104], transporting thiamine pyrophosphate, thiamine monophosphate, and deoxynucleotides in descending order of potency dNDP > dNTP > dNMP. The protein is also capable of nucleotide transport, though less efficiently. The protein in ubiquitously expressed within tissues, with

ions s−1, based on ions flowing down their electrochemical gradient.

antiporter of the mitochondrial or plasma membrane) or electrogenic.

/H+

, succinate, and malonate, accelerated

carrier.

port in a thermodynamically favorable direction, as for example mitochondrial Pi

neutral (the Na+

260 Mitochondrial Diseases

range of 104

/H+

**6.1. Mitochondrial nucleotide transporter**

the highest levels in the kidney and lung [103].

*6.1.1. ADP/ATP translocase*

CoA and S-adenosylmethionine transporters belong in [99–102].

Mitchell and Moyle [91] reported that anions, including Pi

–106

Four ANT isoforms are encoded in human genome on the chromosome X. ANT1–3 are structurally similar and proteins are about 90% identical, and ANT4 only shares 66–68% consistency in the amino acid composition with other isoforms. Isoforms are specifically expressed in different types of cells and tissues. ANT1 (SLC25A4) is expressed in the skeletal muscle, brain, and heart. ANT2 (SLC25A5) is expressed in the liver and proliferating cells and is overexpressed in various types of cancer cell lines. ANT3 (SLC25A6) is ubiquitous in all tissues, and ANT4 (SLC25A31) is specific to the testis and germ cells [105]. The translocase is highly selective of the adenine nucleotide and provides a continuous shift of ADP to the mitochondria required to maintain oxidative phosphorylation and membrane potential. ANT is also implicated in leakage of protons and inducible proton leakage [106, 107].

Impaired translocase activity affects the energy metabolism of the cell by decreasing mitochondrial ATP synthesis and increasing mitochondrial membrane potential [108], thus contributing to the promotion of apoptosis. The rate-limiting factor of apoptosis is mtPTP formation, which is actually increased permeabilization of the mitochondrial membrane for all the solvents up to 1.5 kDa. It is a nonspecific pore, where ANT, VDAC, cyclophilin D, hexokinase, creatine kinase, and peripheral benzodiazepine receptor are effective but not as direct components or core structures. Moreover, there is an evidence for apoptosis regulators of the Bcl-2 family, Bak and Bax, requirement for mtPTP-dependent MOM permeabilization [109]. PTP opening is linked to mitochondrial dysfunction because its occurrence leads to the set of consequences that will arise, as mitochondrial depolarization, cessation of ATP synthesis, Ca2+ release, pyridine nucleotide depletion, inhibition of respiration and matrix swelling, MOM rupture, and release of pro-apoptotic proteins such as cytochrome c, endonuclease G, and AIF [110, 111]. Detrimental effects are seen for long-lasting mtPTP opening, while shortterm effects are involved in physiological regulation of Ca2+ and ROS homeostasis [112, 113].

Cancer cells are able to survive suppression of mitochondrial oxidative phosphorylation under hypoxic conditions through higher rate of glycolysis; however, it depends on ATP uptake especially for mitochondrial potential generation and Ca2+ exchange [114]. The expression of ANT isoforms is related to the adaptation of metabolic properties of cancer cells. ANT2 is overexpressed in various types of human cancer cells and in several hormone-dependent cancers [115, 116]. It was found that ANT2 proves properties allowing the import of ATP into mitochondria (in coexpression with hexokinase II and a subunit of mitochondrial F0F1-ATPase, ATPsynβ), increased glycolysis, maintaining mitochondrial membrane potential, and finally prevention of apoptosis [115]. The effect of hexokinase has been described in Section 4.1. Similarly, the ATP synthasome, a complex of ANT, F0F1-ATPase, and phosphate carrier (PiC), facilitates a mechanism for adenine nucleotide and pyrophosphate release. Under pathological conditions, the imported ATP may also be hydrolyzed by F0F1-ATPase to maintain mitochondrial membrane potential [115]. This type of hydrolysis has also been reported in order to prevent neurodegeneration [117] and in activated macrophages [118].

**6.3. Mitochondrial pyruvate carrier**

concurrently with oxidation of NADH to NAD+

production in glycolysis by regenerating NAD+

of one proton, or exchange with one hydroxide ion [137].

tion is limited when NAD+

Important one is another of the mentioned carriers. Pyruvate is the end product of glycolysis in the cytosol. In mitochondria, pyruvate entering the tricarboxylic acid cycle supports the ATP generation but also serves as a link to anabolic pathways for lipid, amino acid biosynthesis, and gluconeogenesis. The main sources of pyruvate in the cytoplasm are reactions catalyzed by pyruvate kinase. Two more sources are lactate dehydrogenase (LDH) and alanine aminotransferase (ALT), which are important to mention in terms of linking metabolic pathways between tissues. Reversible transamination of pyruvate and glutamate to alanine and α-ketoglutarate catalyzed by ALT converts the pyruvate from muscles into a transport form, alanine, which is reused in the liver for gluconeogenesis. LDH reversibly catalyzes reduction of pyruvate to lactate

Nuclear Encoded Mitochondrial Proteins in Metabolite Transport and Oxidation Pathway…

tissue in conditions requiring excessive energy production and in cancer cells. During increased need for ATP for muscle activity, the energy requirements to support continued muscle activity

dosis becomes limiting, glycolysis can produce ATP to support work rates exceeding those that could be supported by oxidative phosphorylation alone [132]. The lactate is transported to the circulatory system from where it is taken up by the liver and converted back into pyruvate.

The cross-connection of pyruvate with catabolic and anabolic pathway in mitochondria depends on its passage through mitochondrial membranes. Pyruvates cross MOM through VDAC; however, transport through MIM requires specific carrier. Although the existence of carrier was known earlier, the existence of genes on chromosome 6q27 encoding of mitochondrial pyruvate carrier (MPC) formed by hetero-oligomeric complex of two proteins, MPC1 and MPC2, has been revealed recently. Both proteins are needed for sufficient activity [133, 134]. Proteins do not contain any sequence homology to other mitochondrial carriers. Instead, they have been proposed to belong to the PQ-loop/MtN3/MPC superfamily [135]. Members of PQ-loop family are located in a variety of organelles performing diverse functions. They combine common characteristic features that are seven transmembrane domains and two conserved glutamine motifs. Subunits MPC1 and 2 contain three of seven transmembrane domains and are only half size of other PQ-loop family members [136]. Pyruvate uptake has been proposed to be coupled with the electrochemical gradient, occurring with the symport

MPC activity increases in response to glucagon and decreases in response to insulin. Adrenaline and cortisol also have been found to increase pyruvate carboxylation by increasing mitochondrial pyruvate import [138]. The MPC2 transcript levels have been found to increase up to 1.5-fold under fasting conditions. In a physiological response to fasting (e.g., excessive exercise or prolonged food deprivation), hepatic mitochondrial ketone export and pyruvate import through MPC allows to enhance hepatic gluconeogenesis and maintains membrane potential [132]. Conversely, unregulated rate of gluconeogenesis contributes to chronic hyperglycemia in diabetes. Except for some substances (e.g., α-cyano-4-hydroxy cinnamate, UK-5099, and several thiazolidinediones) acting as specific MPC inhibitors, inhibitory

exceed mitochondrial capacity for ATP production. As glycolysis requires NAD+

. Enzyme gains importance especially in muscle

http://dx.doi.org/10.5772/intechopen.72937

depletes and NADH accumulates. In this case, LDH ensures ATP

. With a steady supply of NAD<sup>+</sup>

, ATP produc-

263

, and until aci-

Roussel et al. [119] found isoforms ANT1 and 2 to mediate uncoupling by fatty acids and to lower mitochondrial membrane potential in heart and skeletal muscle in rats. ANT is inhibited by competitive displacement of the nucleotide by long-chain acyl-carnitines (LCAC) [120]. LCAC may accumulate under pathological conditions with excess lipid supply, obesity, and mitochondrial β-oxidation defects [121]. ANT inhibition is thought to contribute to mitochondrial defects in metabolic syndrome [122].

The ANT function in subcellular compartment energy supply is important and therefore it is not surprising that the altered structure and subsequently properties of this solute carrier protein associated with DNA mutations are also associated with serious clinical conditions. Mutations linked to mitochondrial disorders with autosomal recessive inheritance cause mitochondrial myopathy and cardiomyopathy presented in childhood or early adulthood. It is characterized by lactic acidosis, fatigue, proximal muscle weakness, and exercise intolerance [123, 124]. Several different autosomal-dominant mutations in ANT1 gene have been associated with an adulthood-onset disorder, autosomal-dominant progressive external ophthalmoplegia, characterized by ptosis, restriction of eye movement, and accumulation of clonally expanded mtDNA deletions in postmitotic tissues [125, 126]. Thompson et al. [127] have recently described recurrent *de novo*–dominant mutation with severe early-onset of mitochondrial disease. Mutations in the gene encoding ANT1 are associated with the presence of mtDNA deletions. The most likely mechanism of how the defective carrier affects the appearance of mtDNA mutations is the unsufficient adenine nucleotide availability for dATP synthesis and consequent imbalance in dNTP pools [127].
