Biography

The mitochondrion (plural mitochondria) is a double-membrane-bound organelle found in most eukaryotic organisms. Some cells in some multicellular organisms may, however, lack them (for example, mature mammalian red blood cells). A number of unicellular organisms, such as microsporidia, parabasalids, and diplomonads, have also reduced or transformed their mitochondria into other structures.[1] To date, only one eukaryote, Monocercomonoides, is known to have completely lost its mitochondria.[2] The word mitochondrion comes from the Greek μίτος, mitos, "thread", and χονδρίον, chondrion, "granule"[3] or "grain-like". Mitochondria generate most of the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy.[4] Mitochondria are commonly between 0.75 and 3 μm in diameter[5] but vary considerably in size and structure. Unless specifically stained, they are not visible. In addition to supplying cellular energy, mitochondria are involved in other tasks, such as signaling, cellular differentiation, and cell death, as well as maintaining control of the cell cycle and cell growth.[6] Mitochondrial biogenesis is in turn temporally coordinated with these cellular processes.[7][8] Mitochondria have been implicated in several human diseases, including mitochondrial disorders,[9] cardiac dysfunction,[10] heart failure[11] and autism.[12] The number of mitochondria in a cell can vary widely by organism, tissue, and cell type. For instance, red blood cells have no mitochondria, whereas liver cells can have more than 2000.[13][14] The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Although most of a cell's DNA is contained in the cell nucleus, the mitochondrion has its own independent genome that shows substantial similarity to bacterial genomes.[15] Mitochondrial proteins (proteins transcribed from mitochondrial DNA) vary depending on the tissue and the species. In humans, 615 distinct types of protein have been identified from cardiac mitochondria,[16] whereas in rats, 940 proteins have been reported.[17] The mitochondrial proteome is thought to be dynamically regulated.[18] History The first observations of intracellular structures that probably represented mitochondria were published in the 1840s.[19] Richard Altmann, in 1890, established them as cell organelles and called them "bioblasts".[19][20] The term "mitochondria" was coined by Carl Benda in 1898.[19][21] Leonor Michaelis discovered that Janus green can be used as a supravital stain for mitochondria in 1900. In 1904, Friedrich Meves, made the first recorded observation of mitochondria in plants in cells of the white waterlily, Nymphaea alba[19][22] and in 1908, along with Claudius Regaud, suggested that they contain proteins and lipids. Benjamin F. Kingsbury, in 1912, first related them with cell respiration, but almost exclusively based on morphological observations.[19] In 1913, particles from extracts of guinea-pig liver were linked to respiration by Otto Heinrich Warburg, which he called "grana". Warburg and Heinrich Otto Wieland, who had also postulated a similar particle mechanism, disagreed on the chemical nature of the respiration. It was not until 1925, when David Keilin discovered cytochromes, that the respiratory chain was described.[19] In 1939, experiments using minced muscle cells demonstrated that cellular respiration using one oxygen atom can form two adenosine triphosphate (ATP) molecules, and, in 1941, the concept of the phosphate bonds of ATP being a form of energy in cellular metabolism was developed by Fritz Albert Lipmann. In the following years, the mechanism behind cellular respiration was further elaborated, although its link to the mitochondria was not known.[19] The introduction of tissue fractionation by Albert Claude allowed mitochondria to be isolated from other cell fractions and biochemical analysis to be conducted on them alone. In 1946, he concluded that cytochrome oxidase and other enzymes responsible for the respiratory chain were isolated to the mitochondria. Eugene Kennedy and Albert Lehninger discovered in 1948 that mitochondria are the site of oxidative phosphorylation in eukaryotes. Over time, the fractionation method was further developed, improving the quality of the mitochondria isolated, and other elements of cell respiration were determined to occur in the mitochondria.[19] The first high-resolution electron micrographs appeared in 1952, replacing the Janus Green stains as the preferred way of visualising the mitochondria.[19] This led to a more detailed analysis of the structure of the mitochondria, including confirmation that they were surrounded by a membrane. It also showed a second membrane inside the mitochondria that folded up in ridges dividing up the inner chamber and that the size and shape of the mitochondria varied from cell to cell. The popular term "powerhouse of the cell" was coined by Philip Siekevitz in 1957.[23] In 1967, it was discovered that mitochondria contained ribosomes.[24] In 1968, methods were developed for mapping the mitochondrial genes, with the genetic and physical map of yeast mitochondrial DNA being completed in 1976.[19] Origin and evolution Main article: Endosymbiotic theory There are two hypotheses about the origin of mitochondria: endosymbiotic and autogenous. The endosymbiotic hypothesis suggests that mitochondria were originally prokaryotic cells, capable of implementing oxidative mechanisms that were not possible for eukaryotic cells; they became endosymbionts living inside the eukaryote.[25] In the autogenous hypothesis, mitochondria were born by splitting off a portion of DNA from the nucleus of the eukaryotic cell at the time of divergence with the prokaryotes; this DNA portion would have been enclosed by membranes, which could not be crossed by proteins. Since mitochondria have many features in common with bacteria, the endosymbiotic hypothesis is more widely accepted.[25][26] A mitochondrion contains DNA, which is organized as several copies of a single, usually circular, chromosome. This mitochondrial chromosome contains genes for redox proteins, such as those of the respiratory chain. The CoRR hypothesis proposes that this co-location is required for redox regulation. The mitochondrial genome codes for some RNAs of ribosomes, and the 22 tRNAs necessary for the translation of mRNAs into protein. The circular structure is also found in prokaryotes. The proto-mitochondrion was probably closely related to the Rickettsia.[27][28] However, the exact relationship of the ancestor of mitochondria to the alphaproteobacteria and whether the mitochondrion was formed at the same time or after the nucleus, remains controversial.[29] For example, it has been suggested that the SAR11 clade of bacteria shares a relatively recent common ancestor with the mitochondria,[30] while phylogenomic analyses indicates that mitochondria evolved from a proteobacteria lineage that branched off before the divergence of all sampled alphaproteobacteria.[31] The ribosomes coded for by the mitochondrial DNA are similar to those from bacteria in size and structure.[33] They closely resemble the bacterial 70S ribosome and not the 80S cytoplasmic ribosomes, which are coded for by nuclear DNA. The endosymbiotic relationship of mitochondria with their host cells was popularized by Lynn Margulis.[34] The endosymbiotic hypothesis suggests that mitochondria descended from bacteria that somehow survived endocytosis by another cell, and became incorporated into the cytoplasm. The ability of these bacteria to conduct respiration in host cells that had relied on glycolysis and fermentation would have provided a considerable evolutionary advantage. This symbiotic relationship probably developed 1.7 to 2 billion years ago.[35][36] A few groups of unicellular eukaryotes have only vestigial mitochondria or derived structures: the microsporidians, metamonads, and archamoebae.[37] These groups appear as the most primitive eukaryotes on phylogenetic trees constructed using rRNA information, which once suggested that they appeared before the origin of mitochondria. However, this is now known to be an artifact of long-branch attraction—they are derived groups and retain genes or organelles derived from mitochondria (e.g., mitosomes and hydrogenosomes).[1] Monocercomonoides appear to have lost their mitochondria completely and at least some of the mitochondrial functions seem to be carried out by cytoplasmic proteins now.[38] Structure Mitochondrion structure labels.svg Mitochondrion structure 1 Outer membrane 1.1 Porins 2 Intermembrane space 2.1 Intracristal space 2.2 Peripheral space 3 Lamellæ 3.1 Inner membrane 3.11 Inner boundary membrane 3.12 Cristal membrane 3.2 Matrix 3.3 Cristæ 4 Mitochondrial DNA 5 Matix granule 6 Ribosome 7 ATP synthase Edit · Source image Mitochondrion ultrastructure (interactive diagram) A mitochondrion has a double membrane; the inner one contains its chemiosmotic apparatus and has deep grooves which increase its surface area. While commonly depicted as an "orange sausage with a blob inside of it" (like it is here), mitochondria can take many shapes[39] and their intermembrane space is quite thin. A mitochondrion contains outer and inner membranes composed of phospholipid bilayers and proteins.[13] The two membranes have different properties. Because of this double-membraned organization, there are five distinct parts to a mitochondrion. They are: the outer mitochondrial membrane, the intermembrane space (the space between the outer and inner membranes), the inner mitochondrial membrane, the cristae space (formed by infoldings of the inner membrane), and the matrix (space within the inner membrane). Mitochondria stripped of their outer membrane are called mitoplasts. Outer membrane The outer mitochondrial membrane, which encloses the entire organelle, is 60 to 75 angstroms (Å) thick. It has a protein-to-phospholipid ratio similar to that of the eukaryotic plasma membrane (about 1:1 by weight). It contains large numbers of integral membrane proteins called porins. These porins form channels that allow molecules of 5000 daltons or less in molecular weight to freely diffuse from one side of the membrane to the other.[13] Larger proteins can enter the mitochondrion if a signaling sequence at their N-terminus binds to a large multisubunit protein called translocase of the outer membrane, which then actively moves them across the membrane.[40] Mitochondrial pro-proteins are imported through specialised translocation complexes. The outer membrane also contains enzymes involved in such diverse activities as the elongation of fatty acids, oxidation of epinephrine, and the degradation of tryptophan. These enzymes include monoamine oxidase, rotenone-insensitive NADH-cytochrome c-reductase, kynurenine hydroxylase and fatty acid Co-A ligase. Disruption of the outer membrane permits proteins in the intermembrane space to leak into the cytosol, leading to certain cell death.[41] The mitochondrial outer membrane can associate with the endoplasmic reticulum (ER) membrane, in a structure called MAM (mitochondria-associated ER-membrane). This is important in the ER-mitochondria calcium signaling and is involved in the transfer of lipids between the ER and mitochondria. [42] Outside the outer membrane there are small (diameter: 60Å) particles named sub-units of Parson. Intermembrane space The intermembrane space is the space between the outer membrane and the inner membrane. It is also known as perimitochondrial space. Because the outer membrane is freely permeable to small molecules, the concentrations of small molecules, such as ions and sugars, in the intermembrane space is the same as in the cytosol.[13] However, large proteins must have a specific signaling sequence to be transported across the outer membrane, so the protein composition of this space is different from the protein composition of the cytosol. One protein that is localized to the intermembrane space in this way is cytochrome c.[41] Inner membrane Main article: Inner mitochondrial membrane The inner mitochondrial membrane contains proteins with five types of functions:[13] Those that perform the redox reactions of oxidative phosphorylation ATP synthase, which generates ATP in the matrix Specific transport proteins that regulate metabolite passage into and out of the matrix Protein import machinery Mitochondrial fusion and fission protein It contains more than 151 different polypeptides, and has a very high protein-to-phospholipid ratio (more than 3:1 by weight, which is about 1 protein for 15 phospholipids). The inner membrane is home to around 1/5 of the total protein in a mitochondrion.[13] In addition, the inner membrane is rich in an unusual phospholipid, cardiolipin. This phospholipid was originally discovered in cow hearts in 1942, and is usually characteristic of mitochondrial and bacterial plasma membranes.[43] Cardiolipin contains four fatty acids rather than two, and may help to make the inner membrane impermeable.[13] Unlike the outer membrane, the inner membrane doesn't contain porins, and is highly impermeable to all molecules. Almost all ions and molecules require special membrane transporters to enter or exit the matrix. Proteins are ferried into the matrix via the translocase of the inner membrane (TIM) complex or via Oxa1.[40] In addition, there is a membrane potential across the inner membrane, formed by the action of the enzymes of the electron transport chain. Cristae Cross-sectional image of cristae in rat liver mitochondrion to demonstrate the likely 3D structure and relationship to the inner membrane Main article: Cristae The inner mitochondrial membrane is compartmentalized into numerous cristae, which expand the surface area of the inner mitochondrial membrane, enhancing its ability to produce ATP. For typical liver mitochondria, the area of the inner membrane is about five times as large as the outer membrane. This ratio is variable and mitochondria from cells that have a greater demand for ATP, such as muscle cells, contain even more cristae. These folds are studded with small round bodies known as F1 particles or oxysomes. These are not simple random folds but rather invaginations of the inner membrane, which can affect overall chemiosmotic function.[44] One recent mathematical modeling study has suggested that the optical properties of the cristae in filamentous mitochondria may affect the generation and propagation of light within the tissue.[45] Matrix Main article: Mitochondrial matrix The matrix is the space enclosed by the inner membrane. It contains about 2/3 of the total protein in a mitochondrion.[13] The matrix is important in the production of ATP with the aid of the ATP synthase contained in the inner membrane. The matrix contains a highly concentrated mixture of hundreds of enzymes, special mitochondrial ribosomes, tRNA, and several copies of the mitochondrial DNA genome. Of the enzymes, the major functions include oxidation of pyruvate and fatty acids, and the citric acid cycle.[13] Mitochondria have their own genetic material, and the machinery to manufacture their own RNAs and proteins (see: protein biosynthesis). A published human mitochondrial DNA sequence revealed 16,569 base pairs encoding 37 genes: 22 tRNA, 2 rRNA, and 13 peptide genes.[46] The 13 mitochondrial peptides in humans are integrated into the inner mitochondrial membrane, along with proteins encoded by genes that reside in the host cell's nucleus. Mitochondria-associated ER membrane (MAM) Main article: Mitochondria associated membranes (MAM) The mitochondria-associated ER membrane (MAM) is another structural element that is increasingly recognized for its critical role in cellular physiology and homeostasis. Once considered a technical snag in cell fractionation techniques, the alleged ER vesicle contaminants that invariably appeared in the mitochondrial fraction have been re-identified as membranous structures derived from the MAM—the interface between mitochondria and the ER.[47] Physical coupling between these two organelles had previously been observed in electron micrographs and has more recently been probed with fluorescence microscopy.[47] Such studies estimate that at the MAM, which may comprise up to 20% of the mitochondrial outer membrane, the ER and mitochondria are separated by a mere 10–25 nm and held together by protein tethering complexes.[47][42][48] Purified MAM from subcellular fractionation has been shown to be enriched in enzymes involved in phospholipid exchange, in addition to channels associated with Ca2+ signaling.[47][48] These hints of a prominent role for the MAM in the regulation of cellular lipid stores and signal transduction have been borne out, with significant implications for mitochondrial-associated cellular phenomena, as discussed below. Not only has the MAM provided insight into the mechanistic basis underlying such physiological processes as intrinsic apoptosis and the propagation of calcium signaling, but it also favors a more refined view of the mitochondria. Though often seen as static, isolated 'powerhouses' hijacked for cellular metabolism through an ancient endosymbiotic event, the evolution of the MAM underscores the extent to which mitochondria have been integrated into overall cellular physiology, with intimate physical and functional coupling to the endomembrane system. Phospholipid transfer The MAM is enriched in enzymes involved in lipid biosynthesis, such as phosphatidylserine synthase on the ER face and phosphatidylserine decarboxylase on the mitochondrial face.[49][50] Because mitochondria are dynamic organelles constantly undergoing fission and fusion events, they require a constant and well-regulated supply of phospholipids for membrane integrity.[51][52] But mitochondria are not only a destination for the phospholipids they finish synthesis of; rather, this organelle also plays a role in inter-organelle trafficking of the intermediates and products of phospholipid biosynthetic pathways, ceramide and cholesterol metabolism, and glycosphingolipid anabolism.[50][52] Such trafficking capacity depends on the MAM, which has been shown to facilitate transfer of lipid intermediates between organelles.[49] In contrast to the standard vesicular mechanism of lipid transfer, evidence indicates that the physical proximity of the ER and mitochondrial membranes at the MAM allows for lipid flipping between opposed bilayers.[52] Despite this unusual and seemingly energetically unfavorable mechanism, such transport does not require ATP.[52] Instead, in yeast, it has been shown to be dependent on a multiprotein tethering structure termed the ER-mitochondria encounter structure, or ERMES, although it remains unclear whether this structure directly mediates lipid transfer or is required to keep the membranes in sufficiently close proximity to lower the energy barrier for lipid flipping.[52][53] The MAM may also be part of the secretory pathway, in addition to its role in intracellular lipid trafficking. In particular, the MAM appears to be an intermediate destination between the rough ER and the Golgi in the pathway that leads to very-low-density lipoprotein, or VLDL, assembly and secretion.[50][54] The MAM thus serves as a critical metabolic and trafficking hub in lipid metabolism. Calcium signaling A critical role for the ER in calcium signaling was acknowledged before such a role for the mitochondria was widely accepted, in part because the low affinity of Ca2+ channels localized to the outer mitochondrial membrane seemed to fly in the face of this organelle's purported responsiveness to changes in intracellular Ca2+ flux.[47][55] But the presence of the MAM resolves this apparent contradiction: the close physical association between the two organelles results in Ca2+ microdomains at contact points that facilitate efficient Ca2+ transmission from the ER to the mitochondria.[47] Transmission occurs in response to so-called "Ca2+ puffs" generated by spontaneous clustering and activation of IP3R, a canonical ER membrane Ca2+ channel.[47][42] The fate of these puffs—in particular, whether they remain restricted to isolated locales or integrated into Ca2+ waves for propagation throughout the cell—is determined in large part by MAM dynamics. Although reuptake of Ca2+ by the ER (concomitant with its release) modulates the intensity of the puffs, thus insulating mitochondria to a certain degree from high Ca2+ exposure, the MAM often serves as a firewall that essentially buffers Ca2+ puffs by acting as a sink into which free ions released into the cytosol can be funneled.[47][56][57] This Ca2+ tunneling occurs through the low-affinity Ca2+ receptor VDAC1, which recently has been shown to be physically tethered to the IP3R clusters on the ER membrane and enriched at the MAM.[47][42][58] The ability of mitochondria to serve as a Ca2+ sink is a result of the electrochemical gradient generated during oxidative phosphorylation, which makes tunneling of the cation an exergonic process.[58] Normally, mild calcium influx from cytosol into the mitochondrial matrix causes transient depolarization that is corrected by pumping out protons. But transmission of Ca2+ is not unidirectional; rather, it is a two-way street.[55] The properties of the Ca2+ pump SERCA and the channel IP3R present on the ER membrane facilitate feedback regulation coordinated by MAM function. In particular, the clearance of Ca2+ by the MAM allows for spatio-temporal patterning of Ca2+ signaling because Ca2+ alters IP3R activity in a biphasic manner.[47] SERCA is likewise affected by mitochondrial feedback: uptake of Ca2+ by the MAM stimulates ATP production, thus providing energy that enables SERCA to reload the ER with Ca2+ for continued Ca2+ efflux at the MAM.[56][58] Thus, the MAM is not a passive buffer for Ca2+ puffs; rather it helps modulate further Ca2+ signaling through feedback loops that affect ER dynamics. Regulating ER release of Ca2+ at the MAM is especially critical because only a certain window of Ca2+ uptake sustains the mitochondria, and consequently the cell, at homeostasis. Sufficient intraorganelle Ca2+ signaling is required to stimulate metabolism by activating dehydrogenase enzymes critical to flux through the citric acid cycle.[59] However, once Ca2+ signaling in the mitochondria passes a certain threshold, it stimulates the intrinsic pathway of apoptosis in part by collapsing the mitochondrial membrane potential required for metabolism.[47] Studies examining the role of pro- and anti-apoptotic factors support this model; for example, the anti-apoptotic factor Bcl-2 has been shown to interact with IP3Rs to reduce Ca2+ filling of the ER, leading to reduced efflux at the MAM and preventing collapse of the mitochondrial membrane potential post-apoptotic stimuli.[47] Given the need for such fine regulation of Ca2+ signaling, it is perhaps unsurprising that dysregulated mitochondrial Ca2+ has been implicated in several neurodegenerative diseases, while the catalogue of tumor suppressors includes a few that are enriched at the MAM.[58] Molecular basis for tethering Recent advances in the identification of the tethers between the mitochondrial and ER membranes suggest that the scaffolding function of the molecular elements involved is secondary to other, non-structural functions. In yeast, ERMES, a multiprotein complex of interacting ER- and mitochondrial-resident membrane proteins, is required for lipid transfer at the MAM and exemplifies this principle. One of its components, for example, is also a constituent of the protein complex required for insertion of transmembrane beta-barrel proteins into the lipid bilayer.[52] However, a homologue of the ERMES complex has not yet been identified in mammalian cells. Other proteins implicated in scaffolding likewise have functions independent of structural tethering at the MAM; for example, ER-resident and mitochondrial-resident mitofusins form heterocomplexes that regulate the number of inter-organelle contact sites, although mitofusins were first identified for their role in fission and fusion events between individual mitochondria.[47] Glucose-related protein 75 (grp75) is another dual-function protein. In addition to the matrix pool of grp75, a portion serves as a chaperone that physically links the mitochondrial and ER Ca2+ channels VDAC and IP3R for efficient Ca2+ transmission at the MAM.[47][42] Another potential tether is Sigma-1R, a non-opioid receptor whose stabilization of ER-resident IP3R may preserve communication at the MAM during the metabolic stress response.[60][61] ERMES tethering complex. Model of the yeast multimeric tethering complex, ERMES Perspective The MAM is a critical signaling, metabolic, and trafficking hub in the cell that allows for the integration of ER and mitochondrial physiology. Coupling between these organelles is not simply structural but functional as well and critical for overall cellular physiology and homeostasis. The MAM thus offers a perspective on mitochondria that diverges from the traditional view of this organelle as a static, isolated unit appropriated for its metabolic capacity by the cell. Instead, this mitochondrial-ER interface emphasizes the integration of the mitochondria, the product of an endosymbiotic event, into diverse cellular processes. Organization and distribution Typical mitochondrial network (green) in two human cells (HeLa cells) Mitochondria (and related structures) are found in all eukaryotes (except one—the Oxymonad Monocercomonoides sp.).[2][62] Although commonly depicted as bean-like structures they form a highly dynamic network in the majority of cells where they constantly undergo fission and fusion. Mitochondria vary in number and location according to cell type. A single mitochondrion is often found in unicellular organisms. Conversely, numerous mitochondria are found in human liver cells, with about 1000–2000 mitochondria per cell, making up 1/5 of the cell volume.[13] The population of all the mitochondria of a given cell constitutes the chondriome. The mitochondrial content of otherwise similar cells can vary substantially in size and membrane potential,[63] with differences arising from sources including uneven partitioning at cell divisions, leading to extrinsic differences in ATP levels and downstream cellular processes.[64] The mitochondria can be found nestled between myofibrils of muscle or wrapped around the sperm flagellum.[13] Often, they form a complex 3D branching network inside the cell with the cytoskeleton. The association with the cytoskeleton determines mitochondrial shape, which can affect the function as well:[65] different structures of the mitochondrial network may afford the population a variety of physical, chemical, and signalling advantages or disadvantages.[66] Mitochondria in cells are always distributed along microtubules and the distribution of these organelles is also correlated with the endoplasmic reticulum.[67] Recent evidence suggests that vimentin, one of the components of the cytoskeleton, is also critical to the association with the cytoskeleton.[68] Function The most prominent roles of mitochondria are to produce the energy currency of the cell, ATP (i.e., phosphorylation of ADP), through respiration, and to regulate cellular metabolism.[14] The central set of reactions involved in ATP production are collectively known as the citric acid cycle, or the Krebs cycle. However, the mitochondrion has many other functions in addition to the production of ATP. Energy conversion A dominant role for the mitochondria is the production of ATP, as reflected by the large number of proteins in the inner membrane for this task. This is done by oxidizing the major products of glucose: pyruvate, and NADH, which are produced in the cytosol.[14] This type of cellular respiration known as aerobic respiration, is dependent on the presence of oxygen. When oxygen is limited, the glycolytic products will be metabolized by anaerobic fermentation, a process that is independent of the mitochondria.[14] The production of ATP from glucose has an approximately 13-times higher yield during aerobic respiration compared to fermentation.[69] Plant mitochondria can also produce a limited amount of ATP without oxygen by using the alternate substrate nitrite.[70] ATP crosses out through the inner membrane with the help of a specific protein, and across the outer membrane via porins. ADP returns via the same route. Pyruvate and the citric acid cycle Main articles: Pyruvate dehydrogenase, Pyruvate carboxylase, and Citric acid cycle Pyruvate molecules produced by glycolysis are actively transported across the inner mitochondrial membrane, and into the matrix where they can either be oxidized and combined with coenzyme A to form CO2, acetyl-CoA, and NADH,[14] or they can be carboxylated (by pyruvate carboxylase) to form oxaloacetate. This latter reaction ”fills up” the amount of oxaloacetate in the citric acid cycle, and is therefore an anaplerotic reaction, increasing the cycle’s capacity to metabolize acetyl-CoA when the tissue's energy needs (e.g. in muscle) are suddenly increased by activity.[71] In the citric acid cycle, all the intermediates (e.g. citrate, iso-citrate, alpha-ketoglutarate, succinate, fumarate, malate and oxaloacetate) are regenerated during each turn of the cycle. Adding more of any of these intermediates to the mitochondrion therefore means that the additional amount is retained within the cycle, increasing all the other intermediates as one is converted into the other. Hence, the addition of any one of them to the cycle has an anaplerotic effect, and its removal has a cataplerotic effect. These anaplerotic and cataplerotic reactions will, during the course of the cycle, increase or decrease the amount of oxaloacetate available to combine with acetyl-CoA to form citric acid. This in turn increases or decreases the rate of ATP production by the mitochondrion, and thus the availability of ATP to the cell.[71] Acetyl-CoA, on the other hand, derived from pyruvate oxidation, or from the beta-oxidation of fatty acids, is the only fuel to enter the citric acid cycle. With each turn of the cycle one molecule of acetyl-CoA is consumed for every molecule of oxaloacetate present in the mitochondrial matrix, and is never regenerated. It is the oxidation of the acetate portion of acetyl-CoA that produces CO2 and water, with the energy thus released captured in the form of ATP.[71] In the liver, the carboxylation of cytosolic pyruvate into intra-mitochondrial oxaloacetate is an early step in the gluconeogenic pathway, which converts lactate and de-aminated alanine into glucose,[14][71] under the influence of high levels of glucagon and/or epinephrine in the blood.[71] Here, the addition of oxaloacetate to the mitochondrion does not have a net anaplerotic effect, as another citric acid cycle intermediate (malate) is immediately removed from the mitochondrion to be converted into cytosolic oxaloacetate, which is ultimately converted into glucose, in a process that is almost the reverse of glycolysis.[71] The enzymes of the citric acid cycle are located in the mitochondrial matrix, with the exception of succinate dehydrogenase, which is bound to the inner mitochondrial membrane as part of Complex II.[72] The citric acid cycle oxidizes the acetyl-CoA to carbon dioxide, and, in the process, produces reduced cofactors (three molecules of NADH and one molecule of FADH2) that are a source of electrons for the electron transport chain, and a molecule of GTP (that is readily converted to an ATP).[14] NADH and FADH2: the electron transport chain Main articles: Electron transport chain and Oxidative phosphorylation Electron transport chain in the mitochondrial intermembrane space The redox energy from NADH and FADH2 is transferred to oxygen (O2) in several steps via the electron transport chain. These energy-rich molecules are produced within the matrix via the citric acid cycle but are also produced in the cytoplasm by glycolysis. Reducing equivalents from the cytoplasm can be imported via the malate-aspartate shuttle system of antiporter proteins or feed into the electron transport chain using a glycerol phosphate shuttle.[14] Protein complexes in the inner membrane (NADH dehydrogenase (ubiquinone), cytochrome c reductase, and cytochrome c oxidase) perform the transfer and the incremental release of energy is used to pump protons (H+) into the intermembrane space. This process is efficient, but a small percentage of electrons may prematurely reduce oxygen, forming reactive oxygen species such as superoxide.[14] This can cause oxidative stress in the mitochondria and may contribute to the decline in mitochondrial function associated with the aging process.[73] As the proton concentration increases in the intermembrane space, a strong electrochemical gradient is established across the inner membrane. The protons can return to the matrix through the ATP synthase complex, and their potential energy is used to synthesize ATP from ADP and inorganic phosphate (Pi).[14] This process is called chemiosmosis, and was first described by Peter Mitchell[74][75] who was awarded the 1978 Nobel Prize in Chemistry for his work. Later, part of the 1997 Nobel Prize in Chemistry was awarded to Paul D. Boyer and John E. Walker for their clarification of the working mechanism of ATP synthase.[76] Heat production Under certain conditions, protons can re-enter the mitochondrial matrix without contributing to ATP synthesis. This process is known as proton leak or mitochondrial uncoupling and is due to the facilitated diffusion of protons into the matrix. The process results in the unharnessed potential energy of the proton electrochemical gradient being released as heat.[14] The process is mediated by a proton channel called thermogenin, or UCP1.[77] Thermogenin is a 33 kDa protein first discovered in 1973.[78] Thermogenin is primarily found in brown adipose tissue, or brown fat, and is responsible for non-shivering thermogenesis. Brown adipose tissue is found in mammals, and is at its highest levels in early life and in hibernating animals. In humans, brown adipose tissue is present at birth and decreases with age.[77]