Outer mitochondrial membrane within

GMBS or sulfo-GMBS have been used for studying carnitine palmitoyltransferase-1 in its formation of a complex within the outer mitochondrial membrane (Faye et al., 2007), for investigating protein organization of the postsynaptic density (Liu et al., 2006), and in studying the structure and dynamics of rhodopsin (Jacobsen et al., 2006). 

Faye, A., Esnous, C., Price, N.T., Onfray, M.A., Girard, J., and Prip-Buus, C. (2007) Rat liver carnitine palmitoyltransferase 1 forms an oligomeric complex within the outer mitochondrial membrane.. Biol. Cbem. 10.1074/jbc.M705418200. 

Figure 2 Partitioning SODl between the mitochondria and cytosol via the action of CCS. The double line represents the outer membrane of the mitochondria. Light arrows indicate maturation steps in the metallation of SODl, whereas heavy arrows indicate movement of SODl across the outer mitochondrial membrane. The apo or unmetallated form of SODl can readily traverse the membrane in either direction. Once copper is inserted via CCS, SODl can no longer cross the membrane and becomes trapped within the corresponding compartment, either the cytosol or intermembrane space (IMS) of the mitochondria as indicated. This model shows zinc loading of SODl prior to copper metallation, but the exact order by which this occurs in vivo is not clear...

In muscle, most of the fatty acids undergoing beta oxidation are completely oxidized to C02 and water. In liver, however, there is another major fate for fatty acids this is the formation of ketone bodies, namely acetoacetate and b-hydroxybutyrate. The fatty acids must be transported into the mitochondrion for normal beta oxidation. This may be a limiting factor for beta oxidation in many tissues and ketone-body formation in the liver. The extramitochondrial fatty-acyl portion of fatty-acyl CoA can be transferred across the outer mitochondrial membrane to carnitine by carnitine palmitoyltransferase I (CPTI). This enzyme is located on the inner side of the outer mitochondrial membrane. The acylcarnitine is now located in mitochondrial intermembrane space. The fatty-acid portion of acylcarnitine is then transported across the inner mitochondrial membrane to coenzyme A to form fatty-acyl CoA in the mitochondrial matrix. This translocation is catalyzed by carnitine palmitoyltransferase II (CPTII Fig. 14.1), located on the inner side of the inner membrane. This later translocation is also facilitated by camitine-acylcamitine translocase, located in the inner mitochondrial membrane. The CPTI is inhibited by malonyl CoA, an intermediate of fatty-acid synthesis (see Chapter 15). This inhibition occurs in all tissues that oxidize fatty acids. The level of malonyl CoA varies among tissues and with various nutritional and hormonal conditions. The sensitivity of CPTI to malonyl CoA also varies among tissues and with nutritional and hormonal conditions, even within a given tissue. Thus, fatty-acid oxidation may be controlled by the activity and relative inhibition of CPTI. 

More detailed studies indicate that such mitochondrial fractions are likely to contain another resolvable compartment, the mitochondria-associated membrane (MAM) that appears to be a specialized domain of the ER (J.E. Vance, 1990). Evidence obtained using CHO-Kl cells (Y. Shiao, 1995) indicates that nascent PE (made via CDP-ethanolamine) is transported to the MAM but not to the inner mitochondrial membrane. It remains unclear whether some of this PE is transported to the outer mitochondrial membrane. The results are consistent with little import of PE derived from ethanolamine into mitochondria. Furthermore, yeast mutants lacking a functional allele for PS decarboxylase 1 (psdIA strains) are markedly deficient in mitochondrial PE (P.J. Trotter, 1995). The reduced PE in mitochondria cannot be fully restored by PE synthesized in the ER from an ethanolamine precursor, or that made in the Golgi or vacuole by PS decarboxylase 2 (R. Bimer, 2001 M. Storey, 2001). These latter findings clearly demonstrate that there is compartmentation and restricted transport of different pools of PE within cells. 

Carnitine palmitoyltransferase I (CPTI also called carnitine acyltransferase I, CATI), the enzyme that transfers long-chain fatty acyl groups from CoA to carnitine, is located on the outer mitochondrial membrane (Fig. 23.5). Fatty acylcamitine crosses the inner mitochondrial membrane with the aid of a translocase. The fatty acyl group is transferred back to CoA by a second enzyme, carnitine palmitoyl-transferase II (CPTII or CATII). The carnitine released in this reaction returns to the cytosolic side of the mitochondrial membrane by the same translocase that brings fatty acylcamitine to the matrix side. Long-chain fatty acyl CoA, now located within the mitochondrial matrix, is a substrate for (3-oxidation. 

Within the peroxisome, the acetyl groups can be transferred from CoA to carnitine by an acetylcarnitine transferase, or they can enter the cytosol. A similar reaction converts medium-chain-length acyl CoAs and the short-chain butyryl CoA to acyl carnitine derivatives. These acylcarnitines diffuse from the peroxisome to the mitochondria, pass through the outer mitochondrial membrane, and are transported through the inner mitochondrial membrane via the carnitine translocase system. 

Within the liver, they bind to fatty acid-binding proteins and are then activated on the outer mitochondrial membrane, the peroxisomal membrane, and the smooth endoplasmic reticulum by fatty acyl CoA synthetases. The fatty acyl group is transferred from CoA to carnitine for transport through the inner mitochondrial membrane, where it is reconverted back into fatty acyl CoA and oxidized to acetyl CoA in the (3-oxidation spiral (see Chapter 23). 

A key event in preventing apoptosis is thus the retention of cytochrome c within mitochondria. The permeability transition pore complex is formed between the inner and outer mitochondrial membranes and is reported to control protein release from the intermembrane space. The permeabihty transition pore complex comprises the adenine nucleotide transporter, the voltage-dependent anion channel and possibly other proteins such as the benzodiazepine receptor and cyclophilin D [65]. Thus, cells possess specialised systems and processes for retaining cytochrome c within mitochondria to ensure survival, as well as systems that can rapidly mobilise this molecule when the apoptotic pathway is triggered. 

It would also seem of value to explore further the molecular basis for the action of neurogenic amines capable of serving as transmitters at the neuromuscular or intersynaptic regions. It is known that these amines are catabolized (with superoxide production) within the outer mitochondrial membrane. The now well-recognized occurrence of flavin-linked superoxide-generating redox reactions within cellular plasma membranes raises the possibility that amine-induced signal transmission may be dependent on superoxide-linked transmitter metabolism after the transmitter binds to specific receptor sites. 

Freshly prepared mitochondria contain ascorbate, as do mitoplasts, that lack the outer mitochondrial membrane (Li etal. 2001). Both mitochondria and mitoplasts rapidly take up oxidised ascorbate as dehydroascorbic acid and reduce it to ascorbate. Ascorbate concentrations in mitochondria and mitoplasts rise into the low micromolar range during dehydroascorbic acid uptake, although uptake and reduction are opposed by ascorbate efflux. Mitochondrial dehydroascorbic acid reduction depends mainly on GSH, but mitochondrial thioredoxin reductase may also contribute. Reactive oxygen species generated within mitochondria oxidise ascorbate more readily than they do GSH and a-tocopherol. 

Carnitine serves as a cofactor for several enzymes, including carnitine translo-case and acyl carnitine transferases I and II, which are essential for the movement of activated long-chain fatty acids from the cytoplasm into the mitochondria (Figure 11.2). The translocation of fatty acids (FAs) is critical for the genaation of adenosine triphosphate (ATP) within skeletal muscle, via 3-oxidation. These activated FAs become esterified to acylcamitines with carnitine via camitine-acyl-transferase I (CAT I) in the outer mitochondrial membrane. Acylcamitines can easily permeate the membrane of the mitochondria and are translocated across the membrane by carnitine translocase. Carnitine s actions are not yet complete because the mitochondrion has two membranes to cross thus, through the action of CAT II, the acylcar-nitines are converted back to acyl-CoA and carnitine. Acyl-CoA can be used to generate ATP via 3-oxidation, Krebs cycle, and the electron transport chain. Carnitine is recycled to the cytoplasm for fumre use. 

The long chain acyl-CoA synthetases are firmly membrane bound and can only be solubilized by the use of detergents. Within the cell, activity has been detected in endoplasmic reticulum and the outer mitochondrial membrane with small amounts in peroxisomes (when the latter are present). There is some dispute as to whether the activity present in mitochondrial and microsomal fractions is due to the same enzyme. Because long chain fatty acid activation is needed for both catabolism ( -oxidation) and for synthesis (acylation of complex lipids) it would be logical if the long chain acyl-CoA synthetases of mitochondria and the endoplasmic reticulum formed different pools of cellular acyl-CoA. This compartmentation has been demonstrated with yeast mutants where it plays a regulatory role in lipid metabolism (section 3.2.7) and, perhaps, in other organisms. 

A second very important eukaryotic organelle is the mitochondrion, which, like the nucleus, has a double membrane (Figure 1.13). The outer membrane has a fairly smooth surface, but the inner membrane exhibits many folds called cristae. The space within the inner membrane is called the matrix. Oxidation processes that occur in mitochondria yield energy for the cell. Most of the enzymes responsible for these important reactions are associated with the inner mitochondrial membrane. Other enzymes needed for oxidation reactions, as well as DNA that differs from that found in the nucleus, are found in the internal mitochondrial matrix. Mitochondria also contain ribosomes similar to those found in bacteria. Mitochondria are approximately the size of many bacteria, typically about 1 pm in diameter and 2 to 8 pm in length. In theory, they may have arisen from the absorption of aerobic bacteria by larger host cells. 

The third complex is an integral part of the inner mitochondrial membrane. Goenzyme Q is soluble in the lipid component of the mitochondrial membrane. It is separated from the complex in the fractionation process that resolves the electron transport apparatus into its component parts, but the coenzyme is probably close to respiratory complexes in the intact membrane (Figure 20.8). Gytochrome c itself is not part of the complex but is loosely bound to the outer surface of the inner mitochondrial membrane, facing the intermembrane space. It is noteworthy that these two important electron carriers, coenzyme Q and cytochrome c, are not part of die respiratory complexes but can move freely in the membrane. The respiratory complexes themselves move within the membrane (recall lateral motion within membranes from Section 8.3), and electron transport occurs when one complex encounters the next complex in the respiratory chain as they move. 

The protons are released to one side of an otherwise generally proton-impermeable inner mitochondrial membrane to collect the protons in the space between the inner and outer membranes of the mitochondrion. The resulting proton concentration gradient then drives formation of ATP by the quintessential protein-based machine, ATP synthase, as the protons flow back through the inner mitochondrial membrane by means of another special path effecting proton permeability. Thus there are two fundamental questions. The first is, how does electron flow within the membrane achieve unidirectional proton flow across the membrane The second is, how does the return flow of protons result in the formation of ATP, the energy coin of biology ... 

Figure 8.5. The mitochondrion, the energy factory of the cell. (Top) Electron micrographs (A),(B),and (C) of the inner mitochondrial membrane studded with stalks and headpieces that are the extramembrane components of ATP synthase with the remainder contained within the inner membrane. (Bottom) Drawing of a mitochondrion with an outer membrane and a folded inner mitochondrial membrane enclos-...

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