Mitochondrial Membrane-Transport Systems

As examples of inner mitochondrial membrane functions, several transport systems have recently been analyzed in our laboratory. 

---

Cycles in the Function of Mitochondrial Membrane Transport Systems Albert L. Lehninger and Baltazar Reynafarje... 

In conclusion, it is very likely that pantethine stimulated fatty acid oxidation through activation of fatty acyl CoA synthetase after conversion to phosphopantetheine and through stimulation of the mitochondrial membrane transport system of acyl CoA which is the key step of fatty acid oxidation [ 12 ]. Further studies are now in progress along this line. 

The oxaloacetate formed in the mitochondrial matrix is initially reduced to ma-late, which can leave the mitochondria via inner membrane transport systems (see p. 212). 

Figure 15.11b shows the malate/aspartate shuttle system, which is particularly active in liver and heart. It uses malate, aspartate, and oxaloacetate to shuttle cytoplasmic electrons from NADH into the mitochondrial matrix. In this shuttle, NADH reduces oxaloacetate to malate, which travels through an inner membrane transport system that ultimately exchanges the malate for an ot-ketoglutarate. To do... 

FAD is bound covalently to the enzyme protein through a specific histidine residue. Succinate dehydrogenase is tightly bound to the mitochondrial inner membrane. The importance of this binding is that the reduced flavin, which must be reoxidized for the enzyme to act again, becomes reoxidized through interaction with the mitochondrial electron transport system, also bound to the membrane. 

These enzymes contain FAD, and the reduced coenzyme FADH2 that is formed is reoxidized by an electron transferring flavoprotein (Chapter 15), which also contains FAD. This protein carries the electrons abstracted in the oxidation process to the inner membrane of the mitochondrion where they enter the mitochondrial electron transport system, as depicted in Fig. 10-5 and as discussed in detail in... 

Not all endogenous membrane transport systems are affected by the herbicides. For example, spontaneous mitochondrial swelling in isotonic solutions of ammonium phosphate or neutral amino acids, was affected only marginally by the compounds (data not shown). Swelling in these systems involves the endogenous Pi /0H anti-porter (26) and amino acid porter (27), respectively. At this... 

The existence of membrane-bound enzymes has been known for many years and it has been proposed that most particulate enzymes are associated with the lipid matrix of biological membranes. Fleischer Klouwen (1961) were the first to show the lipid requirement in enz3nne reactions of the mitochondrial electron transport system. It is now recognized that there are a number of membrane-bound enzymes which require phospholipid for function. Any lipid-requiring enzyme must fulfill two minimal experimental criteria for proof of lipid involvement, namely (a) removal of lipid by mild procedures result in loss of enzymic activity, and (b) the activity is restored when lipids are added in a suitable physical state to the delipidized enz3mies. Point (b) entails the demonstration that activity is restored after rebinding of phospholipid to the enzyme. 

As with Complex 1, passage of electrons through the Q cycle of Complex 111 is accompanied by proton transport across the inner mitochondrial membrane. The postulated pathway for electrons in this system is shown in Figure 21.12. A large pool of UQ and UQHg exists in the inner mitochondrial membrane. The Q cycle is initiated when a molecule of UQHg from this pool diffuses to a site (called Q, ) on Complex 111 near the cytosolic face of the membrane. 

It should be emphasized here that the four major complexes of the electron transport chain operate quite independently in the inner mitochondrial membrane. Each is a multiprotein aggregate maintained by numerous strong associations between peptides of the complex, but there is no evidence that the complexes associate with one another in the membrane. Measurements of the lateral diffusion rates of the four complexes, of coenzyme Q, and of cytochrome c in the inner mitochondrial membrane show that the rates differ considerably, indicating that these complexes do not move together in the membrane. Kinetic studies with reconstituted systems show that electron transport does not operate by means of connected sets of the four complexes. 

Most of the NADH used in electron transport is produced in the mitochondrial matrix space, an appropriate site because NADH is oxidized by Complex I on the matrix side of the inner membrane. Furthermore, the inner mitochondrial membrane is impermeable to NADH. Recall, however, that NADH is produced in glycolysis by glyceraldehyde-3-P dehydrogenase in the cytosol. If this NADH were not oxidized to regenerate NAD, the glycolytic pathway would cease to function due to NAD limitation. Eukaryotic cells have a number of shuttle systems that harvest the electrons of cytosolic NADH for delivery to mitochondria without actually transporting NADH across the inner membrane (Figures 21.33 and 21.34). 

Figure 12-10. Transporter systems in the inner mitochondrial membrane. , phosphate transporter ...

Carnitine (p-hydroxy-y-trimethylammonium butyrate), (CHjljN"—CH2—CH(OH)—CH2—COO , is widely distributed and is particularly abundant in muscle. Long-chain acyl-CoA (or FFA) will not penetrate the inner membrane of mitochondria. However, carnitine palmitoyltransferase-I, present in the outer mitochondrial membrane, converts long-chain acyl-CoA to acylcarnitine, which is able to penetrate the inner membrane and gain access to the P-oxidation system of enzymes (Figure 22-1). Carnitine-acylcar-nitine translocase acts as an inner membrane exchange transporter. Acylcarnitine is transported in, coupled with the transport out of one molecule of carnitine. The acylcarnitine then reacts with CoA, cat-... 

Defects of mitochondrial transport interfere with the movement of molecules across the inner mitochondrial membrane, which is tightly regulated by specific translocation systems. The carnitine cycle is shown in Figure 42-2 and is responsible for the translocation of acyl-CoA thioesters from the cytosol into the mitochondrial matrix. The carnitine cycle involves four elements the plasma membrane carnitine transporter system, CPT I, the carnitine-acyl carnitine translocase system in the inner mitochondrial membrane and CPT II. Genetic defects have been described for each of these four steps, as discussed previously [4,8,9]. 

To explain how H+ transfer occurred across the membrane Mitchell suggested the protons were translocated by redox loops with different reducing equivalents in their two arms. The first loop would be associated with flavoprotein/non-heme iron interaction and the second, more controversially, with CoQ. Redox loops required an ordered arrangement of the components of the electron transport system across the inner mitochondrial membrane, which was substantiated from immunochemical studies with submitochondrial particles. Cytochrome c, for example, was located at the intermembranal face of the inner membrane and cytochrome oxidase was transmembranal. The alternative to redox loops, proton pumping, is now known to be a property of cytochrome oxidase. 

---