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Cell_Biology_Alberts. Control of membrane traffic thus has a major role in maintaining the composition of the various membranes of the cell. To maintain each membrane-enclosed compartment in the secretory and endocytic pathways at a constant size, the balance between the outward and inward flows of membrane needs to be precisely regulated. For cells to grow, however, the forward flow needs to be greater than the retrograde flow, so that the membrane can increase in area. For cells to maintain a constant size, the forward and retrograde flows must be equal. We still know very little about the mechanisms that coordinate these flows. some regulated exocytosis events serve to enlarge the plasma Membrane


Cell_Biology_Alberts

Cell_Biology_Alberts. some regulated exocytosis events serve to enlarge the plasma Membrane An important task of regulated exocytosis is to deliver more membrane to enlarge the surface area of a cell’s plasma membrane when such a need arises. A spectacular example is the plasma membrane expansion that occurs during the cellularization process in a fly embryo, which initially is a syncytium—a single cell containing about 6000 nuclei surrounded by a single plasma membrane (see Figure 21–15). Within tens of minutes, the embryo is converted into the same number of cells. This process of cellularization requires a vast amount of new plasma membrane, which is added by a carefully orchestrated fusion of cytoplasmic vesicles, eventually forming the plasma membranes that enclose the separate cells. Similar vesicle fusion events are required to enlarge the plasma membrane when other animal cells or plant cells divide during cytokinesis (discussed in Chapter 17).


Cell_Biology_Alberts

Cell_Biology_Alberts. Many animal cells, especially those subjected to mechanical stresses, frequently experience small ruptures in their plasma membrane. In a remarkable process thought to involve both homotypic vesicle–vesicle fusion and exocytosis, a temporary cell-surface patch is quickly fashioned from locally available internal-membrane sources, such as lysosomes. In addition to providing an emergency barrier against leaks, the patch reduces membrane tension over the wounded area, allowing the bilayer to flow back together to restore continuity and seal the puncture. This membrane repair process, the fusion and exocytosis of vesicles is triggered by the sudden increase of Ca2+, which is abundant in the extracellular space and rushes into the cell as soon as the plasma membrane is punctured. Figure 13–70 shows four examples in which regulated exocytosis leads to plasma membrane expansion. polarized cells direct proteins from the Trans golgi network to the appropriate domain of the plasma Membrane


Cell_Biology_Alberts

Cell_Biology_Alberts. polarized cells direct proteins from the Trans golgi network to the appropriate domain of the plasma Membrane Most cells in tissues are polarized, with two or more molecularly and functionally distinct plasma membrane domains. This raises the general problem of how the Figure 13–70 Four examples of regulated exocytosis leading to plasma membrane enlargement. The vesicles fusing with the plasma membrane during cytokinesis (a) and phagocytosis (b) are thought to be derived from endosomes, whereas those involved in wound repair (c) may be derived from plasma membranes. The vast amount of new plasma membrane inserted during cellularization in a fly embryo occurs by the fusion of cytoplasmic vesicles (d).


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Cell_Biology_Alberts. delivery of membrane from the Golgi apparatus is organized so as to maintain the differences between one cell-surface domain and another. A typical epithelial cell, for example, has an apical domain, which faces either an internal cavity or the outside world and often has specialized features such as cilia or a brush border of microvilli. It also has a basolateral domain, which covers the rest of the cell. The two domains are separated by a ring of tight junctions (see Figure 19–21), which prevent proteins and lipids (in the outer leaflet of the lipid bilayer) from diffusing between the two domains, so that the differences between the two domains are maintained.


Cell_Biology_Alberts

Cell_Biology_Alberts. In principle, differences between plasma membrane domains need not depend on the targeted delivery of the appropriate membrane components. Instead, membrane components could be delivered to all regions of the cell surface indiscriminately but then be selectively stabilized in some locations and selectively eliminated in others. Although this strategy of random delivery followed by selective retention or removal seems to be used in certain cases, deliveries are often specifically directed to the appropriate membrane domain. Epithelial cells lining the gut, for example, secrete digestive enzymes and mucus at their apical surface and components of the basal lamina at their basolateral surface. Such cells must have ways of directing vesicles carrying different cargoes to different plasma membrane domains. Proteins from the ER destined for different domains travel together until they reach the TGN, where they are separated and dispatched in secretory or transport vesicles to the


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Cell_Biology_Alberts. membrane domains. Proteins from the ER destined for different domains travel together until they reach the TGN, where they are separated and dispatched in secretory or transport vesicles to the appropriate plasma membrane domain (Figure 13–71).


Cell_Biology_Alberts

Cell_Biology_Alberts. The apical plasma membrane of most epithelial cells is greatly enriched in glycosphingolipids, which help protect this exposed surface from damage—for example, from the digestive enzymes and low pH in sites such as the gut or stomach, respectively. Similarly, plasma membrane proteins that are linked to the lipid bilayer by a GPI anchor (see Figure 12–52) are found predominantly in the apical plasma membrane. If recombinant DNA techniques are used to attach a GPI anchor to a protein that would normally be delivered to the basolateral surface, the protein is usually delivered to the apical surface instead. GPI-anchored proteins are thought to be directed to the apical membrane because they associate with glycosphingolipids in lipid rafts that form in the membrane of the TGN. As discussed in Chapter 10, lipid rafts form in the TGN and plasma membrane when glycosphingolipids and cholesterol molecules self-associate (see Figure 10–13).


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 13–71 Two ways of sorting plasma membrane proteins in a polarized epithelial cell. (a) In the direct pathway, proteins destined for different plasma membrane domains are sorted and packaged into different transport vesicles. The lipid-raft-dependent delivery system to the apical domain described in the text is an example of the direct pathway. (b) In the indirect pathway, a protein is retrieved from the inappropriate plasma membrane domain by endocytosis and then transported to the correct domain via early endosomes—that is, by transcytosis. The indirect pathway, for example, is used in liver hepatocytes to deliver proteins to the apical domain that lines bile ducts.


Cell_Biology_Alberts

Cell_Biology_Alberts. Having selected a unique set of cargo molecules, the rafts then bud from the TGN whaT we don’T know into transport vesicles destined for the apical plasma membrane. This process is similar to the selective partitioning of some membrane proteins into the special-• how are targeting and fusion ized lipid domains in caveolae at the plasma membrane discussed earlier. proteins such as snares regulated, Membrane proteins destined for delivery to the basolateral membrane con-so that they can be returned to their tain sorting signals in their cytosolic tail. When present in an appropriate struc-respective donor compartments in an inactive state?


Cell_Biology_Alberts

Cell_Biology_Alberts. tural context, these signals are recognized by coat proteins that package them into appropriate transport vesicles in the TGN. The same basolateral signals that are recognized in the TGN also function in early endosomes to redirect the proteins and endocytic events to keep its back to the basolateral plasma membrane after they have been endocytosed. plasma membrane a constant size? summary • can newly formed daughter cells generate a golgi apparatus de novo, or do they have to inherit it? ion. Whereas the regulated pathways operate only in specialized secretory cells, a constitutive secretory pathway operates in all eukaryotic cells, characterized by continual vesicle transport from the TGN to the plasma membrane. In the regulated their own membranes?


Cell_Biology_Alberts

Cell_Biology_Alberts. pathways, the molecules are stored either in secretory vesicles or in synaptic vesicles, which do not fuse with the plasma membrane to release their contents until they • how does a cell maintain the receive an appropriate signal. Secretory vesicles containing proteins for secretion right amount of every component bud from the TGN. The secretory proteins become concentrated during the forma-(organelles, molecules), and how does tion and maturation of the secretory vesicles. Synaptic vesicles, which are confined it change these amounts as needed to nerve cells and some endocrine cells, form from both endocytic vesicles and from (for example, to greatly expand the endoplasmic reticulum when the cell endosomes, and they mediate the regulated secretion of small-molecule neurotrans needs to produce large amounts of mitters at the axon terminals of nerve cells. secreted proteins)?


Cell_Biology_Alberts

Cell_Biology_Alberts. secreted proteins)? Proteins are delivered from the TGN to the plasma membrane by the constitutive pathway unless they are diverted into other pathways or retained in the Golgi apparatus. In polarized cells, the transport pathways from the TGN to the plasma membrane operate selectively to ensure that different sets of membrane proteins, secreted proteins, and lipids are delivered to the different domains of the plasma membrane. Which statements are true? Explain why or why not. 13–1 In all events involving fusion of a vesicle to a target membrane, the cytosolic leaflets of the vesicle and target bilayers always fuse together, as do the leaflets that are not in contact with the cytosol. 13–2 There is one strict requirement for the exit of a protein from the ER: it must be correctly folded.


Cell_Biology_Alberts

Cell_Biology_Alberts. 13–2 There is one strict requirement for the exit of a protein from the ER: it must be correctly folded. 13–3 All the glycoproteins and glycolipids in intracellular membranes have oligosaccharide chains facing the lumenal side, and all those in the plasma membrane have oligosaccharide chains facing the outside of the cell. Discuss the following problems. 13–4 In a nondividing cell such as a liver cell, why must the flow of membrane between compartments be balanced, so that the retrieval pathways match the outward flow? Would you expect the same balanced flow in a gut epithelial cell, which is actively dividing? 13–5 Enveloped viruses, which have a membrane coat, gain access to the cytosol by fusing with a cell membrane. Why do you suppose that these viruses encode their own special fusion protein, rather than making use of a cell's SNAREs?


Cell_Biology_Alberts

Cell_Biology_Alberts. 13–6 For fusion of a vesicle with its target membrane to occur, the membranes have to be brought to within 1.5 nm so that the two bilayers can join (Figure Q13–1). Assuming that the relevant portions of the two membranes at the fusion site are circular regions 1.5 nm in diameter, calculate the number of water molecules that would remain between the membranes. (Water is 55.5 M and the volume of a cylinder is πr2h.) Given that an average phospholipid 1.5 nm Figure Q13–1 Close approach of a vesicle and its target membrane in preparation for fusion (problem 13–6).


Cell_Biology_Alberts

Cell_Biology_Alberts. occupies a membrane surface area of 0.2 nm2, how many (A) strain A strain B phospholipids would be present in each of the opposing monolayers at the fusion site? Are there sufficient water molecules to bind to the hydrophilic head groups of this number of phospholipids? (It is estimated that 10–12 water molecules are normally associated with each phospholipid head group at the exposed surface of a membrane.) 13–7 SNAREs exist as complementary partners that carry out membrane fusions between appropriate ves icles and their target membranes. In this way, a vesicle with a particular variety of v-SNARE will fuse only with a membrane that carries the complementary t-SNARE. In some instances, however, fusions of identical membranes (homotypic fusions) are known to occur. For example, when a yeast cell forms a bud, vesicles derived from the mother cell’s vacuole move into the bud where they fuse with one another to form a new vacuole. These vesicles carry both v-SNAREs and t-SNAREs. Are both


Cell_Biology_Alberts

Cell_Biology_Alberts. cell forms a bud, vesicles derived from the mother cell’s vacuole move into the bud where they fuse with one another to form a new vacuole. These vesicles carry both v-SNAREs and t-SNAREs. Are both types of


Cell_Biology_Alberts

Cell_Biology_Alberts. SNAREs essential for this homotypic fusion event? To test this point, you have developed an ingenious assay for fusion of vacuolar vesicles. You prepare vesicles from two different mutant strains of yeast: strain B has a defective gene for vacuolar alkaline phosphatase (Pase); strain A is defective for the protease that converts the pre cursor of alkaline phosphatase (pro-Pase) into its active form (Pase) (Figure Q13–2A). Neither strain has active alkaline phosphatase, but when extracts of the strains are mixed, vesicle fusion generates active alkaline phosphatase, which can be easily measured (Figure Q13–2). Now you delete the genes for the vacuolar v-SNARE, t-SNARE, or both in each of the two yeast strains. You prepare vacuolar vesicles from each and test them for their ability to fuse, as measured by the alkaline phosphatase assay (Figure Q13–2B).


Cell_Biology_Alberts

Cell_Biology_Alberts. What do these data say about the requirements for v-SNAREs and t-SNAREs in the fusion of vacuolar vesicles? Does it matter which kind of SNARE is on which vesicle? 13–8 If you were to remove the ER retrieval signal from protein disulfide isomerase (PDI), which is normally a soluble resident of the ER lumen, where would you expect the modified PDI to be located?


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Cell_Biology_Alberts. 13–9 The KDEL receptor must shuttle back and forth between the ER and the Golgi apparatus to accomplish its task of ensuring that soluble ER proteins are retained in the ER lumen. In which compartment does the KDEL receptor bind its ligands more tightly? In which compartment does it bind its ligands more weakly? What is thought to be the basis for its different binding affinities in the two compartments? If you were designing the system, in which compartment would you have the highest concentration of KDEL receptor? Would you predict that the KDEL receptor, which is a transmembrane protein, would itself possess an ER retrieval signal? 13–10 How does the low pH of lysosomes protect the rest of the cell from lysosomal enzymes in case the lysosome breaks? Figure Q13–2 SNARE requirements for vesicle fusion (problem 13–7).


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Cell_Biology_Alberts. 13–10 How does the low pH of lysosomes protect the rest of the cell from lysosomal enzymes in case the lysosome breaks? Figure Q13–2 SNARE requirements for vesicle fusion (problem 13–7). (A) Scheme for measuring the fusion of vacuolar vesicles. (B) Results of fusions of vesicles with different combinations of v-SNAREs and t-SNAREs. The SNAREs present on the vesicles of the two strains are indicated as v (v-SNARE) and t (t-SNARE).


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Cell_Biology_Alberts. 13–11 Melanosomes are specialized lysosomes that store pigments for eventual release by exocytosis. Various cells such as skin and hair cells then take up the pigment, which accounts for their characteristic pigmentation. Mouse mutants that have defective melanosomes often have pale or unusual coat colors. One such light-colored mouse, the Mocha mouse (Figure Q13–3), has a defect in the gene for one of the subunits of the adaptor protein complex AP3, which is associated with coated vesicles budding from the trans Golgi network. How might the loss of AP3 cause a defect in melanosomes? Figure Q13–3 A normal mouse and the Mocha mouse (problem 13–11). In addition to its light coat color, the Mocha mouse has a poor sense of balance. (Courtesy of Margit Burmeister.) harrison sc & kirchhausen T (2010) structural biology: conservation in vesicle coats. nature 466, 1048–1049. pfeffer sr (2013) a prize for membrane magic. Cell 155, 1203–1206.


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Cell_Biology_Alberts. Margit Burmeister.) harrison sc & kirchhausen T (2010) structural biology: conservation in vesicle coats. nature 466, 1048–1049. pfeffer sr (2013) a prize for membrane magic. Cell 155, 1203–1206. Thor f, gautschi M, geiger r & helenius a (2009) bulk flow revisited: transport of a soluble protein in the secretory pathway. Traffic 10, 1819–1830. The Molecular Mechanisms of Membrane Transport and the Maintenance of Compartmental Diversity antonny b (2011) Mechanisms of membrane curvature sensing. annu. rev. Biochem. 80, 101–123. ferguson sM & de camilli p (2012) dynamin, a membrane-remodelling gTpase. nat. rev. Mol. Cell Biol. 13, 75–88. frost a, unger vM & de camilli p (2009) The bar domain superfamily: membrane-molding macromolecules. Cell 137, 191–196. grosshans bl, ortiz d & novick p (2006) rabs and their effectors: achieving specificity in membrane traffic. Proc. natl acad. sci. Usa 103, 11821–11827.


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Cell_Biology_Alberts. grosshans bl, ortiz d & novick p (2006) rabs and their effectors: achieving specificity in membrane traffic. Proc. natl acad. sci. Usa 103, 11821–11827. hughson fM (2010) copy coats: copI mimics clathrin and copII. Cell 142, 19–21. Jackson lp, kelly bT, Mccoy aJ et al. (2010) a large-scale conformational change couples membrane recruitment to cargo binding in the ap2 clathrin adaptor complex. Cell 141, 1220–1229. Jahn r & scheller rh (2006) snares—engines for membrane fusion. nat. rev. Mol. Cell Biol. 7, 631–643. Jean s & kiger aa (2012) coordination between rab gTpase and phosphoinositide regulation and functions. nat. rev. Mol. Cell Biol. 13, 463–470. Jin l, pahuja kb, wickliffe ke et al. (2012) ubiquitin-dependent regulation of copII coat size and function. nature 482, 495–500. Martens s & McMahon hT (2008) Mechanisms of membrane fusion: disparate players and common principles. nat. rev. Mol. Cell Biol. 9, 543–556.


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Cell_Biology_Alberts. Martens s & McMahon hT (2008) Mechanisms of membrane fusion: disparate players and common principles. nat. rev. Mol. Cell Biol. 9, 543–556. Mcnew Ja, parlati f, fukuda r et al. (2000) compartmental specificity of cellular membrane fusion encoded in snare proteins. nature 407, 153–159. Miller ea & schekman r (2013) copII – a flexible vesicle formation system. Curr. Opin. Cell Biol. 25, 420–427. pfeffer sr (2013) rab gTpase regulation of membrane identity. Curr. Opin. Cell Biol. 25, 414–419. saito k, chen M, bard f et al. (2009) Tango1 facilitates cargo loading at endoplasmic reticulum exit sites. Cell 136, 891–902. seaman Mn (2005) recycle your receptors with retromer. Trends Cell Biol. 15, 68–75. Transport from the ER Through the Golgi Apparatus ellgaard l & helenius a (2003) Quality control in the endoplasmic reticulum. nat. rev. Mol. Cell Biol. 4, 181–191.


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Cell_Biology_Alberts. Transport from the ER Through the Golgi Apparatus ellgaard l & helenius a (2003) Quality control in the endoplasmic reticulum. nat. rev. Mol. Cell Biol. 4, 181–191. emr s, glick bs, linstedt ad et al. (2009) Journeys through the golgi— taking stock in a new era. J. Cell Biol. 187, 449–453. farquhar Mg & palade ge (1998) The golgi apparatus: 100 years of progress and controversy. Trends Cell Biol. 8, 2–10. ladinsky Ms, Mastronarde dn, McIntosh Jr et al. (1999) golgi structure in three dimensions: functional insights from the normal rat kidney cell. J. Cell Biol. 144, 1135–1149. Munro s (2011) The golgin coiled-coil proteins of the golgi apparatus. Cold spring Harb. Perspect. Biol. 3, a005256. pfeffer s (2010) how the golgi works: a cisternal progenitor model. Proc. natl acad. sci. Usa 107, 19614–19618. varki a (2011) evolutionary forces shaping the golgi glycosylation machinery: why cell surface glycans are universal to living cells. Cold spring Harb. Perspect. Biol. 3, a005462.


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Cell_Biology_Alberts. varki a (2011) evolutionary forces shaping the golgi glycosylation machinery: why cell surface glycans are universal to living cells. Cold spring Harb. Perspect. Biol. 3, a005462. Transport from the Trans Golgi Network to Lysosomes andrews nw (2000) regulated secretion of conventional lysosomes. Trends Cell Biol. 10, 316–321. bonifacino Js & rojas r (2006) retrograde transport from endosomes to the trans-golgi network. nat. rev. Mol. Cell Biol. 7, 568–579. de duve c (2005) The lysosome turns fifty. nat. Cell Biol. 7, 847–849. futerman ah & van Meer g (2004) The cell biology of lysosomal storage disorders. nat. rev. Mol. Cell Biol. 5, 554–565. kraft c & Martens s (2012) Mechanisms and regulation of autophagosome formation. Curr. Opin. Cell Biol. 24, 496–501. Mizushima n, yoshimori T & ohsumi y (2011) The role of atg proteins in autophagosome formation. annu. rev. Cell Dev. Biol. 27, 107–132.


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Cell_Biology_Alberts. Mizushima n, yoshimori T & ohsumi y (2011) The role of atg proteins in autophagosome formation. annu. rev. Cell Dev. Biol. 27, 107–132. parzych kr & klionsky dJ (2014) an overview of autophagy: morphology, mechanism, and regulation. antioxid. redox signal. 20, 460–473. Transport into the Cell from the Plasma Membrane: Endocytosis bonifacino Js & Traub lM (2003) signals for sorting of transmembrane proteins to endosomes and lysosomes. annu. rev. Biochem. 72, 395–447. brown Ms & goldstein Jl (1986) a receptor-mediated pathway for cholesterol homeostasis. science 232, 34–47. conner sd & schmid sl (2003) regulated portals of entry into the cell. nature 422, 37–44. doherty gJ & McMahon hT (2009) Mechanisms of endocytosis. annu. rev. Biochem. 78, 857–902. howes MT, Mayor s & parton rg (2010) Molecules, mechanisms, and cellular roles of clathrin-independent endocytosis. Curr. Opin. Cell Biol. 22, 519–527. huotari J & helenius a (2011) endosome maturation. EMBO J. 30, 3481–3500.


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Cell_Biology_Alberts. huotari J & helenius a (2011) endosome maturation. EMBO J. 30, 3481–3500. hurley Jh & hanson pI (2010) Membrane budding and scission by the escrT machinery: it’s all in the neck. nat. rev. Mol. Cell Biol. 11, 556–566. kelly bT & owen dJ (2011) endocytic sorting of transmembrane protein cargo. Curr. Opin. Cell Biol. 23, 404–412. Maxfield fr & Mcgraw Te (2004) endocytic recycling. nat. rev. Mol. Cell Biol. 5, 121–132. McMahon hT & boucrot e (2011) Molecular mechanism and physiological functions of clathrin-mediated endocytosis. nat. rev. Mol. Cell Biol. 12, 517–533. Mercer J & helenius a (2012) gulping rather than sipping: macropinocytosis as a way of virus entry. Curr. Opin. Microbiol. 15, 490–499. sorkin a & von Zastrow M (2009) endocytosis and signalling: intertwining molecular networks. nat. rev. Mol. Cell Biol. 10, 609–622. Tjelle Te, lovdal T & berg T (2000) phagosome dynamics and function. BioEssays 22, 255–263.


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Cell_Biology_Alberts. Tjelle Te, lovdal T & berg T (2000) phagosome dynamics and function. BioEssays 22, 255–263. Transport from the Trans Golgi Network to the Cell Exterior: Exocytosis burgess Tl & kelly rb (1987) constitutive and regulated secretion of proteins. annu. rev. Cell Biol. 3, 243–293. li f, pincet f, perez e et al. (2011) complexin activates and clamps snarepins by a common mechanism involving an intermediate energetic state. nat. struct. Mol. Biol. 18, 941–946. Martin Tf (1997) stages of regulated exocytosis. Trends Cell Biol. 7, 271–276. Mellman I & nelson wJ (2008) coordinated protein sorting, targeting and distribution in polarized cells. nat. rev. Mol. Cell Biol. 9, 833–845. Mostov k, su T & ter beest M (2003) polarized epithelial membrane traffic: conservation and plasticity. nat. Cell Biol. 5, 287–293. pang Zp & südhof Tc (2010) cell biology of ca2+-triggered exocytosis. Curr. Opin. Cell Biol. 22, 496–505.


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Cell_Biology_Alberts. pang Zp & südhof Tc (2010) cell biology of ca2+-triggered exocytosis. Curr. Opin. Cell Biol. 22, 496–505. schuck s & simons k (2004) polarized sorting in epithelial cells: raft clustering and the biogenesis of the apical membrane. J. Cell sci. 117, 5955–5964. Energy Conversion: Mitochondria and Chloroplasts 14


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Cell_Biology_Alberts. To maintain their high degree of organization in a universe that is constantly drifting toward chaos, cells have a constant need for a plentiful supply of ATP, as we have explained in Chapter 2. In eukaryotic cells, most of the ATP that powers life processes is produced by specialized, membrane-enclosed, energy-converting organelles. These are of two types. Mitochondria, which occur in virtually all cells of animals, plants, and fungi, burn food molecules to produce ATP by oxidative phosphorylation. Chloroplasts, which occur only in plants and green algae, harness solar energy to produce ATP by photosynthesis. In electron micrographs, the most striking features of both mitochondria and chloroplasts are their extensive internal membrane systems. These internal membranes contain sets of membrane protein complexes that work together to produce most of the cell’s ATP. In bacteria, simpler versions of essentially the same protein complexes produce ATP, but they are located in the cell’s


Cell_Biology_Alberts

Cell_Biology_Alberts. protein complexes that work together to produce most of the cell’s ATP. In bacteria, simpler versions of essentially the same protein complexes produce ATP, but they are located in the cell’s plasma membrane (Figure 14–1).


Cell_Biology_Alberts

Cell_Biology_Alberts. Comparisons of DNA sequences indicate that the energy-converting organelles in present-day eukaryotes originated from prokaryotic cells that were endocytosed during the evolution of eukaryotes (discussed in Chapter 1). This explains why mitochondria and chloroplasts contain their own DNA, which still encodes a subset of their proteins. Over time, these organelles have lost most of their own genomes and become heavily dependent on proteins that are encoded by genes in the nucleus, synthesized in the cytosol, and then imported into the organelle. And the eukaryotic cells now rely on these organelles not only for the ATP they need for biosynthesis, solute transport, and movement, but also for many important biosynthetic reactions that occur inside each organelle.


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Cell_Biology_Alberts. The common evolutionary origin of the energy-converting machinery in mitochondria, chloroplasts, and prokaryotes (archaea and bacteria) is reflected in the fundamental mechanism that they share for harnessing energy. This is known as chemiosmotic coupling, signifying a link between the chemical bond-forming reactions that generate ATP (“chemi”) and membrane transport processes


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Cell_Biology_Alberts. Figure 14–1 The membrane systems of bacteria, mitochondria, and chloroplasts are related. Mitochondria and chloroplasts are cell organelles that have originated from bacteria and have retained the bacterial energy-conversion mechanisms. Like their bacterial ancestors, mitochondria and chloroplasts have an outer and an inner membrane. Each of the membranes colored in this diagram contains energy-harvesting electron-transport chains. the deep invaginations of the mitochondrial inner membrane and the internal membrane system of the chloroplast harbor the machinery for cellular respiration and photosynthesis, respectively.


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Cell_Biology_Alberts. (“osmotic”). The chemiosmotic process occurs in two linked stages, both of which are performed by protein complexes in a membrane. Stage 1: High-energy electrons (derived from the oxidation of food molecules, from pigments excited by sunlight, or from other sources described later) are transferred along a series of electron-transport protein complexes that form an electron-transport chain embedded in a membrane. Each electron transfer releases a small amount of energy that is used to pump protons (H+) and thereby generate a large electrochemical gradient across the membrane (Figure 14–2). As discussed in Chapter 11, such an electrochemical gradient provides a way of storing energy, and it can be harnessed to do useful work when ions flow back across the membrane.


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Cell_Biology_Alberts. Stage 2: The protons flow back down their electrochemical gradient through an elaborate membrane protein machine called ATP synthase, which catalyzes the production of ATP from ADP and inorganic phosphate (Pi). This ubiquitous enzyme works like a turbine in the membrane, driven by protons, to synthesize ATP (Figure 14–3). In this way, the energy derived from food or sunlight in stage 1 is converted into the chemical energy of a phosphate bond in ATP.


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Cell_Biology_Alberts. Electrons move through protein complexes in biological systems via tightly bound metal ions or other carriers that take up and release electrons easily, or by special small molecules that pick electrons up at one location and deliver them to another. For mitochondria, the first of these electron carriers is NAD+, a water-soluble small molecule that takes up two electrons and one H+ derived from food molecules (fats and carbohydrates) to become NADH. NADH transfers these electrons from the sites where the food molecules are degraded to the inner mitochondrial membrane. There, the electrons from the energy-rich NADH are passed from one membrane protein complex to the next, passing to a lower-energy compound at each step, until they reach a final complex in which they combine with molecular oxygen (O2) to produce water. The energy released at each step as the electrons flow down this path from the energy-rich NADH to the low-energy water molecule drives H+ pumps in the inner


Cell_Biology_Alberts

Cell_Biology_Alberts. molecular oxygen (O2) to produce water. The energy released at each step as the electrons flow down this path from the energy-rich NADH to the low-energy water molecule drives H+ pumps in the inner mitochondrial membrane, utilizing three different membrane protein complexes. Together, these complexes generate the proton-motive force harnessed by ATP synthase to produce the ATP that serves as the universal energy currency throughout the cell (see Chapter 2).


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 14–4 compares the electron-transport processes in mitochondria, which harness energy from food molecules, with those in chloroplasts, which harness energy from sunlight. The energy-conversion systems of mitochondria and chloroplasts can be described in similar terms, and we shall see later in the chapter that two of their key components are closely related. One of these is the ATP synthase, and the other is a proton pump (colored green in Figure 14–4). Among the crucial constituents that are unique to photosynthetic organisms are the two photosystems. These use the green pigment chlorophyll to capture Figure 14–2 Stage 1 of chemiosmotic coupling. Energy from sunlight or the oxidation of food compounds is captured to generate an electrochemical proton gradient across a membrane. the electrochemical gradient serves as a versatile energy store that drives energy-requiring reactions in mitochondria, chloroplasts, and bacteria.


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Cell_Biology_Alberts. Figure 14–3 Stage 2 of chemiosmotic coupling. an atp synthase (yellow) embedded in the lipid bilayer of a membrane harnesses the electrochemical proton gradient across the membrane, using it as a local energy store to drive atp synthesis. the red arrows show the direction of proton movement through the atp synthase.


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Cell_Biology_Alberts. Figure 14–4 Electron-transport processes. (a) the mitochondrion converts energy from chemical fuels. (B) the chloroplast converts energy from sunlight. In both cases, electron flow is indicated by blue arrows. Each of the protein complexes (green) is embedded in a membrane. In the mitochondrion, fats and carbohydrates from food molecules are fed into the citric acid cycle and provide electrons to generate the energy-rich compound NaDh from NaD+. these electrons then flow down an energy gradient as they pass from one complex to the next in the electron-transport chain, until they combine with molecular O2 in the last complex to produce water. the energy released at each stage is harnessed to pump h+ across the membrane. In the chloroplast, by contrast, electrons are extracted from water through the action of light in the photosystem II complex and molecular O2 is released. the electrons pass on to the next complex in the chain, which uses some of their energy to pump protons across the


Cell_Biology_Alberts

Cell_Biology_Alberts. the action of light in the photosystem II complex and molecular O2 is released. the electrons pass on to the next complex in the chain, which uses some of their energy to pump protons across the membrane, before passing to photosystem I, where sunlight generates high-energy electrons that combine with NaDp+ to produce NaDph. NaDph then enters the carbon-fixation cycle along with CO2 to generate carbohydrates.


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Cell_Biology_Alberts. light energy and power the transfer of electrons, not unlike a photocell in a solar panel. The chloroplasts drive electron transfer in the direction opposite to that in mitochondria: electrons are taken from water to produce O2, and these electrons are used (via NADPH, a molecule closely related to the NADH used in mitochondria) to synthesize carbohydrates from CO2 and water. These carbohydrates then serve as the source for all other compounds a plant cell needs. Thus, both mitochondria and chloroplasts make use of an electron-transfer chain to produce an H+ gradient that powers reactions that are critical for the cell. However, chloroplasts generate O2 and take up CO2, whereas mitochondria consume O2 and release CO2 (see Figure 14–4).


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Cell_Biology_Alberts. Mitochondria occupy up to 20% of the cytoplasmic volume of a eukaryotic cell. Although they are often depicted as short, bacterium-like bodies with a diameter of 0.5–1 μm, they are in fact remarkably dynamic and plastic, moving about the cell, constantly changing shape, dividing, and fusing (Movie 14.1). Mitochondria are often associated with the microtubular cytoskeleton (Figure 14–5), which determines their orientation and distribution in different cell types. Thus, in highly polarized cells such as neurons, mitochondria can move long distances (up to a meter or more in the extended axons of neurons), being propelled along the tracks of the microtubular cytoskeleton. In other cells, mitochondria remain fixed at points of high energy demand; for example, in skeletal or cardiac muscle cells, they pack between myofibrils, and in sperm cells they wrap tightly around the flagellum (Figure 14–6).


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Cell_Biology_Alberts. Mitochondria also interact with other membrane systems in the cell, most notably the endoplasmic reticulum (ER). Contacts between mitochondria and ER define specialized domains thought to facilitate the exchange of lipids between the two membrane systems. These contacts also appear to induce mitochondrial fission, which, as we discuss later, is involved in the distribution and partitioning of mitochondria within cells (Figure 14–7).


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Cell_Biology_Alberts. The acquisition of mitochondria was a prerequisite for the evolution of complex animals. Without mitochondria, present-day animal cells would have had to generate all of their ATP through anaerobic glycolysis. When glycolysis converts glucose to pyruvate, it releases only a small fraction of the total free energy that is potentially available from glucose oxidation (see Chapter 2). In mitochondria, the metabolism of sugars is complete: pyruvate is imported into the mitochondrion and ultimately oxidized by O2 to CO2 and H2O, which allows 15 times more ATP to be made from a sugar than by glycolysis alone. As explained later, this became possible only when enough molecular oxygen accumulated in the Earth’s atmosphere to allow organisms to take full advantage, via respiration, of the large amounts of energy potentially available from the oxidation of organic compounds.


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Cell_Biology_Alberts. Mitochondria are large enough to be seen in the light microscope, and they were first identified in the nineteenth century. Real progress in understanding their internal structure and function, however, depended on biochemical procedures developed in 1948 for isolating intact mitochondria, and on electron microscopy, which was first used to look at cells at about the same time. Figure 14–5 The relationship between mitochondria and microtubules. (a) a light micrograph of chains of elongated mitochondria in a living mammalian cell in culture. the cell was stained with a fluorescent dye (rhodamine 123) that specifically labels mitochondria in living cells. (B) an immunofluorescence micrograph of the same cell stained (after fixation) with fluorescent antibodies that bind to microtubules. Note that the mitochondria tend to be aligned along microtubules. (Courtesy of Lan Bo Chen.)


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Cell_Biology_Alberts. Figure 14–6 Localization of mitochondria near sites of high ATP demand in cardiac muscle and a sperm tail. (a) Cardiac muscle in the wall of the heart is the most heavily used muscle in the body, and its continual contractions require a reliable energy supply. It has limited built-in energy stores and has to depend on a steady supply of atp from the copious mitochondria aligned close to the contractile myofibrils (see Figure 16–32). (B) During sperm development, microtubules wind helically around the flagellar axoneme, where they are thought to help localize the mitochondria in the tail to produce the structure shown.


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Cell_Biology_Alberts. Figure 14–7 Interaction of mitochondria with the endoplasmic reticulum. 0 sec 20 sec 30 sec (a) Fluorescence light microscopy shows that tubules of the Er (green) wrap around parts of the mitochondrial network (red) in mammalian cells. the mitochondria then divide at the contact sites. after contact is established, fission occurs within less than a minute, as indicated by time-lapse microscopy. (B) Schematic drawing of an Er tubule wrapped around part of the mitochondrial reticulum. It is thought that Er–mitochondrial contacts also mediate the exchange of lipids between the two membrane systems. (a, adapted from J.r. Friedman et al., Science 334:358–362, 2011.) the Mitochondrion has an Outer Membrane and an Inner Membrane


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Cell_Biology_Alberts. Like the bacteria from which they originated, mitochondria have an outer and an inner membrane. The two membranes have distinct functions and properties, and delineate separate compartments within the organelle. The inner membrane, which surrounds the internal mitochondrial matrix compartment (Figure 14–8), is highly folded to form invaginations known as cristae (the singular is crista), which contain in their membranes the proteins of the electron-transport chain. Where the inner membrane runs parallel to the outer membrane, between the cristae, it is known as the inner boundary membrane. The narrow (20–30 nm) gap between the inner boundary membrane and the outer membrane is known as the intermembrane space. The cristae are about 20 nm-wide membrane discs or tubules that protrude deeply into the matrix and enclose the crista space. The crista membrane is continuous with the inner boundary membrane, and where their membranes join, the membrane forms narrow membrane tubes or slits,


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Cell_Biology_Alberts. into the matrix and enclose the crista space. The crista membrane is continuous with the inner boundary membrane, and where their membranes join, the membrane forms narrow membrane tubes or slits, known as crista junctions.


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Cell_Biology_Alberts. Like the bacterial outer membrane, the outer mitochondrial membrane is freely permeable to ions and to small molecules as large as 5000 daltons. This


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Cell_Biology_Alberts. Figure 14–8 Structure of a mitochondrion. (a) tomographic slice through a three-dimensional map of a mouse heart mitochondrion determined by electron-microscope tomography. the outer membrane envelops the inner boundary membrane. the inner membrane is highly folded into tubular or lamellar cristae, which crisscross the matrix. the dense matrix, which contains most of the mitochondrial protein, appears dark in the electron microscope, whereas the intermembrane space and the crista space appear light due to their lower protein content. the inner boundary membrane follows the outer membrane closely at a distance of ≈20 nm. the inner membrane turns sharply at the cristae junctions, where the cristae join the inner boundary membrane. (B) tomographic surface-rendered portion of a yeast mitochondrion, showing how flattened cristae project into the matrix from the inner membrane (Movie 14.2). (C) Schematic drawing of a mitochondrion showing the outer membrane (gray), and the inner membrane


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Cell_Biology_Alberts. showing how flattened cristae project into the matrix from the inner membrane (Movie 14.2). (C) Schematic drawing of a mitochondrion showing the outer membrane (gray), and the inner membrane (yellow). Note that the inner membrane is compartmentalized into the inner boundary membrane and the crista membrane. there are three distinct spaces: the inner membrane space, the crista space, and the matrix. (a, courtesy of tobias Brandt; B, from K. Davies et al., Proc. Natl Acad. Sci. USA 109:13602–13607, 2012. With permission from the National academy of Sciences.)


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Cell_Biology_Alberts. Figure 14–9 Biochemical fractionation of purified mitochondria into separate components. Large numbers of mitochondria are isolated from homogenized tissue by centrifugation and then suspended in a medium of low osmotic strength. In such a medium, water flows into mitochondria and greatly expands the matrix space (yellow). While the cristae of the inner membrane unfold to accommodate the swelling, the outer membrane— which has no folds—breaks, releasing structures composed of the inner membrane surrounding the matrix. these techniques have made it possible to study the protein composition of the inner membrane (comprising a mixture of cristae, boundary membranes, and cristae junctions), the outer membrane, and the matrix.


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Cell_Biology_Alberts. is because it contains many porin molecules, a special class of β-barrel-type membrane protein that creates aqueous pores across the membrane (see Figure 10–23). As a consequence, the intermembrane space between the outer and inner membrane has the same pH and ionic composition as the cytoplasm, and there is no electrochemical gradient across the outer membrane. If purified mitochondria are gently disrupted and then fractionated (Figure 14–9), the biochemical composition of membranes and mitochondrial compartments can be determined. the Inner Membrane Cristae Contain the Machinery for Electron transport and atp Synthesis Unlike the outer mitochondrial membrane, the inner mitochondrial membrane is a diffusion barrier to ions and small molecules, just like the bacterial inner membrane. However, selected ions, most notably protons and phosphate, as well as essential metabolites such as ATP and ADP, can pass through it by means of special transport proteins.


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Cell_Biology_Alberts. The inner mitochondrial membrane is highly differentiated into functionally distinct regions with different protein compositions. As discussed in Chapter 10, the lateral segregation of membrane regions with different protein and lipid compositions is a key feature of cells. In the inner mitochondrial membrane, the boundary membrane region is thought to contain the machinery for protein import, new membrane insertion, and assembly of the respiratory-chain complexes. The membranes of the cristae, which are continuous with the boundary membrane, contain the ATP synthase enzyme that produces most of the cell’s ATP; they also contain the large protein complexes of the respiratory chain—the name given to the mitochondrion’s electron-transport chain.


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Cell_Biology_Alberts. At the cristae junctions, where the membranes of the cristae join the boundary membrane, special protein complexes provide a diffusion barrier that segregates the membrane proteins in the two regions of the inner membrane; these complexes are also thought to anchor the cristae to the outer membrane, thus maintaining the highly folded topology of the inner membrane. Cristae membranes have one of the highest protein densities of all biological membranes, with a lipid content of 25% and a protein content of 75% by weight. The folding of the inner membrane into cristae greatly increases the membrane area available for oxidative phosphorylation. In highly active cardiac muscle cells, for example, the total area of cristae membranes can be up to 20 times larger than the area of the cell’s plasma membrane. In total, the surface area of cristae membranes in each human body adds up to roughly the size of a football field. the Citric acid Cycle in the Matrix produces NaDh


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Cell_Biology_Alberts. the Citric acid Cycle in the Matrix produces NaDh Together with the cristae that project into it, the matrix is the major working part of the mitochondrion. Mitochondria can use both pyruvate and fatty acids as fuel. Pyruvate is derived from glucose and other sugars, whereas fatty acids are derived from fats. Both of these fuel molecules are transported across the inner mitochondrial membrane by specialized transport proteins, and they are then converted to the crucial metabolic intermediate acetyl CoA by enzymes located in the mitochondrial matrix (see Chapter 2). in medium of low osmolarity the infux of water causes the mitochondrion to swell and the outer membrane to rupture, releasing the contents of the intermembrane space; the inner membrane remains intact FOOD MOLECULES FROM CYTOSOL


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Cell_Biology_Alberts. The acetyl groups in acetyl CoA are oxidized in the matrix via the citric acid cycle, also called the Krebs cycle (see Figure 2–57 and Movie 2.6). The oxidation of these carbon atoms in acetyl CoA produces CO2, which diffuses out of the mitochondrion to be released to the environment as a waste product. More importantly, the citric acid cycle saves a great deal of the bond energy released by this oxidation in the form of electrons carried by NADH. This NADH transfers its electrons from the matrix to the electron-transport chain in the inner mitochondrial membrane, where—through the chemiosmotic coupling process described previously (see Figures 14–2 and 14–3)—the energy that was carried by NADH electrons is converted into phosphate-bond energy in ATP. Figure 14–10 outlines this sequence of reactions schematically.


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Cell_Biology_Alberts. The matrix contains the genetic system of the mitochondrion, including the mitochondrial DNA and the ribosomes. The mitochondrial DNA (see section on genetic systems, p. 800) is organized into compact bodies—the nucleoids—by special scaffolding proteins that also function as transcription regulatory proteins. The large number of enzymes required for the maintenance of the mitochondrial genetic system, as well as for many other essential reactions to be outlined next, accounts for the very high protein concentration in the matrix; at more than 500 mg/mL, this concentration is close to that in a protein crystal. Mitochondria not only generate most of the cell’s ATP; they also provide many other essential resources for biosynthesis and cell growth. Before describing in detail the remarkable machinery of the respiratory chain, we diverge briefly to touch on some of these important roles. Figure 14–10 A summary of the energy-converting metabolism in mitochondria.


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Cell_Biology_Alberts. Figure 14–10 A summary of the energy-converting metabolism in mitochondria. pyruvate and fatty acids enter the mitochondrion (top of the figure) and are broken down to acetyl Coa. the acetyl Coa is metabolized by the citric acid cycle, which reduces NaD+ to NaDh, which then passes its high-energy electrons to the first complex in the electron-transport chain. In the process of oxidative phosphorylation, these electrons pass along the electron-transport chain in the inner membrane cristae to oxygen (O2). this electron transport generates a proton gradient, which drives the production of atp by the atp synthase (see Figure 14–3). Electrons from the oxidation of succinate, a reaction intermediate in the citric acid cycle (see panel 2–9, pp. 106–107), take a separate path to enter this electron-transport chain (not shown, see p. 772).


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Cell_Biology_Alberts. the membranes that comprise the mitochondrial inner membrane—the inner boundary membrane and the crista membrane—contain different mixtures of proteins and they are therefore shaded differently in this diagram.


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Cell_Biology_Alberts. Mitochondria are critical for buffering the redox potential in the cytosol. Cells need a constant supply of the electron acceptor NAD+ for the central reaction in glycolysis that converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate (see Figure 2–48). This NAD+ is converted to NADH in the process, and the NAD+ needs to be regenerated by transferring the high-energy NADH electrons somewhere. The NADH electrons will eventually be used to help drive oxidative phosphorylation inside the mitochondrion. But the inner mitochondrial membrane is impermeable to NADH. The electrons are therefore passed from the NADH to smaller molecules in the cytosol that can move through the inner mitochondrial membrane. Once in the matrix, these smaller molecules transfer their electrons to NAD+ to form mitochondrial NADH, after which they are returned to the cytosol for recharging—creating a so-called shuttle system for the NADH electrons.


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Cell_Biology_Alberts. In addition to ATP, biosynthesis in the cytosol requires both a constant supply of reducing power in the form of NADPH and small carbon-rich molecules to serve as building blocks (discussed in Chapter 2). Descriptions of biosynthesis often state that the needed carbon skeletons come directly from the breakdown of sugars, whereas the NADPH is produced in the cytosol by a side pathway for the breakdown of sugars (the pentose phosphate pathway, an alternative to glycolysis). But under conditions where nutrients abound and plenty of ATP is available, mitochondria help to generate both the reducing power and the carbon-rich building blocks (the “carbon skeletons” in Panel 2–1, pp. 90–91) needed for cell growth. For this purpose, excess citrate produced in the mitochondrial matrix by the citric acid cycle (see Panel 2–9, pp. 106–107) is transported down its electrochemical gradient to the cytosol, where it is metabolized to produce essential components of the cell. Thus, for example, as


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Cell_Biology_Alberts. acid cycle (see Panel 2–9, pp. 106–107) is transported down its electrochemical gradient to the cytosol, where it is metabolized to produce essential components of the cell. Thus, for example, as part of a cell’s response to growth signals, large amounts of acetyl CoA are produced in the cytosol from citrate exported from mitochondria, accelerating the production of the fatty acids and sterols that build new membranes (described in Chapter 10). Cancer cells are frequently mutated in ways that enhance this pathway, as part of their program of abnormal growth (see Figure 20–26).


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Cell_Biology_Alberts. The urea cycle is a central metabolic pathway in mammals that converts the ammonia (NH4+) produced by the breakdown of nitrogen-containing compounds (such as amino acids) to the urea excreted in urine. Two critical steps of the urea cycle are carried out in the mitochondria of liver cells, while the remaining steps occur in the cytosol. Mitochondria also play an essential part in the metabolic adaptation of cells to different nutritional conditions. For example, under conditions of starvation, proteins in our bodies are broken down to amino acids, and the amino acids are imported into mitochondria and oxidized to produce NADH for ATP production.


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Cell_Biology_Alberts. The biosynthesis of heme groups—which, as we shall see in the next section, play a central part in electron transfer—is another critical process that is shared between the mitochondrion and the cytoplasm. Iron–sulfur clusters, which are essential not only for electron transfer in the respiratory chain (see p. 766), but also for the maintenance and stability of the nuclear genome, are produced in mitochondria (and chloroplasts). Nuclear genome instability, a hallmark of cancer, can sometimes be linked to the decreased function of cellular proteins that contain iron–sulfur clusters.


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Cell_Biology_Alberts. Mitochondria also have a central role in membrane biosynthesis. Cardiolipin is a two-headed phospholipid (Figure 14–11) that is confined to the inner mitochondrial membrane, where it is also produced. But mitochondria are also a major source of phospholipids for the biogenesis of other cell membranes. Phosphatidylethanolamine, phosphatidylglycerol, and phosphatidic acid are synthesized in the mitochondrion, while phosphatidylinositol, phosphatidylcholine, and phosphatidylserine are primarily synthesized in the endoplasmic reticulum (ER). As described in Chapter 12, most of the cell’s membranes are assembled in the ER. The exchange of lipids between the ER and mitochondria is thought to occur at special sites of close contact (see Figure 14–7) by an as-yet unknown mechanism.


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Cell_Biology_Alberts. Figure 14–11 The structure of cardiolipin. Cardiolipin consists of two covalently linked phospholipid units, with a total of four rather than the usual two fatty acid chains (see Figure 10–3). Cardiolipin is only produced in the mitochondrial inner membrane, where it interacts closely with membrane proteins involved in oxidative phosphorylation and atp transport. In cristae, its two juxtaposed phosphate groups may act as a local proton trap on the membrane surface. Finally, mitochondria are important calcium buffers, taking up calcium from the ER and sarcoplasmic reticulum at special membrane junctions. Cellular calcium levels control muscle contraction (see Chapter 16) and alterations are implicated in neurodegeneration and apoptosis. Clearly, cells and organisms depend on mitochondria in many different ways. We now return to the central function of the mitochondrion in respiratory ATP generation. a Chemiosmotic process Couples Oxidation Energy to atp production


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Cell_Biology_Alberts. Although the citric acid cycle that takes place in the mitochondrial matrix is considered to be part of aerobic metabolism, it does not itself use oxygen. Only the final step of oxidative metabolism consumes molecular oxygen (O2) directly (see Figure 14–10). Nearly all the energy available from metabolizing carbohydrates, fats, and other foodstuffs in earlier stages is saved in the form of energy-rich compounds that feed electrons into the respiratory chain in the inner mitochondrial membrane. These electrons, most of which are carried by NADH, finally combine with O2 at the end of the respiratory chain to form water. The energy released during the complex series of electron transfers from NADH to O2 is harnessed in the inner membrane to generate an electrochemical gradient that drives the conversion of ADP + Pi to ATP. For this reason, the term oxidative phosphorylation is used to describe this final series of reactions (Figure 14–12).


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Cell_Biology_Alberts. The total amount of energy released by biological oxidation in the respiratory chain is equivalent to that released by the explosive combustion of hydrogen when it combines with oxygen in a single step to form water. But the combustion of hydrogen in a single-step chemical reaction, which has a strongly negative ∆G, releases this large amount of energy unproductively as heat. In the respiratory chain, the same energetically favorable reaction H2 + ½ O2 →H2O is divided into small steps (Figure 14–13). This stepwise process allows the cell to store nearly half of the total energy that is released in a useful form. At each step, the electrons, which can be thought of as having been removed from a hydrogen molecule to


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Cell_Biology_Alberts. Figure 14–12 The major net energy conversion catalyzed by the mitochondrion. In the process of oxidative phosphorylation, the mitochondrial inner membrane serves as a device that changes one form of chemical-bond energy to another, converting a major part of the energy of NaDh oxidation into phosphate-bond energy in atp. Figure 14–13 A comparison of biological oxidation with combustion. (a) If hydrogen were simply burned, nearly all of the energy would be released in the form of heat. (B) In biological oxidation reactions, about half of the released energy is stored in a form useful to the cell by means of the electron-transport chain (the respiratory chain) in the crista membrane of the mitochondrion. Only the rest of the energy is released as heat. In the cell, the protons and electrons shown here as being derived from h2 are removed from hydrogen atoms that are covalently linked to NaDh molecules.


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Cell_Biology_Alberts. produce two protons, pass through a series of electron carriers in the inner mitochondrial membrane. At each of three distinct steps along the way (marked by the three electron-transport complexes of the respiratory chain, see below), much of the energy is utilized for pumping protons across the membrane. At the end of the electron-transport chain, the electrons and protons recombine with molecular oxygen into water. Water is a very low-energy molecule and is thus very stable; it can serve as an electron donor only when a large amount of energy from an external source is spent on splitting it into protons, electrons, and molecular oxygen. This is exactly what happens in oxygenic photosynthesis, where the external energy source is the sun, as we shall see later in the section on chloroplasts (p. 782). the Energy Derived from Oxidation Is Stored as an Electrochemical Gradient


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Cell_Biology_Alberts. the Energy Derived from Oxidation Is Stored as an Electrochemical Gradient In mitochondria, the process of electron transport begins when two electrons and a proton are removed from NADH (to regenerate NAD+). These electrons are passed to the first of about 20 different electron carriers in the respiratory chain. The electrons start at a large negative redox potential (see Panel 14–1, p. 765)— that is, at a high energy level—which gradually drops as they pass along the chain. The proteins involved are grouped into three large respiratory enzyme complexes, each composed of protein subunits that sit in the inner mitochondrial membrane. Each complex in the chain has a higher affinity for electrons than its predecessor, and electrons pass sequentially from one complex to the next until they are finally transferred to molecular oxygen, which has the highest electron affinity of all.


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Cell_Biology_Alberts. The net result is the pumping of H+ out of the matrix across the inner membrane, driven by the energetically favorable flow of electrons. This transmembrane movement of H+ has two major consequences: 1. It generates a pH gradient across the inner mitochondrial membrane, with a high pH in the matrix (close to 8) and a lower pH in the intermembrane space. Since ions and small molecules equilibrate freely across the outer mitochondrial membrane, the pH in the intermembrane space is the same as in the cytosol (generally around pH 7.4). 2. It generates a voltage gradient across the inner mitochondrial membrane, creating a membrane potential with the matrix side negative and the crista space side positive.


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Cell_Biology_Alberts. 2. It generates a voltage gradient across the inner mitochondrial membrane, creating a membrane potential with the matrix side negative and the crista space side positive. The pH gradient (∆pH) reinforces the effect of the membrane potential (∆V ), because the latter acts to attract any positive ion into the matrix and to push any negative ion out. Together, ∆pH and ∆V make up the electrochemical gradient, which is measured in units of millivolts (mV). This gradient exerts a protonmotive force, which tends to drive H+ back into the matrix (Figure 14–14). force due to proton-motive force due to INTERMEMBRANE membrane potential + + + + + + + + + pH 7.2 inner mitochondrial membrane pH 7.9


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Cell_Biology_Alberts. force due to proton-motive force due to INTERMEMBRANE membrane potential + + + + + + + + + pH 7.2 inner mitochondrial membrane pH 7.9 Figure 14–14 The electrochemical proton gradient across the inner mitochondrial membrane. this gradient is composed of a large force due to the membrane potential (∆V) and a smaller force due to the h+ concentration gradient—that is, the ph gradient (∆ph). Both forces combine to generate the proton-motive force, which pulls h+ back into the mitochondrial matrix. the exact relationship between these forces is expressed by the Nernst equation (see panel 11–1, p. 616).


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Cell_Biology_Alberts. The electrochemical gradient across the inner membrane of a respiring mitochondrion is typically about 180 mV (inside negative), and it consists of a membrane potential of about 150 mV and a pH gradient of about 0.5 to 0.6 pH units (each ∆pH of 1 pH unit is equivalent to a membrane potential of about 60 mV). The electrochemical gradient drives not only ATP synthesis but also the transport of selected molecules across the inner mitochondrial membrane, including the import of selected proteins from the cytoplasm (discussed in Chapter 12).


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Cell_Biology_Alberts. The mitochondrion performs most cellular oxidations and produces the bulk of the animal cell’s ATP. A mitochondrion has two separate membranes: the outer membrane and the inner membrane. The inner membrane surrounds the innermost space (the matrix) of the mitochondrion and forms the cristae, which project into the matrix. The matrix and the inner membrane cristae are the major working parts of the mitochondrion. The membranes that form cristae account for a major part of the membrane surface area in most cells, and they contain the mitochondrion’s electron-transport chain (the respiratory chain).


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Cell_Biology_Alberts. The mitochondrial matrix contains a large variety of enzymes, including those that convert pyruvate and fatty acids to acetyl CoA and those that oxidize this acetyl CoA to CO2 through the citric acid cycle. These oxidation reactions produce large amounts of NADH, whose high-energy electrons are passed to the respiratory chain. The respiratory chain then uses the energy derived from transporting electrons from NADH to molecular oxygen to pump H+ out of the matrix. This produces a large electrochemical proton gradient across the inner mitochondrial membrane, composed of contributions from both a membrane potential and a pH difference. This electrochemical gradient exerts a force to drive H+ back into the matrix. This proton-motive force is harnessed both to produce ATP and for the selective transport of metabolites across the inner mitochondrial membrane.


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Cell_Biology_Alberts. Having considered in general terms how a mitochondrion uses electron transport to generate a proton-motive force, we now turn to the molecular mechanisms that underlie this membrane-based energy-conversion process. In describing the respiratory chain of mitochondria, we accomplish the larger purpose of explaining how an electron-transport process can pump protons across a membrane. As stated at the beginning of this chapter, mitochondria, chloroplasts, archaea, and bacteria use very similar chemiosmotic mechanisms. In fact, these mechanisms underlie the function of all living organisms—including anaerobes that derive all their energy from electron transfers between two inorganic molecules, as we shall see later. We start with some of the basic principles on which all of these processes depend. the redox potential Is a Measure of Electron affinities


Cell_Biology_Alberts

Cell_Biology_Alberts. We start with some of the basic principles on which all of these processes depend. the redox potential Is a Measure of Electron affinities In chemical reactions, any electrons removed from one molecule are always passed to another, so that whenever one molecule is oxidized, another is reduced. As with any other chemical reaction, the tendency of such redox reactions to proceed spontaneously depends on the free-energy change (∆G) for the electron transfer, which in turn depends on the relative affinities of the two molecules for electrons.


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Cell_Biology_Alberts. Because electron transfers provide most of the energy for life, it is worth taking the time to understand them. As discussed in Chapter 2, acids donate protons and bases accept them (see Panel 2–2, p. 93). Acids and bases exist in conjugate acid–base pairs, in which the acid is readily converted into the base by the loss of a proton. For example, acetic acid (CH3COOH) is converted into its conjugate base, the acetate ion (CH3COO–), in the reaction: In an exactly analogous way, pairs of compounds such as NADH and NAD+ are called redox pairs, since NADH is converted to NAD+ by the loss of electrons in the reaction:


Cell_Biology_Alberts

Cell_Biology_Alberts. In an exactly analogous way, pairs of compounds such as NADH and NAD+ are called redox pairs, since NADH is converted to NAD+ by the loss of electrons in the reaction: NADH is a strong electron donor: because two of its electrons are engaged in a covalent bond which releases energy when broken, the free-energy change for passing these electrons to many other molecules is favorable. Energy is required to form this bond from NAD+, two electrons, and a proton (the same amount of energy that was released when the bond was broken). Therefore NAD+, the redox partner of NADH, is of necessity a weak electron acceptor.


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Cell_Biology_Alberts. We can measure the tendency to transfer electrons from any redox pair experimentally. All that is required is the formation of an electrical circuit linking a 1:1 (equimolar) mixture of the redox pair to a second redox pair that has been arbitrarily selected as a reference standard, so that we can measure the voltage difference between them (Panel 14–1). This voltage difference is defined as the redox potential; electrons move spontaneously from a redox pair like NADH/NAD+ with a lower redox potential (a lower affinity for electrons) to a redox pair like O2/H2O with a higher redox potential (a higher affinity for electrons). Thus, NADH is a good molecule for donating electrons to the respiratory chain, while O2 is well suited to act as the “sink” for electrons at the end of the chain. As explained in Panel 14–1, the difference in redox potential, ∆Eʹ0, is a direct measure of the standard free-energy change (∆G°) for the transfer of an electron from one molecule to another.


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Cell_Biology_Alberts. Electron transfers release Large amounts of Energy


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Cell_Biology_Alberts. As just discussed, those pairs of compounds that have the most negative redox potentials have the weakest affinity for electrons and therefore are useful as carriers with a strong tendency to donate electrons. Conversely, those pairs that have the most positive redox potentials have the greatest affinity for electrons and therefore are useful as carriers with a strong tendency to accept electrons. A 1:1 mixture of NADH and NAD+ has a redox potential of –320 mV, indicating that NADH has a strong tendency to donate electrons; a 1:1 mixture of H2O and ½O2 has a redox potential of +820 mV, indicating that O2 has a strong tendency to accept electrons. The difference in redox potential is 1140 mV, which means that the transfer of each electron from NADH to O2 under these standard conditions is enormously favorable, since ∆G° = –109 kJ/mole, and twice this amount of energy is gained for the two electrons transferred per NADH molecule (see Panel 14–1). If we compare this free-energy change


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Cell_Biology_Alberts. enormously favorable, since ∆G° = –109 kJ/mole, and twice this amount of energy is gained for the two electrons transferred per NADH molecule (see Panel 14–1). If we compare this free-energy change with that for the formation of the phosphoanhydride bonds in ATP, where ∆G° = 30.6 kJ/mole (see Figure 2–50), we see that, under standard conditions, the oxidation of one NADH molecule releases more than enough energy to synthesize seven molecules of ATP from ADP and Pi. (In the cell, the number of ATP molecules generated will be lower because the standard conditions are far from the physiological ones; in addition, small amounts of energy are inevitably dissipated as heat along the way.)


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Cell_Biology_Alberts. The electron-transport properties of the membrane protein complexes in the respiratory chain depend upon electron-carrying cofactors, most of which are transition metals such as Fe, Cu, Ni, and Mn, bound to proteins in the complexes. These metals have special properties that allow them to promote both enzyme catalysis and electron-transfer reactions. Most relevant here is the fact that their ions exist in several different oxidation states with closely spaced redox potentials, which enables them to accept or give up electrons readily; this property is


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Cell_Biology_Alberts. HOW REDOX POTENTIALS ARE MEASURED voltmeter salt bridge Areduced and Aoxidized in equimolar amounts 1 M H+ and 1 atmosphere H2 gas examples of redox reactions standard redox potential E0 ° c c e – One beaker (left) contains substance A with an equimolar mixture of the reduced (Areduced) and oxidized (Aoxidized) members of its redox pair. The other beaker contains the hydrogen reference standard (2H+ + 2e – H2), whose redox potential is arbitrarily assigned as zero by international agreement. (A salt bridge formed from a concentrated KCl solution allows K+ and Cl– to move between the beakers, as required to neutralize the charges when electrons fow between the beakers.) The metal wire (dark blue) provides a resistance-free path for electrons, and a voltmeter then measures the redox potential of substance A. If electrons fow from Areduced to H+, as indicated here, the redox pair formed by substance A is said to have a negative redox potential. If they instead fow from H2 to Aoxidized,


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Cell_Biology_Alberts. of substance A. If electrons fow from Areduced to H+, as indicated here, the redox pair formed by substance A is said to have a negative redox potential. If they instead fow from H2 to Aoxidized, the redox pair is said to have a positive redox potential. THE STANDARD REDOX POTENTIAL, E˜0 The standard redox potential for a redox pair, defned as E0 , is measured for a standard state where all of the reactants are at a concentration of 1 M, including H+ . Since biological reactions occur at pH 7, biologists instead defne the standard state as Areduced = Aoxidized and H+ = 10–7 M. This standard redox potential is designated by the symbol E0 , in place of E0. ° PANEL 14–1: Redox Potentials 765


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Cell_Biology_Alberts. CALCULATION OF ˜Go FROM REDOX POTENTIALS E0 ° E0 ° To determine the energy change for an electron transfer, the ˜G o of the reaction (kJ/mole) is calculated as follows: ˜G o = –n(0.096) ˜E0 , where n is the number of electrons transferred across a redox potential change of ˜E0 millivolts (mV), and ˜E0 = (acceptor) – (donor) EXAMPLE: ° ° ° EFFECT OF CONCENTRATION CHANGES As explained in Chapter 2 (see p. 60), the actual free-energy change for a reaction, ˜G, depends on the concentration of the reactants and generally will be different from the standard free-energy change, ˜Go. The standard redox potentials are for a 1:1 mixture of the redox pair. For example, the standard redox potential of –320 mV is for a 1:1 mixture of NADH and NAD+. But when there is an excess of NADH over NAD+, electron transfer from NADH to an electron acceptor becomes more favorable. This is refected by a more negative redox potential and a more negative ˜G for electron transfer.


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Cell_Biology_Alberts. ˜E0 = +30 – (–320) = +350 mV The same calculation reveals that the transfer of one electron from ubiquinone to oxygen has an even more favorable ˜Go of –76 kJ/mole. The ˜Go value for the transfer of one electron from NADH to oxygen is the sum of these two values, –110 kJ/mole. ˜Go = –n(0.096) = –1(0.096)(350) = –34 kJ/mole ° °˜E0 ° ° For the transfer of one electron from NADH to ubiquinone: Figure 14–15 The structure of the heme group attached covalently to cytochrome c. the porphyrin ring of the heme is shown in red. there are six different cytochromes in the respiratory chain. Because the hemes in different cytochromes have slightly different structures and are kept in different local environments by their respective proteins, each has a different affinity for an electron, and a slightly different spectroscopic signature. exploited by the membrane protein complexes in the respiratory chain to move electrons both within and between complexes.


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Cell_Biology_Alberts. exploited by the membrane protein complexes in the respiratory chain to move electrons both within and between complexes. Unlike the colorless atoms H, C, N, and O that constitute the bulk of biological molecules, transition metal ions are often brightly colored, which makes the proteins that contain them easy to study by spectroscopic methods using visible light. One family of such colored proteins, the cytochromes, contains a bound heme group, in which an iron atom is tightly held by four nitrogen atoms at the corners of a square in a porphyrin ring (Figure 14–15). Similar porphyrin rings are responsible both for the red color of blood and for the green color of leaves, binding an iron in hemoglobin or a magnesium in chlorophyll, respectively.


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Cell_Biology_Alberts. Iron–sulfur proteins contain a second major family of electron-transfer cofactors. In this case, either two or four iron atoms are bound to an equal number of sulfur atoms and to cysteine side chains, forming iron–sulfur clusters in the protein (Figure 14–16). Like the cytochrome hemes, these clusters carry one electron at a time. The simplest of the electron-transfer cofactors in the respiratory chain—and the only one that is not always bound to a protein—is a quinone (called ubiquinone, or coenzyme Q). A quinone (Q) is a small hydrophobic molecule that is freely mobile in the lipid bilayer. This electron carrier can accept or donate either one or two electrons. Upon reduction (note that reduced quinones are called quinols), it picks up a proton from water along with each electron (Figure 14–17).


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Cell_Biology_Alberts. In the mitochondrial electron-transport chain, six different cytochrome hemes, eight iron–sulfur clusters, three copper atoms, a flavin mononucleotide (another electron-transfer cofactor), and ubiquinone work in a defined sequence to carry electrons from NADH to O2. In total, this pathway involves more than 60 different polypeptides arranged in three large membrane protein complexes, each of which binds several of the above electron-carrying cofactors.


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Cell_Biology_Alberts. As we would expect, the electron-transfer cofactors have increasing affinities for electrons (higher redox potentials) as the electrons move along the respiratory chain. The redox potentials have been fine-tuned during evolution by the protein environment of each cofactor, which alters the cofactor’s normal affinity for electrons. Because iron–sulfur clusters have a relatively low affinity for electrons, they predominate in the first half of the respiratory chain; in contrast, the heme cytochromes predominate further down the chain, where a higher electron affinity is required. NaDh transfers Its Electrons to Oxygen through three Large Enzyme Complexes Embedded in the Inner Membrane


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Cell_Biology_Alberts. NaDh transfers Its Electrons to Oxygen through three Large Enzyme Complexes Embedded in the Inner Membrane Membrane proteins are difficult to purify because they are insoluble in aqueous solutions, and they are easily disrupted by the detergents that are required to solubilize them. But by using mild nonionic detergents, such as octylglucoside or dodecyl maltoside (see Figure 10–28), they can be solubilized and purified in their native form, and even crystallized for structure determination. Each of the three different detergent-solubilized respiratory-chain complexes can be re-inserted


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Cell_Biology_Alberts. Figure 14–16 The structure of an iron–sulfur cluster. these dark brown clusters consist either of four iron and four sulfur atoms, as shown here, or of two irons and two sulfurs linked to cysteines in the polypeptide chain via covalent sulfur bridges, or to histidines. although they contain several iron atoms, each iron–sulfur cluster can carry only one electron at a time. Nine different iron– sulfur clusters participate in electron transport in the respiratory chain. into artificial lipid bilayer vesicles and shown to pump protons across the membrane as electrons pass through them. In the mitochondrion, the three complexes are linked in series, serving as electron-transport-driven H+ pumps that pump protons out of the matrix to acidify the crista space (Figure 14–18): 1.


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Cell_Biology_Alberts. In the mitochondrion, the three complexes are linked in series, serving as electron-transport-driven H+ pumps that pump protons out of the matrix to acidify the crista space (Figure 14–18): 1. The NADH dehydrogenase complex (often referred to as Complex I) is the largest of these respiratory enzyme complexes. It accepts electrons from NADH and passes them through a flavin mononucleotide and eight iron–sulfur clusters to the lipid-soluble electron carrier ubiquinone. The reduced ubiquinol then transfers its electrons to cytochrome c reductase. 2. The cytochrome c reductase (also called the cytochrome b-c1 complex) is a large membrane protein assembly that functions as a dimer. Each monomer contains three cytochrome hemes and an iron–sulfur cluster. The complex accepts electrons from ubiquinol and passes them on to the small, soluble protein cytochrome c, which is located in the crista space and carries electrons one at a time to cytochrome c oxidase. 3.


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Cell_Biology_Alberts. 3. The cytochrome c oxidase complex contains two cytochrome hemes and three copper atoms. The complex accepts electrons one at a time from cytochrome c and passes them to molecular oxygen. In total, four electrons and four protons are needed to convert one molecule of oxygen to water. We have previously discussed how the redox potential reflects electron affinities. Figure 14–19 presents an outline of the redox potentials measured along the respiratory chain. These potentials change in three large steps, one across each proton-translocating respiratory complex. The change in redox potential between any two electron carriers is directly proportional to the free energy released when an electron transfers between them. Each complex acts as an energy-conversion device by harnessing some of this free-energy change to pump H+ across the inner membrane, thereby creating an electrochemical proton gradient as electrons pass along the chain.


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Cell_Biology_Alberts. Figure 14–17 Quinone electron carriers. Ubiquinone in the lipid bilayer picks up one h+ (red) from the aqueous environment for each electron (blue) it accepts, in two steps, from respiratory-chain complexes. the first step involves the acquisition of a proton and an electron and converts the ubiquinone into an unstable ubisemiquinone radical. In the second step, it becomes a fully reduced ubiquinone (called ubiquinol), which is freely mobile as an electron carrier in the lipid bilayer of the membrane. When the ubiquinol donates its electrons to the next complex in the chain, the two protons are released. the long hydrophobic tail (green) that confines ubiquinone to the membrane consists of 6–10 five-carbon isoprene units, depending on the organism. the corresponding electron carrier in the photosynthetic membranes of chloroplasts is plastoquinone, which has almost the same structure and works in the same way. For simplicity, we refer to both ubiquinone and plastoquinone in this


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Cell_Biology_Alberts. in the photosynthetic membranes of chloroplasts is plastoquinone, which has almost the same structure and works in the same way. For simplicity, we refer to both ubiquinone and plastoquinone in this chapter as quinone (abbreviated as Q).


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Cell_Biology_Alberts. Figure 14–18 The path of electrons through the three respiratory-chain proton pumps. (Movie 14.3) the approximate size and shape of each complex is shown. During the transfer of electrons from NaDh to oxygen (blue arrows), ubiquinone and cytochrome c serve as mobile carriers that ferry electrons from one complex to the next. During the electron-transfer reactions, protons are pumped across the membrane by each of the respiratory enzyme complexes, as indicated (red arrows).


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Cell_Biology_Alberts. For historical reasons, the three proton pumps in the respiratory chain are sometimes denoted as Complex I, Complex III, and Complex IV, according to the order in which electrons pass through them from NaDh. Electrons from the oxidation of succinate by succinate dehydrogenase (designated as Complex II) are fed into the electron-transport chain in the form of reduced ubiquinone. although embedded in the crista membrane, succinate dehydrogenase does not pump protons and thus does not contribute to the proton-motive force; it is therefore not considered to be an integral part of the respiratory chain.


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Cell_Biology_Alberts. along the mitochondrial electron _400 transport chain. the redox potential _300 (designated Eʹ0) increases as electrons flow down the respiratory chain to oxygen. the _200 standard free-energy change in kilojoules, _100 ∆G°, for the transfer of each of the two can be obtained from the left-hand ordinate [∆G° = –n(0.096) ∆Eʹ0, where n is the number of electrons transferred across a redox potential change of ∆Eʹ0 mV]. Electrons flow through a respiratory through the multiple electron carriers in 500 each complex (blue arrows). as indicated, part of the favorable free-energy change 600 is harnessed by each enzyme complex to 700 pump h+ across the inner mitochondrial membrane (red arrows). the NaDh 800 dehydrogenase pumps up to four h+ per electron, the cytochrome c reductase complex pumps two, whereas the direction of electron fow


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Cell_Biology_Alberts. X-ray crystallography has elucidated the structure of each of the three respiratory-chain complexes in great detail, and we next examine each of them in turn to see how they work. the NaDh Dehydrogenase Complex Contains Separate Modules for Electron transport and proton pumping The NADH dehydrogenase complex is a massive assembly of membrane and nonmembrane proteins that receives electrons from NADH and passes them to ubiquinone. In animal mitochondria, it consists of more than 40 different protein subunits, with a molecular mass of nearly a million daltons. The x-ray structures of the NADH dehydrogenase complex from fungi and bacteria show that it is L-shaped, with both a hydrophobic membrane arm and a hydrophilic arm that projects into the mitochondrial matrix (Figure 14–20).


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Cell_Biology_Alberts. Electron transfer and proton pumping are physically separated in the NADH dehydrogenase complex, with electron transfer occurring in the matrix arm and proton pumping in the membrane arm. The NADH docks near the tip of the matrix arm, where it transfers its electrons via a bound flavin mononucleotide to a string of iron–sulfur clusters that runs down the arm, acting like a wire to carry electrons to a protein-bound molecule of ubiquinone. Electron transfer to the quinone is thought to trigger proton translocation in a set of proton pumps in the membrane arm, and for this to happen the two processes must be energetically and mechanically linked. A mechanical link is thought to be provided by a 6-nm long, amphipathic α helix that runs parallel to the membrane surface on the matrix side of the membrane arm. This helix may act like the connecting rod in a steam engine to generate a mechanical, energy-transducing power stroke that links the quinone-binding site to the proton-translocating


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Cell_Biology_Alberts. membrane arm. This helix may act like the connecting rod in a steam engine to generate a mechanical, energy-transducing power stroke that links the quinone-binding site to the proton-translocating modules in the membrane (see Figure 14–20).


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Cell_Biology_Alberts. The reduction of each quinone by the transfer of two electrons can cause four protons to be pumped out of the matrix into the crista space. In this way, NADH dehydrogenase generates roughly half of the total proton-motive force in mitochondria. Cytochrome c reductase takes Up and releases protons on the Opposite Side of the Crista Membrane, thereby pumping protons As described previously, when a quinone molecule (Q) accepts its two electrons, it also takes up two protons to form a quinol (QH2; see Figure 14–17). In cytochrome c oxidase complex pumps one per electron. Note that NaDh is not the only source of electrons for the respiratory chain. the flavin FaDh2, which is generated by fatty acid oxidation (see Figure 2–56) and by succinate dehydrogenase in the citric acid cycle (see Figure 2–57), also contributes. Its two electrons are passed directly to ubiquinone, bypassing NaDh dehydrogenase.


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Cell_Biology_Alberts. Figure 14–20 The structure of NADH dehydrogenase. (a) the model of the mitochondrial complex shown here is based on the x-ray structure of the smaller bacterial complex, which works in the same way. the matrix arm of NaDh dehydrogenase (also known as Complex I) contains eight iron–sulfur (FeS) clusters that appear to participate in electron transport. the membrane contains more than 70 transmembrane helices, forming three distinct proton-pumping modules, while the matrix arm contains the electron-transport cofactors. (B) NaDh donates two electrons, via a bound flavin mononucleotide (FMN; yellow), to a chain of seven iron–sulfur clusters (red and yellow spheres). From the terminal iron–sulfur cluster, the electrons pass to ubiquinone (orange). Electron transfer results in conformational changes (black arrows) that are thought to be transmitted to a long amphipathic α helix (purple) on the matrix side of the membrane arm, which pulls on discontinuous transmembrane helices (red) in three


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Cell_Biology_Alberts. (black arrows) that are thought to be transmitted to a long amphipathic α helix (purple) on the matrix side of the membrane arm, which pulls on discontinuous transmembrane helices (red) in three membrane subunits, each of which resembles an antiporter (see Chapter 11). this movement is thought to change the conformation of charged residues in the three proton channels, resulting in the translocation of three protons out of the matrix. a fourth proton may be translocated at the interface of the two arms (dotted line). (C) this shows the symbol for NaDh dehydrogenase used throughout this chapter. (adapted from r.G. Efremov, r. Baradaran and L.a. Sazanov, Nature 465:441–445, 2010. pDB code: 3M9S.) the respiratory chain, ubiquinol tranfers electrons from NADH dehydrogenase to cytochrome c reductase. Because the protons in this QH2 molecule are taken up from the matrix and released on the opposite side of the crista membrane, two protons are transferred from the matrix into the crista


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Cell_Biology_Alberts. reductase. Because the protons in this QH2 molecule are taken up from the matrix and released on the opposite side of the crista membrane, two protons are transferred from the matrix into the crista space per pair of electrons transferred (Figure 14–21). This vectorial transfer of protons supplements the electrochemical proton gradient that is created by the NADH dehydrogenase proton pumping just discussed.


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Cell_Biology_Alberts. Cytochrome c reductase is a large assembly of membrane protein subunits. Three subunits form a catalytic core that passes electrons from ubiquinol to cytochrome c, with a structure that has been highly conserved from bacterial ancestors (Figure 14–22). It pumps protons by a vectorial transfer of protons that involves a binding site for a second molecule of ubiquinone; the elaborate Figure 14–21 How a directional release and uptake of protons by a quinone pumps protons across a membrane.


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Cell_Biology_Alberts. Figure 14–21 How a directional release and uptake of protons by a quinone pumps protons across a membrane. two protons are picked up on the matrix side of the inner mitochondrial membrane when the reaction Q + 2e – + 2h+ → Qh2 is catalyzed by the NaDh dehydrogenase complex. this molecule of ubiquinol (Qh2) diffuses rapidly in the plane of the membrane, becoming bound to the crista side of cytochrome c reductase. When its oxidation by cytochrome c reductase generates two protons and two electrons (see Figure 14–17), the two protons are released into the crista space. the flow of electrons is not shown in this diagram. 770 Chapter 14: Energy Conversion: Mitochondria and Chloroplasts electrons out to Figure 14–22 The structure of cytochrome c reductase. Cytochrome c reductase (also known as the cytochrome b-c1 complex) is a dimer of two identical 240,000-dalton halves, each composed of 11 different protein molecules in mammals.


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Cell_Biology_Alberts. A structure graphic of the entire dimer, showing in color the three proteins that form the functional core of the enzyme complex: cytochrome b(green) and cytochrome c1(blue) are colored in one half, and the Rieske protein (purple) containing an Fe2S2 iron–sulfur cluster (red and yellow) is colored in the other. These three protein subunits interact across the two halves. Transfer of electrons through cytochrome c reductase to the small, soluble carrier protein cytochrome c. Electrons entering from ubiquinol near the matrix side of the membrane are captured by the iron–sulfur cluster of the Rieske protein, which moves its iron–sulfur group back and forth to transfer these electrons to heme c(red). Heme c then transfers them to the carrier molecule cytochrome c.


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Cell_Biology_Alberts. As detailed in Figure 14–23, only one of the two electrons from each ubiquinol is transferred through this path. To increase proton pumping, the second ubiquinol electron is passed to a molecule of ubiquinone bound to cytochrome c reductase on the opposite side of the membrane—near the matrix. (C) This shows the symbol for cytochrome c reductase used throughout this chapter. (PDB code: 1EZV.) redox loop mechanism used is called the Q cycle because while one of the electrons received from each QH2 molecule istransferred from ubiquinone through the complex to the carrier protein cytochrome c, the other electron is recycled back into the quinone pool. Through the mechanism illustrated in Figure 14–23, the Q cycle increases the total amount of redox energy that can be stored in the electrochemical proton gradient. As a result, two protons are pumped across the crista membrane for every electron that is transferred from NADH dehydrogenase to cytochrome c.


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Cell_Biology_Alberts. The Cytochrome c Oxidase Complex Pumps Protons and Reduces O2 Using a Catalytic Iron–Copper Center The final link in the mitochondrial electron-transport chain is cytochrome c oxidase. The cytochrome c oxidase complex accepts electrons from the soluble electron carrier cytochrome c, and it uses yet a different, third mechanism to pump protons across the inner mitochondrial membrane. The structure of the mammalian complex is illustrated in Figure 14–24. The atomic-resolution structures, combined with studies of the effect of mutations introduced into the enzyme by genetic engineering of the yeast and bacterial proteins, have revealed the detailed mechanisms of this electron-driven proton pump.


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Cell_Biology_Alberts. Because oxygen has a high affinity for electrons, it can release a large amount of free energy when it is reduced to form water. Thus, the evolution of cellular respiration, in which O2 is converted to water, enabled organisms to harness much more energy than can be derived from anaerobic metabolism. As we discuss later, the availability of the large amount of energy released by the reduction of molecular oxygen to form water is thought to have been essential to the emergence of multicellular life: this would explain why all large organisms respire. The ability of biological systems to use O2 in this way, however, requires sophisticated chemistry. Once a molecule of O2 has picked up one electron, it forms a superoxide radical anion (O2•–) that is dangerously reactive and rapidly takes up an additional three electrons wherever it can get them, with destructive effects on its immediate environment. We can tolerate oxygen in the air we breathe only because the uptake of the first


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Cell_Biology_Alberts. up an additional three electrons wherever it can get them, with destructive effects on its immediate environment. We can tolerate oxygen in the air we breathe only because the uptake of the first electron by the O2 molecule is slow, allowing cells to use enzymes to control electron uptake by oxygen. Thus, cytochrome c oxidase holds


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Cell_Biology_Alberts. Figure 14–23 The two-step mechanism of the cytochrome c reductase Q-cycle. In step 1, ubiquinol reduced by NaDh dehydrogenase docks to the cytochrome c reductase complex. Oxidation of the quinol produces two protons and two electrons. the protons are released into the cristae space. One electron passes via an iron-sulfur cluster to heme c1, and then to the soluble electron carrier protein cytochrome c on the membrane surface. the second electron passes via hemes bL and bh to a ubiquinone (red Q) bound at a separate site near the matrix side of the protein. Uptake of a proton from the matrix produces an ubisemiquinone radical (see Figure 14–17), which remains bound to this site (red Qh• in B).


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Cell_Biology_Alberts. In step 2, a second ubiquinol (blue Qh2) docks and releases two protons and two electrons, as described for step 1. One electron is passed to a second cytochrome c, whereas the other electron is accepted by the ubisemiquinone. the ubisemiquinone takes up a proton from the matrix and is released into the lipid bilayer as fully reduced ubiquinol (red Qh2). On balance, the oxidation of one ubiquinol in the Q cycle pumps two protons through the membrane by a directional release and uptake of protons (see Figure 14–21), while releasing another two into the cristae space. In addition, in each of the two steps and (B), one electron is transferred to a cytochrome c carrier (Movie 14.4).


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Cell_Biology_Alberts. Figure 14–24 The structure of cytochrome c oxidase. the final complex in the mitochondrial electron-transfer chain consists of 13 different protein subunits, with a total mass of 204,000 daltons. (a) the entire dimeric complex is shown, positioned in the crista membrane. the highly conserved subunits I (green), II (purple), and III (blue) are encoded by the mitochondrial genome, and they form the functional core of the enzyme.


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Cell_Biology_Alberts. (B) the functional core of the complex. Electrons pass through this structure from cytochrome c via bound copper ions (blue spheres) and hemes (red) to an O2 molecule bound between heme a3 and a copper ion. the four protons needed to reduce O2 to water are taken up from the matrix; see also Figure 14–25. (C) this shows the symbol for cytochrome c oxidase used throughout this chapter. (pDB code: 2OCC.) 772 Chapter 14: Energy Conversion: Mitochondria and Chloroplasts on to oxygen at a special bimetallic center, where it remains clamped between a heme-linked iron atom and a copper ion until it has picked up a total of four electrons. Only then are the two oxygen atoms of the oxygen molecule safely released as two molecules of water (Figure 14–25).


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Cell_Biology_Alberts. The cytochrome c oxidase reaction accounts for about 90% of the total oxygen uptake in most cells. This protein complex is therefore crucial for all aerobic life. Cyanide and azide are extremely toxic because they bind to the heme iron atoms in cytochrome c oxidase much more tightly than does oxygen, thereby greatly reducing ATP production. the respiratory Chain Forms a Supercomplex in the Crista Membrane