Chapter 5: Aerobic Respiration and the Mitochondrion

1. Chapter 5: Aerobic Respiration and the Mitochondrion

2. Mitochondrial outer membrane • ~50% lipid by weight • Contains many enzymes involved in diverse activities: epinephrine oxidation, tryptophan degradation, fatty acid elongation, etc. • Porin channel is surrounded by a barrel of β strands • If porin channels wide open, outer membrane is freely permeable to molecules like ATP, NAD & coenzyme A

3. Porins • Molecules up to ~5,000 daltons to penetrate • The intermembrane space & cytoplasm are basically continuous with respect to ATP, NAD, CoA, etc.

4. Mitochondrial inner membrane • Very high protein/lipid ratio (3:1 by weight; ~1 protein/every 15 phospholipids) • >100 different polypeptides; devoid of cholesterol • Rich in the unusual phospholipid cardiolipin • Both the presence of cardiolipin & the absence of cholesterol are characteristic of bacterial plasma membranes

5. Mitochondrial inner membrane • Ca2+-ATPase • Electron transport chain • ATP synthase

6. Mitochondrial matrix • Enzymes • Ribosomes • Circular double-stranded DNAs (encode inner membrane proteins; nuclear DNA codes for some, too) • Humans mitochondrial DNA encodes • 13 mitochondrial polypeptides • rRNAs and 22 tRNAs that are used in protein synthesis within the organelle

7. The role of anaerobic and aerobic metabolism in exercise • Muscle cells contain a store of creatine phosphate (CrP ) • CrP + ADP Cr + ATP • Human skeletal muscles consist of fast-twitch fibers and slow-twitch fibers

8. Fast-twitch fibers • Contract very rapidly; 15 – 40 msec • Nearly devoid of mitochondria • Unable to make much ATP by aerobic respiration

9. Slow-twitch fibers • Contract more slowly; 40 – 100 msec • Have large numbers of mitochondria

10. Aerobic exercise • Energy source • Initially by glucose stored as glycogen in muscles • After a few minutes the muscles depend increasingly on free fatty acids released into blood from adipose (fat) tissue • The longer the exercise period, the greater the dependency on fatty acids

11. Direct evidence for rotation of γ subunit relative to αβ subunits • Prepared a genetically engineered version of working part of ATP synthase (3α, 3β & a γ [α3β3γ]) • Fixed polypeptide complex to glass coverslip by its head & attached short, fluorescently labeled actin filament to γ subunit end jutting into medium • Add ATP & rotation seen (like propellor) • Powered by energy released as ATPs were bound & catalyzed by β subunit catalytic sites

12. The mechanism by which H+ movement drives c ring rotation • Each a subunit has 2 half-channels that are physically separated (offset) from one another • One half-channel leads from intermembrane (cytosolic) space into the middle of the a subunit; the other leads from the middle of the a subunit into the matrix • Each proton moves from the intermembrane space through the half-channel & binds to a negatively charged Asp residue situated at the surface of the adjoining c subunit

13. The mechanism by which H+ movement drives c ring rotation • Binding of H+ to Asp carboxyl group generates a major conformational change in the c subunit that causes the subunit to rotate ~30° in a counterclockwise direction • This movement of the recently protonated c subunit brings the adjoining ring subunit (protonated at an earlier step) into alignment with the second a subunit half-channel

14. The mechanism by which H+ movement drives c ring rotation • The Asp releases its associated proton, which diffuses into the matrix • After proton dissociation, the c subunit then returns to its original conformation & is ready to accept another proton from the intermembrane space & repeat the cycle

15. Peroxisomes • Found in 1954 & called microbody • Simple membrane-bound vesicles with 0.1 - 1.0 µm diameter • Often have dense, crystalline core of an oxidative enzyme(s) & consequently granular appearance • Multifunctional organelles containing >50 enzymes involved in diverse activities like: • Oxidation of very long chain fatty acids (VLCFAs); whose chains typically contain 24 – 26 C

16. Peroxisomes • Synthesis of plasmalogens • Abnormalities in plasmalogen synthesis can lead to severe neurological dysfunction • Luciferase • which generates light emitted by fireflies, is also a peroxisomal enzyme

17. Peroxisomes • Named peroxisomes since they are the site of synthesis & degradation of H2O2 • H2O2 is produced by a number of peroxisomal enzymes • Urate oxidase, glycolate oxidase & amino acid oxidases that utilize molecular oxygen to oxidize their respective substrates • Catalase (at high concentration in peroxisomes) rapidly breaks down H2O2 generated in these reactions

18. Peroxisomes • Form by splitting from preexisting organelles • Import preformed proteins from cytosol • Do similar kinds of oxidative metabolism in mitochondria • Alanine/glyoxylate aminotransferase, is seen in the mitochondria of some mammals (cats, dogs) & peroxisomes of others (rabbits, humans)

19. Glyoxysomes • A specialized type of peroxisome found only in plants • Contain some of same enzymes (catalase, fatty acid oxidase), but others as well • Plant seedlings rely on stored fatty acids to provide energy & material to form new plant • Glyoxylate cycle

20. Glyoxysomes • A primary metabolic activity in these germinating seedlings is the conversion of stored fatty acids to carbohydrate • Stored fatty acid disassembly produces acetyl CoA & it condenses with oxaloacetate to form citrate • Citrate is then converted to glucose by a series of glyoxylate cycle enzymes found in glyoxysomes

22. Diseases result from abnormal mitochondrial or peroxisomal function • Muscle & nerve tissues tend to be most seriously impacted in these disorders since they have the highest demand for ATP • Depending on protein(s) affected, conditions vary in severity from diseases that lead to death during infancy to disorders that produce seizures (中風驟發), blindness, deafness and/or strokelike episodes • Sometimes conditions are mild & characterized by intolerance to exercise or nonmotile sperm

23. Abnormal mitochondria • Closer examination of mitochondria reveals large numbers of abnormal inclusions • A number of common neurological diseases with adult onset (like Parkinson's disease) might be a consequence of degenerative changes in mitochondrial function • The first such disease-causing mutation was reported in 1995 • The mutation occurred in gene encoding the flavoprotein subunit of the TCA enzyme succinate dehydrogenase

24. Mitochondrial disorder inheritance contrasts in several ways with nuclear gene Mendelian inheritance • Mitochondria in cells of human embryo are derived exclusively from mitochondria present in the egg at the time of conception without any contribution from the fertilizing sperm • Mitochondrial disorders are inherited maternally • Mitochondria in cell can contain mixture of normal (wild-type) & mutant mtDNA (heteroplasmy)

25. mtDNA Mutation • Nuclear DNA is protected from damage by a variety of DNA repair systems which are generally lacking in mitochondria • mtDNA may also be subjected to high levels of mutagenic oxygen radicals • mtDNA experiences >10 times the mutation rate of nuclear DNA

26. Abnormal peroxisomes • Zellweger syndrome (ZS) is a rare inherited disease characterized by a variety of neurological, visual & liver abnormalities leading to death during early infancy • Sidney Goldfischer et al. (1973) – reported that liver & renal cells from these patients lacked peroxisomes • Later studies showed that peroxisomes were not entirely absent from the cells of these individuals

27. Zellweger syndrome (ZS) • Peroxisomes were present as empty membranous ghosts (organelles lacking the enzymes normally found in peroxisomes) • These individuals can make peroxisomal enzymes but the enzymes fail to be imported into peroxisomes & stay largely in cytosol where they are unable to carry out their normal functions • Mutations in at least 11 different genes • Encoding proteins involved in uptake of peroxisomal enzymes from cytosol

28. Adrenoleukodystrophy (ALD), subject of the movie Lorenzo's Oil • Absence of a single peroxisomal enzyme • A defect in a membraneprotein that transports very-long-chain-fatty-acids (VLCFAs) into the peroxisomes where they are normally metabolized • In the absence of this protein, VLCFAs accumulate in brain & destroy myelin sheaths that insulate nerve cells • Boys with the disease are typically unaffected until midchildhood, when symptoms of adrenal insufficiency & neurological dysfunction begin

29. Adrenoleukodystrophy (ALD) • A diet rich in certain fatty acids is able to retard the progress of the disease • A number of ALD patients have been successfully treated by bone marrow transplantation, which provides normal cells capable of metabolizing VLCFAs • Administration of drugs (e.g., lovastatin) that may lower VLCFA levels • Clinical studies employing gene therapy are also being planned

30. Chapter 8-1: Cytoplasmic Membrane Systems: Structure, Function, and Membrane Trafficking

31. Endomembrane system • Plasma membrane, vesicles, vacuoles, ER, Golgi apparatus, nuclear membrane, lysosome • Have distinct structures & functions but together form an endomembrane system • Dynamic, integrated network • Materials are shuttled (transport vesicles) between the endomembrane system

32. Transport vesicles in endomembrane system • Transport vesicles form by budding from donor compartment • Transport vesicles move in directed manner, often pulled by motor proteins operating on tracks formed by microtubules & microfilaments of the cytoskeleton • When they reach their destination, they fuse with acceptor compartment

33. Transport in endomembrane system • Endocytic pathway • Exocytotic pathway • Secretory pathway

34. Biosynthetic (secretory) pathway • Synthesis in ER (protein) or Golgi (lipid, carbohydrate) • Many materials made in ER (proteins) & Golgi (complex polysaccharides) fated for secretion from cell • Two types of secretory activity • Constitutive secretion • Regulated secretion

35. Constitutive secretion • Synthesis & secretion into extracellular space occurs in continual, unregulated manner • Form extracellular matrix & plasma membrane

36. Regulated secretion • Secretory materials stored in large, densely packed, membrane-bound secretory granules in cell periphery • Secreted after correct stimulus • Endocrine cells release hormones • Pancreatic acinar cells release digestive enzymes • Nerve cells release neurotransmitters

37. Proteins targeting • Through sorting signals located on proteins & receptors in transport vesicle walls that recognize them • Salivary gland cell protein trafficking • Salivary mucus proteins (made in ER) specifically targeted to secretory granules • Lysosome enzymes (also made in ER) specifically sent to lysosome • Sorting signals are encoded in protein amino acid sequence or in attached oligosaccharides

38. Approaches to the study of cytomembranes • EM micrographs give detailed view of cell cytoplasm, but little insight into functions of the structures • Insights gained from autoradiography • Detect location of radioactively labeled materials in cell • Insights from pulse-chase trials

39. Pulse-chase trials • Expose to hot amino acids briefly (pulse) • Wash to remove excess isotope from tissue • Transferred tissue to medium with unlabeled amino acids (chase), which lasts for varying time periods • See wave of radioactivity moving through cell, discern pathway sequence

40. Use of green fluorescent protein (GFP) reveals the movement of proteins within a living cell • GFP is small protein from certain jellyfish that emits a green fluorescent light • GFP gene fused to DNA encoding protein to be studied • Introduce the chimeric DNA into cells • Chimeric DNA expresses chimeric protein (GFP fused to the protein to be studied)

41. Use of green fluorescent protein (GFP) reveals the movement of proteins within a living cell • Usually, GFP stuck to end of a protein has little or no effect on its movement or function & protein under study has no effect on fluorescence of attached GFP

42. Example: infect a mammalian cell with vesicular stomatitis virus (VSV) strain in which a viral gene (VSVG) is fused to GFP gene • Cell begins to make massive amounts of VSVG protein in RER • VSVG then goes to Golgi complex & eventually to the plasma membrane of the infected cell where they are incorporated into viral envelopes • Can see relatively synchronous wave of protein movement (green fluorescence) soon after infection

43. Infect a mammalian cell with vesicular stomatitis virus (VSV) strain in which a viral gene (VSVG) is fused to GFP gene • Synchrony is enhanced by use of virus with mutant VSVG protein that cannot leave ER of infected cells grown at elevated temperature (40°C). • The green fluorescence is restricted to the ER.

44. When temperature is lowered to 32°C, the fluorescent GFP-VSVG protein that had accumulated in ER moves synchronously to Golgi complex for various processing events & then to membrane • Temperature-sensitive mutants • Permissive temperature Mutants function normally • Restrictive temperatures Mutants function abnormally

45. Cell fractionation • Homogenization • Organelles fractionation by centrifugation