MITOCHONDRIA. Speaker: 陳玉怜 Mar. 2018

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1 MITOCHONDRIA Speaker: 陳玉怜 Mar. 2018

2 1. Structure 2. Genetic system 3. Function: ATP synthesis 4. Dysfunction: cell apoptosis

3 eukaryotic cells mitochondrion mitochondria the specialized membranes inside energy-converting organelles are employed for the production of ATP.

4 * occupy a substantial portion of the cytoplasmic volume * the metabolism of sugars is completed: the pyruvate is imported into the mitochondrion and oxidized by O 2 to CO 2 and H 2 O. (This allows 15 times more ATP to be made than that produced by glycolysis alone).

6 * stiff, elongated cylinders (a diameter of µm) * are remarkably mobile and plastic organelles * move - often seem to be associated with microtubules. * remain fixed in one position where they provide ATP directly to a site of high ATP consumption (packed between adjacent myofibrils in a cardiac muscle cell; wrapped rightly around the flagellum in a sperm)

7 Mitochondrial plasticity -time-lapse microcinematography -rapid changes of shape in a living cell

8 Dynamic mitochondrial reticulum * form a continuous reticulum underlying the plasma membrane * a balance of fission and fusion determines the arrangement of the mitochondria * shape change; the network is constantly remodeled by fission and fusion.

9 Mitochondrial fission and fusion -Involve both outer and inner mitochondrial membrane (movie)

10 The relationship between mitochondria and microtubules Mitochondria microtubules The mitochondria tend to be aligned along microtubules.

11 Localization of mitochondria near sites of high ATP utilization in cardiac muscle

12 Localization of mitochondria near sites of high ATP utilization in a sperm tail

13 The mitochondrion: *an outer membrane *an inner membrane *two internal compartments (matrix and intermembrane space)

$Biochemical fractionation of purified$

14 Biochemical fractionation of purified mitochondria into separate components

16 The outer membrane - a transport protein (porin) -forms large aqueous channels through the lipid bilayer. - a sieve: all molecules of 5000 daltons or less (small proteins). Such molecules can enter the intermembrane space, but most of them cannot pass the impermeable inner membrane.

17 the inner membrane Its lipid bilayer contains a high proportion of the "double" phospholipid cardiolipin (has four fatty acids rather than two and may help to make the membrane especially impermeable to ions)

18 Inner membrane - contains a variety of transport proteins that make it selectively permeable to those small molecules that are metabolized or required by the many mitochondrial enzymes concentrated in the matrix. - enzymes of the respiratory chain, are essential to the process of oxidative phosphorylation, which generates most of the animal cell's ATP.

19 cristae (movie) * The inner membrane is usually highly convoluted, forming a series of infoldings that project into the matrix. increase the area of the inner membrane * a liver cell: constitutes about onethird of the total cell membrane. * cardiac muscle cells: many cristae

20 Matrix The matrix enzymes include those that metabolize pyruvate and fatty acids to produce acetyl CoA and those that oxidize acetyl CoA in the citric acid cycle.

22 Matrix granules * Phospholipid * Glycoprotein * Lipid * Calcium precipitable lipoprotein * Cytochrome c oxidase

25 1. Structure 2. Genetic system 3. Function: ATP synthesis 4. Dysfunction: cell apoptosis

Red: nuclear genome Bright yellow spots: mitochondrial genome Green: mitochondrial matrix space (A) immunogold

26 THE GENETIC SYSTEMS OF MITOCHONDRIA -contain their own genomes, as well as their own biosynthetic machinery for making RNA and organelle proteins. Red: nuclear genome Bright yellow spots: mitochondrial genome Green: mitochondrial matrix space (A) immunogold labelling with anti-dna; gold particles marking mtdna are found near the mitochondrial membrane. (B) Whole mount view of and immunogold labelling with anti-dna; mtdna (marked by gold particles) resists extraction.

27 Mitochondria contain complete genetic systems X

28 the process of DNA replication

29 Why do mitochondria have their own genetic systems?

30 Two separate genetic systems one in the organelle and one in the cell nucleus. Most of the proteins - by nuclear DNA, synthesized in the cytosol, and then imported individually into the organelle. Some organelle proteins and RNAs - the organelle DNA and are synthesized in the organelle itself. The human mitochondrial genome contains about nucleotides and encodes 2 ribosomal RNAs, 22 transfer RNAs, and 13 different polypeptide chains.

31 1. Structure 2. Genetic system 3. Function: ATP synthesis 4. Dysfunction: cell apoptosis

32 Chemiosmotic coupling (chemiosmosis) * the chemical bond-forming reactions that generate ATP ("chemi") * membrane-transport processes ("osmotic"). * The coupling process occurs in two linked stages, both of which are performed by protein complexes embedded in a membrane.

33 proton-motive force

34 High-energy electrons are generated via the citric acid cycle Fuel: pyruvate (from glucose and other sugars) and fatty acids (from fats) -are transported across the inner mitochondrial membrane and then converted to the crucial metabolic intermediate acetyl CoA by enzymes located in the mitochondrial matrix. The acetyl groups in acetyl CoA are then oxidized in the matrix via the citric acid cycle.

35 Energy generating metabolism in mitochondria

36 Pyruvate+HS-CoA+NAD + acetyl CoA+CO 2 +NADH+H + decarboxylation NADH: reduced nicotinamide adenine dinucleotide

37 The tricarboxylic acid cycle (TCA cycle) 三羧酸循環檸檬酸循環 Krebs cycle 2C 菸鹼醯胺腺嘌呤二核苷酸 Nicotinamide adenine dinucleotide 4C 4C 6C 6C 4C 4C 4C 5C flavin adenine dinucleotide reduced 黃素腺嘌呤二核苷酸 Acetyl CoA+2H 2 O+FAD+3NAD++GDP+Pi 2CO 2 +FADH 2 +3NADH+3H + +GTP+HS-CoA

Spectroscopic Methods Have Been Used to Identify Many Electron Carriers in the Respiratory Chain @ cytochromes a, b, and c.

39 Spectroscopic Methods Have Been Used to Identify Many Electron Carriers in the Respiratory cytochromes a, b, and c. - constitute a family of colored proteins that are related by the presence of a bound heme group, whose iron atom changes from the ferric oxidation state (Fe 3+ ) to the ferrous oxidation state (Fe 2+ ) whenever it accepts an electron. heme group = a porphyrin ring +iron atom +four nitrogen atoms

40 @ Iron-sulfur proteins either two or four iron atoms are bound to an equal number of sulfur atoms and to cysteine side chains, forming an iron-sulfur center on the protein.

@ ubiquinone The simplest of the electron carriers in the respiratory chain and the only one that is not part of a protein is a small hydrophobic molecule that is freely

41 @ ubiquinone The simplest of the electron carriers in the respiratory chain and the only one that is not part of a protein is a small hydrophobic molecule that is freely mobile in the lipid bilayer A quinone (Q) can pick up or donate either one or two electrons; upon reduction, it picks up a proton from the medium along with each electron it carries.

42 The Respiratory Chain Includes Three Large Enzyme Complexes Embedded in the Inner Membrane 1.The NADH dehydrogenase complex (complex I) is the largest of the respiratory enzyme complexes, containing more than 40 polypeptide chains. It accepts electrons from NADH and passes them through a flavin and at least seven iron-sulfur centers to ubiquinone. Ubiquinone then transfers its electrons to a second respiratory enzyme complex, the cytochrome b-c 1 complex. 2.The cytochrome b-c 1 complex contains at least 11 different polypeptide chains and functions as a dimer. Each monomer contains three hemes bound to cytochromes and an iron-sulfur protein. The complex accepts electrons from ubiquinone and passes them on to cytochrome c, which carries its electron to the cytochrome oxidase complex. 3.The cytochrome oxidase complex also functions as a dimer; each monomer contains 13 different polypeptide chains, including two cytochromes and two copper atoms. The complex accepts one electron at a time from cytochrome c and passes them four at a time to oxygen.

43 * During the transfer of electrons from NADH to oxygen, ubiquinone and cytochrome c serve as mobile carriers. * Protons are pumped across the membrane by each of the respiratory enzyme complexes.

44 How electrons are donated by NADH A hydride ion (H - a hydrogen atom and extra electron) NADH: reduced nicotinamide adenine dinucleotide NAD + : nicotinamide adenine dinucleotide

45 The process of electron transport - The hydride ion is removed from NADH (to regenerate NAD + ) and is converted into a proton and two electrons (H - H + + 2e - ). - Each of these ions being tightly bound to a protein molecule that alters the electron affinity of the metal ion. Most of the proteins involved are grouped into three large respiratory enzyme complexes.

46 A chemiosmotic process converts oxidation energy into ATP These electrons, carried by NADH and FADH 2, are then combined with O 2 by means of the respiratory chain embedded in the inner mitochondrial membrane. The large amount of energy released is harnessed by the inner membrane to drive the conversion of ADP + Pi to ATP (oxidative phosphorylation).

47 The NADH dehydrogenase complex (complex I) -It accepts electrons from NADH and passes them through a flavin and at least seven iron-sulfur centers to ubiquinone. - Ubiquinone then transfers its electrons to a second respiratory enzyme complex, the cytochrome b-c1 complex.

48 The cytochrome b-c1 complex - Each monomer contains three hemes bound to cytochromes and an ironsulfur protein. - The complex accepts electrons from ubiquinone and passes them on to cytochrome c, which carries its electron to the cytochrome oxidase complex.

49 The cytochrome b-c1 complex contains at least 11 different polypeptide chains and functions as a dimer. Greencytochrome b Blue- Cytochrome c1 Purple- Iron-sulfur center X-ray

50 The atomic structure of cytochrome b-c1

51 The cytochrome oxidase complex -including two cytochromes and two copper atoms. -The complex accepts one electron at a time from cytochrome c and passes them four at a time to oxygen.

52 The molecular structure of cytochrome oxidase - a dimer - each monomer contains 13 different polypeptide chains, including two cytochromes and two copper atoms.

53 An iron-copper center in cytochrome oxidase catalyzes O 2 reduction

55 A general model for H+ pumping

56 Redox potential changes along the mitochondrial electron-transport chain

57 electron transport chain = the entire set of proteins in mem+ small molecules in electron transfer

59 Harnessing energy for life

Chemiosmotic coupling *Electrochemical proton gradient-drive other memembedded protein machines *Special proteins couple the downhill H + flow

60 Chemiosmotic coupling *Electrochemical proton gradient-drive other memembedded protein machines *Special proteins couple the downhill H + flow to the transport of specific metabolites into and out of the organelles *Electrochemical proton gradientrapid rotation of the bacterial flagellum

As electrons move along the respiratory chain, energy stored as an electrochemical proton gradient across the inner membrane - a voltage gradient (the inside

61 As electrons move along the respiratory chain, energy stored as an electrochemical proton gradient across the inner membrane - a voltage gradient (the inside negative and the outside positive) - a ph gradient (the ph higher in the matrix than in the cytosol the ph and the Vconstitute electrochemi cal proton gradient

mitochondrial membrane is used to drive ATP

62 How the Proton Gradient Drives ATP Synthesis The electrochemical proton gradient across the inner mitochondrial membrane is used to drive ATP synthesis in the critical process of oxidative phosphorylation.

63 The major net energy conversion catalyzed by the mitochondrion

64 ATP synthase head portion -F1 ATPase (matrix) F 0 - transmembrane H + carrier -inner mitochondrial membrane

ATP synthase (F 0 F 1 ATPase, more than 500,000 daltons) -a lollipop head and composed of a ring of 6 subunits, projects on the matrix side of the inner mitochondrial

65 ATP synthase (F 0 F 1 ATPase, more than 500,000 daltons) -a lollipop head and composed of a ring of 6 subunits, projects on the matrix side of the inner mitochondrial membrane. a "stator (green) a "rotor" -is formed by a ring of 10 to 14 identical transmembrane protein subunits. (Paul Boyer &John E. Walker: 1997 Nobel Prize in Chemistry)

66 How the Proton Gradient Drives Coupled Transport Across the Inner Membrane In mitochondria, many charged small molecules, such as pyruvate, ADP and Pi, are pumped into the matrix from the cytosol, while others, such as ATP must be moved in the opposite direction. Pyruvate and inorganic phosphate (Pi) are cotransported inward with H + as the H + moves into the matrix. ADP-ATP co-transport is driven by the voltage difference across the membrane.

67 Some of the active transport processes driven by the electrochemical proton gradient across the inner mitochondrial membrane

68 Proton Gradients Produce Most of the Cell's ATP The vast majority of the ATP produced from the oxidation of glucose in an animal cell is produced by chemiosmotic mechanisms in the mitochondrial membrane. Oxidative phosphorylation in the mitochondrion also produces a large amount of ATP from the NADH and the FADH 2 that is derived from the oxidation of fats.

ATP Synthase Can Also Function in Reverse to Hydrolyze ATP and Pump H+ In addition to harnessing the How of H+ down an electrochemical proton gradient to make ATP, the ATP synthase can work in

69 ATP Synthase Can Also Function in Reverse to Hydrolyze ATP and Pump H+ In addition to harnessing the How of H+ down an electrochemical proton gradient to make ATP, the ATP synthase can work in reverse: it can use the energy of ATP hydrolysis to pump H+ across the inner mitochondrial membrane. It thus acts as a reversible coupling device, interconverting electrochemical proton gradient and chemical bond energies.

70 As protons pass through a narrow channel formed at the stator-rotor contact, their movement causes the rotor ring to spin. This spinning also turns a stalk attached to the rotor.

72 1. Structure 2. Genetic system 3. Function: ATP synthesis 4. Dysfunction: cell apoptosis

73 Mitochondrial dysfunction Cause cell death through ATP depletion and Ca 2+ dysregulation. Proteins released from mitochondria (cytochrome c, apoptosis-inducing factor, smac-diablo) Be modulated by regulated targeting of BCL2 family members to the outer mitochondrial membrane.

74 BCL2 family * Antiapoptotic members (Bcl-2, Bcl-x L, Bclw, Mcl-1, A1) * BH1-BH4 * Proapoptotic members (Bax, Bak, Bok, Bcl-rambo, Bcl-GBID) BH1-BH3 * BH3-only protein (numerous pro-apoptotic members)(bad, Bid, Bim, Blk)

75 Schematic drawing of Bcl-2 and its related proteins

76 Activation of Bax/Bak by BH3-only proteins. Once activated, BH-3 only proteins are translocated to the mitochondria to inactivate anti-apototic members of the Bcl-2 family and activate multi-domain members, resulting in an increase of outer mitochondrial membrane permeability. Bad, Bid, Bim, Blk

78 Mechanisms for MOMP during apoptosis Intermembrane space protein ANT: adenine nucleotide transporter PT: permeability transition VDAC: voltage-dependent anion channel Green and Kroemer, Science, 2004

79 Apoptotic signal transduction pathways Apoptotic protease-activating factor 1 Tsujimoto, Journal of cellular physiology, 2003

80 Release of mitochondrial proteins by formation of apoptotic proteins-conducting pores

81 X X X X X Moll et al., 2006 The mitochondrial pro-apoptotic activities of p53

82 EXR: retinoid X receptor The Nur77-Bcl2 apoptotic and survival pathways

83 DBD:DNA binding domain Mechanistic similarities of the transcription-independent pro-death program of p53 and Nut 77 at mitochondria

84 Examples of pathogenic processes involving excessive or deficient MOMP Disease Ischemia reperfusion damage of brain or heart Neurodegenerat ive diseases Liver disease Pathogenic perturbation of MOMP Redox stress, excessive Ca 2+ load, absent adenosine triphosphate and nicotine adenine dinucleotide, and accumulating fatty acids favor PT and MOMP. Respiratory dysfunction affects highly sensitive neurons in the central nervous system, leading to their premature death. Hepatotoxins (including bile acid and ethanol) and hepatitis B or C encoded proteins induce MOMP. Pharmacological correction of deregulated MOMP Bcl-2 inhibitors of the PT pore, as well as mito K ATP channel openers, can exert neuro- or cardioprotective effects. Putative inhibitors of the PT pore (minocyclin, rasagiline, and tauroursodeoxycholic acid) can prevent neurodegeneration. Ursodeoxycholic acid prevents bile acid induced PT and thus exerts hepatoprotective effects.

85 Cancer MOMP-inhibitory proteins from the Bcl-2 family or unrelated proteins (such as Muc1) enhance apoptosis resistance. Cytotoxic agents targeting Bcl- 2 like proteins, PT pore components, and/or mitochondrial lipids enforce MOMP and kill cancer cells.

86 1. Structure 2. Genetic system 3. Function: ATP synthesis 4. Dysfunction: cell apoptosis

CELL BIOLOGY - CLUTCH CH AEROBIC RESPIRATION.

CELL BIOLOGY - CLUTCH CH AEROBIC RESPIRATION. !! www.clutchprep.com CONCEPT: OVERVIEW OF AEROBIC RESPIRATION Cellular respiration is a series of reactions involving electron transfers to breakdown molecules for (ATP) 1. Glycolytic pathway: Glycolysis