醫(yī)學(xué)細(xì)胞生物學(xué)：Chapter 6 Mitochondria and Energy Conversion

1、1Chapter 6 Mitochondria and Energy Conversion自養(yǎng)生物與異養(yǎng)生物Autotroph：light energy+CO2+H2O organic compoundHeterotroph： organic compound H2O + chemical energy (ATP) chloroplasts Mitochondria The sun is the ultimate source of energy all organisms and cellsBrief History of Mitochondria studies Richard Altma

2、nnin(German) 1890, established them as cell organelles and called them bioblasts.The term mitochondria was coined by Carl Benda in 1898.Leonor Michaelis discovered Janus green can be used as a supravital stain for mitochondria in 1900. 1912,Kingsburg confirmed mitochondria is the place of oxidation-

3、reduction reaction.In 19131950, to explore the function of mitochondria. E.g. particles from extracts of guinea-pig liver were linked to respiration by Otto Heinrich Warburg, which he called grana. The first high resolution micrographs appeared by Palade and Sjostrand in 1952-1953, replacing the Jan

4、us Green stains .The popular term powerhouse of the cell was coined by Philip Siekevitz in 1957.1963年，Nass，雞胚線粒體中存在DNA。 Schatz，分離到完整的mtDNA；1981年，測定人mtDNA的DNA序列；1987年，Wallac，mtDNA突變可引起疾??；1988年，首次報(bào)道m(xù)tDNA突變。 Section 1Mitochondrial Basic CharactersMit under Light microscope: tubular or filamentous organ

5、elles1. Shape, size, amounts Mitochondria are often flexible, rod-shaped organelles that are about 0.5 to 1m in diameter and 1.53.0m in length. Mitochondria vary considerably in size & shape, (e.g. they can be round). Their number correlate with the metabolic activities of the cell. Mit. Occupy 15 t

6、o 20 percent of the cells volume, E.g. in liver cells, 800-2000 Mit. In muscle cells which require large amounts of ATP to fuel their contraction.(heart and kidney cells)Part 1 Shape, size, amounts and Structure of MitVariational shapes of MitRelationship between mitochondria and microtubules.The si

7、ze and number of mitochondria reflect the energy requirements of the cell.2. Mitochondrial ultrastructure Mitochondria (false color scanning EM)(1) outer membrane（外膜） 5一7nm thickness,fairly smooth，通透性較高 Contains channel-forming protein, called Porin（孔蛋白）. Permeable to all molecules of 10000 daltons

8、or less. The composition of the intermembrane space is similar to the cytosol.(2) inner membrane and spaces（內(nèi)膜） 4.5nm thickness.高度的選擇通透性a surface area 5 times the outer membrane Contains proteins with three types of functions: Electron-transport chain: Carry out oxidation reactions; ATP synthase: Ma

9、kes ATP in the matrix Transport proteins: Allow the passage of metabolitesPermeable to all molecules under 150 daltons. cristaeThe multiply-folded inner membraneintermembrane space(膜間腔，外腔) matrix space(基質(zhì)腔，內(nèi)腔) Intercristae space嵴間腔，Intracristae space嵴內(nèi)空間 finger-like projections The elementary partic

10、les can be seen on cristae which are the site where ATP is generated (3) Translocation contact site （轉(zhuǎn)位接觸點(diǎn)） 結(jié)構(gòu)：內(nèi)膜轉(zhuǎn)位子 + 外膜轉(zhuǎn)位子Translocon of the outer membrane, TomTranslocon of the inner membrane, Tim(4) The matrix Matrix has a gel-like consistency owing to the presence of a high concentration (up to

11、500 mg/ml ) of water-soluble proteins, the mixture contains :hundreds of enzymes, including those required for the oxidation of pyruvate and fatty acids and for TCA cycle. mtDNA genome, 70s mt-ribosomes, tRNAs, and various enzymes required for expression of the mt-genes.(5) elementary particlesThe e

12、lementary particles can be seen on cristae作用： ADP+Pi ATP Part 2 Chemical composition of Mitochondra低滲膨脹或洋地黃皂甙處理細(xì)胞；離心，分離線粒體；3. 線粒體低滲處理，離心。膜間隙成分分離；4. 置高滲液中，密度梯度離心，使外膜與重的線粒體質(zhì)分離；5. 去垢劑處理，內(nèi)膜小泡與 基質(zhì)分離。1Protein(enzyme):65%-70% 可溶性蛋白，不可溶性蛋白 酶：120余種，氧化還原酶、水解酶、合成酶等，不同部位有標(biāo)志酶；Fat:25% -30% 以磷脂為主，內(nèi)膜主要含心磷脂，膽固醇含量低。D

13、NA、RNA輔酶與維生素：CoQ、FMN、FAD、NAD+；無機(jī)鹽離子：Fe2+、Fe3+、Cu+、Cu2+、Ca2+等；Inner membrane: protein：fat =1：1 outer membrane： protein：fat =4：1Localization of metabolic functions within the mitochondrion Outer membrane: Phospholipid synthesisfatty acid desaturationFatty acid elongationInner membrane:Electron transpo

14、rtOxidative phosphorylationMetabolite transport MatrixPyruvate oxidationTCA cycle oxidation of fatsDNA replication, RNA transcriptionProtein translationIntermembrane spaceNucleotide phosphorylationLocalization of metabolic functions within the mitochondrion 線粒體主要酶的分布o(jì)uter membrane （monoamine oxidase

15、）（cytochrome oxidase）inner membrane（adenylate kinase）（malate dehydrogenase）intermembrane spaceMatrixMarker enzymesPart 3 Mitochondrial genetic system Mitochondria have their own DNA in the matrix (genetic system, which is separate and distinct from the nuclear genome of the cell), and can make many

16、of their own proteins.1251. mtDNA Characteristics of mtDNA genomecircular DNA molecule like bacteria. The mtDNA structure is very tight (No intron).There is no histones combine with mtDNA.mtDNA is asymmetricCodons are not rigidly paired with anticodonsSome codons means different sense.Codes for only

17、 about 11 proteins. Also rRNA and all tRNAs.The structure of Mitochondrial genomecircle dsDNA:Outer circle (H) and inner circle (L ) Mitochondria also have their own ribosomes and tRNA(22 tRNAs ),2rRNAs:16S,12S ( 5S rRNA is imported into mitochondria, but its function is uncertain)Human mtDNAHuman m

18、itochondrial DNA is 16569 bp; encodes 37 genes, including 13 mitochondrial proteinsSubunits 1, 2, and 3 of cytochrome c oxidase Subunits 6, 8 of the Fo ATPase Apocytochrome b subunit of CoQH2-Cytochrome C reductase Seven NADH-CoQ reductase subunits（ND1、ND2、ND4、ND4L、ND5、ND6、ND7）The nucleus encodes th

19、e remaining proteins. 1mtDNA結(jié)構(gòu)16569 bpComplexComplexComplexATPase 8/6rRNAtRNAH鏈編碼：2個(gè)rRNA，14個(gè)tRNA，12個(gè)蛋白質(zhì)L鏈編碼：8個(gè)tRNA，1個(gè)蛋白質(zhì)。 Human mtDNA2. mtDNA transcription The mtDNA is transcribed by RNA polymerases that are encoded in the nuclear DNA. These mitochondrial RNA polymerases are far simpler than the th

20、ree eukaryotic RNA the two mitochondrial promoters(H chain and L chain)重鏈啟動(dòng)子（heavy-strand promoter，HSP） 輕鏈啟動(dòng)子（light-strand promoter，LSP） mitochondrial transcription factor 1,mtTFA mtRNA直接在線粒體的核糖體上合成蛋白質(zhì) 所有線粒體tRNA都是mtDNA編碼1mtDNA具有半自主性ComplexSubunitsNuclearmtDNA413474401110113103ATPastDNA的

21、遺傳特性1mtDNA的遺傳密碼與通用密碼不同MetIleAUAMetMetAUGStopArgAGAStopArgAGGTrpStopUGATrpTrpUGGmtDNAUniversal codeCodonmtDNA的遺傳特性線粒體使用核基因的通用密碼，但也有些例外mitochondrial genomes encodeonly a small number of proteins that are essential components of the oxidative phosphorylation system. all of the ribosomal RNAs and most o

22、f the transfer RNAs needed for translation of these protein-coding sequences within mitochondria. Other mitochondrial proteins are encoded by nuclear genes, which have been transferred to the nucleus from the ancestral mitochondrial genome.3. mtDNA replicationorigin of heavy-strand replication, OHor

23、igin of light-strand replication, OLbidirectional replication(雙向復(fù)制)mtDNA在有絲分裂和減數(shù)分裂間期都要經(jīng)過復(fù)制分離。1Because the growth and proliferation of mitochondria are controlled by both nuclear genome and its own genome. Mitochondria are usually called semiautonomous organelle. 半自主性細(xì)胞器：自身含有遺傳表達(dá)系統(tǒng)(自主性)；但編碼的遺傳信息十分有限，

24、其RNA轉(zhuǎn)錄、蛋白質(zhì)翻譯、自身構(gòu)建和功能發(fā)揮等必須依賴核基因組編碼的遺傳信息(自主性有限)。Part 4 The transport protein into Mitochondria the nucleus contains the genes that encode most of the mitochondrial proteins.The proteins encoded by nucleus import into mt under the help of molecular chaperone.分子伴侶：熱休克蛋白70 （Hsp70） 線粒體基質(zhì)hsp70 （ mtHsp70 ）

25、熱休克蛋白60 （Hsp60）熱休克蛋白10 （Hsp10）39Importing mitochondria proteins The most proteins found in Mit are encoded by the nuclear genome and synthesized on the free cytosolic ribosomes and imported from the cytosol into Mit as completed polypeptide chain (preprotein). Because of the double-membrane structur

26、e of Mit, the import of proteins is more complicated than the transfering of a polypeptide across a single phospholipid bilayer, e.g. ER. at or near special contact sites.1. Nuclear encoded proteins to the mitochondrial matrix Mt precursor proteins - HSP70-unfolding-ATP hydrolyzed- HSP70 releasing-

27、Mt precursor proteins binding to receptors on outer membrane (1) Mt precursor proteins are unfolded out of mt. Mt precursor proteinsremain unfolded by binding hsp70 chaperone proteins after are synthesizedThe *matrix-targeting sequences (15 to35 aa) at the N-terminus of the various Mit proteins (as

28、preproteins) are rich in positively charged amino acids-Arg and Lys.- and hydroxylated ones- Ser and Thr; they also devoid of acidic Asp and Glu. This presequence is capable of folding into a helix.After releasing hsp70, N-terminal signal sequence of Mt precursor protein is recognized by receptors;

29、then the protein binding to receptors (2) Polypeptide chain pass through both mt membrancesUnfolded Mt precursor protein binding to receptor-contact site (pathway on outer/inner membrane)-going into matrix-mtHSP70 binding to leading peptide in case it went back to cytosol(3) Protein refoldingPolypep

30、tide chain -mt matrix-mtHSP70、HSP60、HSP10 working as folding factor- Polypeptide chain refolding- hydrolyzing leading peptide -protein natural conformation1(1) Intermembrane space2. Nuclear encoded proteins to other sites of mitochondria膜間腔導(dǎo)入序列（ISTS）與MTS：二種方式 直接從胞質(zhì)擴(kuò)散方式 1(2)Outer membrane, inner memb

31、ranePart 5 Mitochondria originendosymbiosis hypothesisMargulis，1970年：線粒體的祖先-原線粒體是一種革蘭氏陰性細(xì)菌。1 細(xì)胞生物學(xué)1內(nèi)共生起源學(xué)說的主要論據(jù)基因組在大小、形態(tài)和結(jié)構(gòu)方面與細(xì)菌相似。有自己完整的蛋白質(zhì)合成系統(tǒng)，能獨(dú)立合成蛋白質(zhì)，蛋 白質(zhì)合成機(jī)制有很多類似細(xì)菌而不同于真核生物。兩層被膜有不同的進(jìn)化來源，外膜與細(xì)胞的內(nèi)膜系統(tǒng)相 似，內(nèi)膜與細(xì)菌質(zhì)膜相似。以分裂的方式進(jìn)行繁殖，與細(xì)菌的繁殖方式相同。能在異源細(xì)胞內(nèi)長期生存，說明線粒體具有的自主性 與共生性的特征。線粒體的祖先很可能來自反硝化副球菌或紫色非硫光合 細(xì)菌。 細(xì)胞

32、生物學(xué)12. 非共生起源學(xué)說主要內(nèi)容：真核細(xì)胞的前身是一個(gè)進(jìn)化上比較高等 的好氧細(xì)菌。成功之處：解釋了真核細(xì)胞核被膜的形成與演化的 漸進(jìn)過程。不足之處實(shí)驗(yàn)證據(jù)不多無法解釋為何線粒體與細(xì)菌在DNA分子結(jié)構(gòu)和蛋白質(zhì)合成性能上有那么多相似之處對線粒體的DNA酶、RNA酶和核糖體的來源也很難解釋。真核細(xì)胞的細(xì)胞核能否起源于細(xì)菌的核區(qū)？ 細(xì)胞生物學(xué)Part 6 Mitochondria proliferation and fusionMitochondria replication much like bacterial cells. When they get too large, they unde

33、rgo fission. This involves a furrowing of the inner and then the outer membrane as if someone was pinching the mitochondrion. Then the two daughter mitochondria split. 1 dividing 1551Part 7 Mitochondrial FunctionEnergy Conversion主要涉及因素（1）活性氧的增加（2）MPTP(線粒體內(nèi)膜非特異性通透性孔道，mitichondrial permeability transi

34、tion pore) 細(xì)胞凋亡時(shí)，MPTP開放（3）細(xì)胞色素C滲漏（4）Bcl-2家族：促進(jìn)或抑制（5）P53蛋白的異位表達(dá)凋亡誘導(dǎo)因素線粒體跨膜電位線粒體膜通透性增加 細(xì)胞凋亡的啟動(dòng)因子釋放Cyt.C,Apaf, AIF等釋放增強(qiáng)caspase3的水解活性活化DNase激活Caspases細(xì)胞凋亡通透性轉(zhuǎn)換孔（MPTP）開放核基因編碼的膜間蛋白快速蛋白質(zhì)水解1 細(xì)胞生物學(xué)Mitochondria and ApoptosisApaf-1凋亡蛋白酶激活因子-1（apoptotic protease activating factor-1），線蟲的同源物為ced-4。作用：參與線粒體凋亡途徑，該基

35、因敲除后，小鼠神經(jīng)細(xì)胞過多，腦畸形發(fā)育。Apaf-1與cyt c、ATP/dATP結(jié)合后，召集caspase-9，形成凋亡體（apoptosome），激活caspase-3，啟動(dòng)caspase級聯(lián)反應(yīng)。Section 2Cellular Respiration and Energy ConversionCellular respirationCellular respiration is the process of oxidizing food molecules to carbon dioxide and water. The energy released is trapped in t

36、he form of ATP for use by all the energy-consuming activities of the cell. The process occurs in two phases: Glycolysis: the breakdown of glucose to pyruvic acid oxidation of pyruvic acid to carbon dioxide and water1The characteristics of the cell respiration：Enzymes involved in REDOX reactionThe en

37、ergy stored in the high-energy phosphate bonds of ATPEnergy gradually releaseResponse to constant temperature and constant pressureReaction needs H20 is involved1 ADP+Pi ATP+1.72kJdephosphorylationphosphorylationATP is the energy conversion molecular “energy currency”Cellular Energy Metabolism*Cellu

38、lar aerobic respiration-oxidation metabolism: oxidation of pyruvate, fatty acids and amino acids;TCA and electron transport (ET)-establishment of a proton -motive force and the ATP formation 糖、脂肪細(xì)胞質(zhì)丙酮酸和脂肪酸線粒體乙酰coA（三羧酸循環(huán)）氫通過電子傳遞鏈到達(dá)氧生成水，同時(shí)ADP磷酸化生成ATPCellular Energy conversionFrom glucose to ATP, 3 ste

39、psGlycolysis cytosol tricarboxylic acid cycle mit matrix electron transport and oxidative phosphorylation mit inner membranePart 1 GlycolysisGlucose + 2 ADP + 2 NAD+ + 2 Pi 2 Pyruvate + 2 ATP + 2 NADH + 2 H+ substrate-level phosphorylationPart 2 tricarboxylic acid cycleMost of the usable energy obta

40、ined from the breakdown of carbohydrates or fats within mitochondria. Electrons from NADH and FADH2 are transferred to molecular oxygen, coupled to the formation of ATP moleculesPart 3 oxidative phosphorylationMolecular basis of oxidative phosphorylation11. The Structural foundation of oxidative pho

41、sphorylation CoQcytC complex、 (1) respiratory chainThe Electron Transport Chain2.The structure of elementary particle elementary particle(EP) consists of three parts a spherical or polyhedral head（F1 particle ）: catalyses the synthesis of ATP a cylindrical stalk : junction, no enzyme activitya base

42、piece （F0 particle ）: associated with the cristae and the inner membrane,transports protons ATP synthaseATP synthase function: synthesize ATP energy source:Electron-transport catalyses the synthesis of ATP from ADP and phosphate driven by a flux of protons across the membrane down the proton gradien

43、t generated by electron transfer. F1 particle(頭部,偶聯(lián)因子F1)contains 5 subunits, in a stoichiometry of 3a:3b:1g:1d:1e. Three substrate binding sites are in the b-subunits. The free F1 particle catalyzes ATP hydrolysis F1 particle catalyzes ATP-synthesis when binding to F0 particle . F0 particle(基片,偶聯(lián)因子F

44、0 )consists (in E. coli) of three subunits a, b and c( 1:2:9-12)12. oxidative coupled phosphorylation Two electron chains: Transport of electrons from NADH(I、III、IV)Transport of electrons from FADH2(II、III、IV)3. Electron transport and H+ proton gradients The Mechanism of Oxidative Phosphorylationche

45、miosmotic coupling hypothesis: ATP is generated by the use of energy stored in the form of proton gradients across biological membranes1oxidative phosphorylation chemiosmotic coupling4. electrochemical gradient and ATP generationATP synthase as a reversible coupling deviceElectron-transport and oxidative phosphorylation Binding Change Model and rotational catalysis (F0F1ATP