Mitochondrium – Peroxisome - Chloroplast

1. outer membrane inner membrane matrix crista Mitochondrium – Peroxisome - Chloroplast Dr. habil. Kőhidai László Assoc. Prof. Dept. Genetics, Cell & Immunobology Semmelweis University 2008

2. History • Altmann – describes Mch • Benda -name „Mitochondrium” was given by him • Warburg - invetigated the enzymes of respiratory chain • Lehninger – described oxydative phosphorylation

3. Morphology

5. Network of Mch in a fibroblast cell Detection of ATP-syntase

6. Characteristic data • Size: 7 x 0.5 mm BUT: wide range in different cell types ! • Number: depends on the energy requirements/budget of the cell sperim - 24 WBC. - 300 liver cell - 500-2500 Chaos-Chaos - 500.000 ! ameba

7. Compositioncompartmentalisation • Outer membrane • poor in proteins • characteristic protein: porin • (b-sheet– trimers • form channels) • permeability up to 5000 dalton • Inner membrane • 70% proteins • e- - transporter chaini • ATP synthesis • other point impermeable – 20% cardiolipin

8. Matrix • Pyruvate dehydrogenase complex • Enzymes of citric acid cycle • Enzymes of b-oxydation of fatty acids • Enzymes of amino acid oxydation • DNA, ribosomes • ATP, ADP, Pi • Mg2+, Ca2+, K+

9. Inner membrane of Mch crista tubular fingerprint-like berry-like

10. Localization in the cell Basal striation

11. Mch as osmotic regulator of the cell normal condensed Significant H20 ration of matrix moves to the intermembraneous space and forms a „condensed” comformation

12. Relation of biochemical processes in Mch. pyruvate fatty acid Acethyl-CoA CO2 ATP Citric acid cycle NADH+H FADH2 H2O O2

13. H+ in ATP synthesis ATP cleavage H+ out Terms of Chemiosmotic theory • Mch. Respiratory chain – moves electrons - pumps H+ into intermembrane space • Mch. ATP synthase works also as a H+ pump. • Reversible mechanism: • Several carrier molecules for metabolites, ions – in the inner membrane of Mch. • Other point of the inner membrane of Mch. is impermeable for H+ and OH-.

14. H + H + H + Intermembrane space UQ I. IV. III. II. Matrix • NADH dehydrogenase • Succinyl dehydrogenase • Ubiquinone – cytochrom c oxydoreductase • Cytochrom oxydase

15. Enzyme systems of inner membrane in Mch I. e - II. III. Acidic pH H + IV. Redox potential INCREASING: I. < III. < IV.

16. K+ Resting phase Matrix [H+]=10-9 M [K+] = [Cl-] = 0.1 M [H+]=10-9 M Intermembrane space Ionophore treated (Valinomycin) H+ ATP Matrix [H+]=10-9 M [K+]<[Cl-] [H+]=10-7 M K+ Intermembrane space

17. Electrochemical proton-gradient pH gradient membrane-potential DpH DV ATP synthesis

18. NADH NAD+ NADH dehydrogenase H+ Q b-c1 complex cyt c Electron transport in Mch cytochrome oxydase O2 H2O

19. head basis Knob-like protusions of the inner Mch membrane ATP-synthase proton carrier

20. Structure of ATP-synthase F1 ATP-ase (6 subunits) Transmembrane H+ carriers (9 subunits)

21. ATP-synthase e - rotor a, b, d - stator

22. Experimental evidence

23. Bacterio-rhodopsin ATP synthase H+ ADP + Pi H+ H+ H+ H+ H+ ATP H+

24. ATP ADP + Pi H + ADP + Pi ATP

25. H+ ATP H2PO4- H+ ADP H2PO4- H+ H+ ATP ADP Transports required by ATP-synthase Symport Antiport Phosphate translocase Adenine nucleotie translocase

26. Heat Brown adipose tissue Mch. H+ H+ H+ H+ II. I. IV. III. thermogenin

27. Transports ! Hsp70 ! Signal seq. Receptor Contact-point Translocon GIP Mch. Hsp-k

28. Origin of Mitochondrion • De novo synthesis • Division • Endosymbiont theory Archaic Cyanobacteria – 1.5 x 109 yrs ago • porin (Gram (-) bact.) • electron transport chain • ATP synthase • mt DNS • ribosome BUT:Giardia has NO Mch (anaerob)

29. Origin of Mitochondrion 2 • Composition of outer membrane – eukaryotic type; the inner membrane is composed by prokaryotic components • Mch has own protein synthetic system, the starter amino acid is formyl-Met • Inhibitors of protein synthesis in Mch: antibiotics acting on bacterial protein synthesis

30. Network of Mch in budding S. cerevisiae

31. CELL PROLIFERATION Isotope labelling

32. mt-DNA • ring shape, 5 –10 copies/Mch. • 20 Mch genes are coding proteins • there are no introns • few regulator genes • no histons • repliation, transcription, translation • 22 tRNA, 2 rRNA

33. Human mt-DNA rRNA Cyt b ND1; 2 ND3-6 I. III. II. ATP-syntase

34. Mch myopathy Clusters of fibers Single fibre Crystalline structure in the matrix of Mch Mass of pathologic Mch-s

35. Peroxisome • Single membrane coverage • Selective import of proteins • No genome • Oxydative enzymes: catalase urate oxydase (crystalloid)

36. Origin of peroxisome • O2 producing bacteria – early phase of phylogeny • the O2 is toxic to other cells/organisms • peroxisome could neutralize the O2 and its radicals • in the cytoplasm

37. Functions of peroxisome • RH2 + O2 R + H2O2 (toxic) • H2O2 + R’H2 R’ + 2H2O catalase (liver, kidney) • b-oxydation: alkyl chain - (C2 ac.CoA)n

38. Peroxisomes in plants • In plants • leafs: photorespiration - O2 consumption; CO2 • germination: glyoxylate cycle (glyoxysome) • Fatty acid ac. CoA succinate glucose

39. Peroxisome in plants peroxisome glyoxisome lipid

40. PAF-1 Peroxisome • import of proteins - 3 amino acid signal sequence on C-terminal - PAF-1 – peroxisomal assembly factor-1 • Zellweger syndrome • protein to be impoted is affected - empty peroxisomes (brain, liver, kidney affected; lethal)

41. Gene transfection - Zellweger syndrome PXR1

42. Chloroplast

45. Thylakoid membrane (light reaction) Stroma (dark reaction)

46. Oxygen-requiring bacteria move to regions where oxygen is being liberated by photosynthesis Engelmann-experiment (1894)

47. Photopigments of chloroplast

48. Z-scheme of electron transport in chloroplast

49. Chloroplast – NAPH / ATP synthesis

50. Comparison of ATP generation in Mch - Chloroplast