1. Biogenesis of energy- transducing membranes in chloroplasts and mitochondria Prof. Patrice Hamel Department of Plant Cellular and Molecular Biology and Molecular and Cellular Biochemistry (hamel.16@osu.edu)

4. Mitochondria and chloroplasts are related organelles involved in the conversion of energy Mitochondria Chloroplasts Respiration (NADH, ATP) Photosynthesis (NADPH, ATP)

5. Mitochondria and chloroplasts are related organelles involved in the conversion of energy Mitochondria Chloroplasts Mitochondrial inner membrane Thylakoid membrane nucleus DNA Specialized membranes containing multimeric complexes involved in electron transfer reaction

6. PQ 2 H 2 O 4H + + O 2 3 H + ADP + Pi ATP Fd cyt b 6 f PSI PSII ATP synthase stroma 4H + 2NADP + + 2H + 2NADPH FNR PQ plastocyanin lumen

7. cyt b 6 f PSI PSII stroma lumen plastocyanin cyt f cyt c 6 c-type cytochromes are essential for photosynthesis

8. Cys heme What are c-type cytochromes? CXXCH motif BacteriaMitochondriaPlastids p-side: periplasmic space p-side: intermembrane space p-side: thylakoid lumen

9. The cytochrome c biogenesis question Bacteria Mitochondria Plastids hemeapocyt c holocyt c S S p-side

10. Biochemical requirements to complete cytochrome c maturation Heme transport

11. p-side HS S 3+3+ Biochemical requirements to complete cytochrome c maturation S 2+2+ Heme transport Reduction of heme and cysteine thiols p-side

12. HS S 3+3+ Biochemical requirements to complete cytochrome c maturation S S S 2+2+ HS Heme transport Reduction of heme and cysteine thiols Catalysis of thioether bond linkage (heme lyase) p-side

13. N CP M F Chlamydomonas, green and cool! Fresh water unicellular alga with compatible mating types (mt+ and mt-) Can grow in the light (photosynthesis) or on a carbon source (respiration) Nuclear genome entirely sequenced and annotated Amenable to molecular genetic manipulation of the nuclear, chloroplast and mitochondrial genomes Beat Michigan! Yeahh!

14. WTmutantWTmutant Minimal (high light)Acetate (low light) Chlamydomonas reinhardtii, a model to study plastid cytochrome c biogenesis  Chlamydomonas photosynthetic mutants are viable

15. WTmutantWTmutant Minimal (high light)Acetate (low light)  Chlamydomonas photosynthetic mutants are viable  Two c-type cytochromes function in photosynthesis: membrane-bound cyt f and soluble cyt c 6 thylakoid membrane b6fb6f PSI e-e- e-e- cyt f stroma lumen cyt c 6 Cytochrome f and cytochrome c 6 function in photosynthesis in Chlamydomonas Plastids thylakoid

16. stroma thylakoid lumen cytoplasm apocytochrome f thylakoid lumen Apocytochrome f is encoded by the chloroplast genome Biogenesis of holocytochrome f and holocytochrome c 6 in Chlamydomonas plastids

17. stroma thylakoid lumen cytoplasm apocytochrome f thylakoid lumen apocytochrome c 6 Biogenesis of holocytochrome f and holocytochrome c 6 in Chlamydomonas plastids Apocytochrome c 6 is encoded by the nuclear genome

18. stroma thylakoid lumen cytoplasm apocytochrome f thylakoid lumen heme Biogenesis of holocytochrome f and holocytochrome c 6 in Chlamydomonas plastids apocytochrome c 6 heme is synthesized in the stroma

19. stroma thylakoid lumen cytoplasm lumen holocyt c 6 holocyt f stroma thylakoid lumen cytoplasm lumen apocyt c 6 apocyt f How are apoforms of cyt f and c 6 converted to their holoforms in the thylakoid lumen?

20. WTccsWTccs Minimal (high light)Acetate (low light)  Isolation of photosynthetic mutants that display a dual deficiency in holocyt f and holocyt c 6 Ccs mutants are photosynthetic deficient and do not accumulate holoforms of plastid c-type cytochromes stroma thylakoid lumen cytoplasm lumen holocyt c 6 holocyt f apocyt f apocyt c 6 WT ccs: cytochrome c synthesis WTccs Anti-cyt f cyt f heme stain Anti-cyt c 6 cyt c 6 heme stain

21. The ccs mutants are blocked at heme attachment pre-apocyt c 6 i-apocyt c 6 apocyt c 6 holocyt c 6 X stroma thylakoid lumen cytoplasm thylakoid lumen pre-apocyt c 6 i-apocyt c 6     apocyt c 6

22. The ccs mutants are blocked at heme attachment pre-apocyt c 6 i-apocyt c 6 apocyt c 6 holocyt c 6 X stroma thylakoid lumen cytoplasm thylakoid lumen pre-apocyt c 6 i-apocyt c 6     apocyt c 6 WTccs pulse

23. The ccs mutants are blocked at heme attachment pre-apocyt c 6 i-apocyt c 6 apocyt c 6 holocyt c 6 X stroma thylakoid lumen cytoplasm thylakoid lumen pre-apocyt c 6 i-apocyt c 6     apocyt c 6 chase 0 10 30 60’ ccs WTccs pulse

24. The ccs mutants are blocked at heme attachment pre-apocyt c 6 i-apocyt c 6 apocyt c 6 holocyt c 6 X stroma thylakoid lumen cytoplasm thylakoid lumen pre-apocyt c 6 i-apocyt c 6     apocyt c 6 chase 0 10 30 60’ ccs The ccs mutants synthesize the apoforms of cyt c 6 and cyt f but fail to convert them to their holoforms because of a block at the heme attachment step WTccs pulse

25. CCS2 CCS3 CCS4 CCS5 CCS6 ccsA PlastidNucleus WTccsWTccs Minimal (high light)Acetate (low light) Seven loci are required for the maturation of plastid c-type cytochromes CCS1

26. A CcsA-Ccs1 complex involved in heme delivery? 36 kD H WxW H heme CcsA Ccs1 stroma lumen Blue Native PAGE Ccs1 is part of a ~ 200 kDa complex whose accumulation is dependent upon the presence of CcsA HH Anti-Ccs1

27. Ccs2 Ccs3 Ccs4 A candidate cytochrome c assembly complex 36 kD Ccs1 67 kD H WxW H H H heme stroma lumen CcsA 36 kDa Ccs1 61 kDa CCS1 CCS2 CCS3 CCS4 CCS5 CCS6 ccsA PlastidNucleus Ccs5 Ccs6 Purification of the CCS complex and resolutions of its constituents Molecular cloning of other CCS genes

28. ccs4 and ccs5 are rescued by exogenous thiol compounds cyt f heme stain  -cyt f 0 1 mM ccs5 WT DTT Ccs4 and Ccs5 are involved in redox metabolism? HS S 3+3+ S 2+2+ Reduction of heme and cysteine thiols lumen p-side

29. Complex I is the largest respiratory complex in the mitochondrial inner membrane

30. > 40 subunits (FMN, FeS) Dual genetic origin (5 to 9 subunits encoded in the mitochondrial genome Many human disease are caused by complex I deficiencies and 50% of complex I defects have no molecular explanation Complex I is the largest respiratory complex in the mitochondrial inner membrane

31. Chlamydomonas, a model system to study complex I biogenesis WT Complex I mutant Succinate NADH UQ Cytochrome c O 2 complex III complex IV complex I ATP complex II Acetate in the dark ATP cobnd4 nd5cox1 nd2nd6 nd1rtl 5 mitochondria-encoded subunits and 37 nucleus-encoded subunits

32. Isolation of nuclear mutants deficient for complex I assembly Chlamydomonas wild type cells Transformation with hph gene Selection of transformants on TAP + Hygromycin B plates acetate in the dark acetate in the light Complex I mitochondrial mutant Complex III mitochondrial mutant Complex I nuclear mutant Wild type strain Incubation at 25ºC, high light, 7-10 days

33. Isolation of nuclear mutants deficient for complex I assembly Acetate in the lightAcetate in the dark Biochemical verification of complex I assembly defect (in gel-staining for complex I activity) Molecular analysis of the mutant to identify the disrupted gene WT complex I mutant complex I candidate mutant

34. You said it already!! Chamy is cool! Beat Michigan!