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Biogenesis of energytransducing membranes in chloroplasts and mitochondria Prof. Patrice Hamel Department of Plant Cellular and Molecular Biology and Molecular and Cellular Biochemistry ([email protected]) Mitochondria and chloroplasts are related organelles involved in the conversion of energy Mitochondria Respiration (NADH, ATP) Chloroplasts Photosynthesis (NADPH, ATP) Mitochondria and chloroplasts are related organelles involved in the conversion of energy nucleus DNA DNA Mitochondria Chloroplasts Mitochondrial inner membrane Thylakoid membrane Specialized membranes containing multimeric complexes involved in electron transfer reaction ATP ADP + Pi Fd 2NADP++ 2H+ 4H+ stroma FNR 2NADPH PQ PQ lumen 4H+ 2 H2O 4H+ + O2 3 H+ ATP synthase plastocyanin PSI PSII cyt b6f c-type cytochromes are essential for photosynthesis stroma lumen cyt f cyt c6 plastocyanin PSI PSII cyt b6f What are c-type cytochromes? CXXCH motif Cys Cys heme p-side: periplasmic space Bacteria p-side: intermembrane space p-side: thylakoid lumen Mitochondria Plastids The cytochrome c biogenesis question Bacteria heme apocyt c p-side p-side Mitochondria p-side S S holocyt c Plastids Biochemical requirements to complete cytochrome c maturation p-side Heme transport Biochemical requirements to complete cytochrome c maturation p-side p-side 3+ S S Heme transport 2+ HS HS Reduction of heme and cysteine thiols Biochemical requirements to complete cytochrome c maturation p-side p-side 3+ S p-side S Heme transport HS 2+ HS HS S S HS Reduction of heme and cysteine thiols Catalysis of thioether bond linkage (heme lyase) Beat Michigan! Yeahh! Chlamydomonas, green and cool! F N CP M 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 Chlamydomonas reinhardtii, a model to study plastid cytochrome c biogenesis Minimal (high light) Acetate (low light) Chlamydomonas photosynthetic mutants are viable WT mutant WT mutant Cytochrome f and cytochrome c6 function in photosynthesis in Chlamydomonas 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 c6 WT mutant WT mutant PSI stroma b6f thylakoid membrane thylakoid cyt f ee- Plastids cyt c6 lumen Biogenesis of holocytochrome f and holocytochrome c6 in Chlamydomonas plastids Apocytochrome f is encoded by the chloroplast genome cytoplasm stroma apocytochrome f thylakoid lumen thylakoid lumen Biogenesis of holocytochrome f and holocytochrome c6 in Chlamydomonas plastids Apocytochrome c6 is encoded by the nuclear genome cytoplasm apocytochrome c6 stroma apocytochrome f thylakoid lumen thylakoid lumen Biogenesis of holocytochrome f and holocytochrome c6 in Chlamydomonas plastids heme is synthesized in the stroma cytoplasm apocytochrome c6 stroma apocytochrome f thylakoid lumen thylakoid lumen heme How are apoforms of cyt f and c6 converted to their holoforms in the thylakoid lumen? cytoplasm stroma apocyt f lumen apocyt c6 thylakoid lumen cytoplasm stroma lumen holocyt f holocyt c6 thylakoid lumen Ccs mutants are photosynthetic deficient and do not accumulate holoforms of plastid c-type cytochromes Minimal (high light) Acetate (low light) Isolation of photosynthetic mutants that display a dual deficiency in holocyt f and holocyt c6 WT ccs WT ccs ccs: cytochrome c synthesis cytoplasm apocyt c6 WT ccs stroma lumen holocyt f holocyt c6 thylakoid lumen apocyt f WT ccs Anti-cyt f Anti-cyt c6 cyt f heme stain cyt c6 heme stain The ccs mutants are blocked at heme attachment pre-apocyt c6 cytoplasm pre-apocyt c6 i-apocyt c6 stroma apocyt c6 X holocyt c6 i-apocyt c6 apocyt c6 thylakoid lumen thylakoid lumen The ccs mutants are blocked at heme attachment pre-apocyt c6 cytoplasm pre-apocyt c6 i-apocyt c6 stroma apocyt c6 X holocyt c6 WT ccs pulse i-apocyt c6 apocyt c6 thylakoid lumen thylakoid lumen The ccs mutants are blocked at heme attachment cytoplasm pre-apocyt c6 pre-apocyt c6 i-apocyt c6 stroma apocyt c6 X apocyt c6 thylakoid lumen thylakoid lumen holocyt c6 WT ccs pulse ccs 0 10 30 i-apocyt c6 60’ chase The ccs mutants are blocked at heme attachment cytoplasm pre-apocyt c6 pre-apocyt c6 i-apocyt c6 stroma apocyt c6 X apocyt c6 thylakoid lumen thylakoid lumen holocyt c6 WT ccs pulse i-apocyt c6 ccs 0 10 30 60’ chase The ccs mutants synthesize the apoforms of cyt c6 and cyt f but fail to convert them to their holoforms because of a block at the heme attachment step Seven loci are required for the maturation of plastid c-type cytochromes Minimal (high light) WT ccs Acetate (low light) WT ccs Plastid Nucleus ccsA CCS1 CCS2 CCS3 CCS4 CCS5 CCS6 A CcsA-Ccs1 complex involved in heme delivery? heme stroma H CcsA 36 kD H H H Ccs1 WxW lumen Ccs1 is part of a ~ 200 kDa complex whose accumulation is dependent upon the presence of CcsA Blue Native PAGE Anti-Ccs1 A candidate cytochrome c assembly complex heme Plastid Nucleus ccsA CCS1 CCS2 CCS3 CCS4 CCS5 CCS6 stroma H H CcsA 36 kD Ccs1 Ccs1 36 kDa H WxW Ccs5 6167 kDa kD H Ccs2 Ccs3 lumen Ccs4 Ccs6 Purification of the CCS complex and resolutions of its constituents Molecular cloning of other CCS genes p-side Ccs4 and Ccs5 are involved in redox metabolism? 3+ S 2+ HS S HS lumen Reduction of heme and cysteine thiols ccs4 and ccs5 are rescued by exogenous thiol compounds WT ccs5 0 cyt f heme stain a-cyt f 1 mM 0 1 mM DTT Complex I is the largest respiratory complex in the mitochondrial inner membrane Complex I is the largest respiratory complex in the mitochondrial inner membrane > 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 Chlamydomonas, a model system to study complex I biogenesis Acetate in the dark NADH complex I WT ATP Complex I mutant UQ complex III complex II Cytochrome c complex IV ATP ATP Succinate cob nd4 nd5 cox1 nd2 nd6 nd1 rtl 5 mitochondria-encoded subunits and 37 nucleus-encoded subunits O2 Isolation of nuclear mutants deficient for complex I assembly Transformation with hph gene Selection of transformants on TAP + Hygromycin B plates Chlamydomonas wild type cells Wild type strain acetate in the dark acetate in the light Complex III mitochondrial mutant Complex I nuclear mutant Complex I mitochondrial mutant Incubation at 25ºC, high light, 7-10 days Isolation of nuclear mutants deficient for complex I assembly Acetate in the light Acetate in the dark WT complex I mutant complex I candidate mutant Biochemical verification of complex I assembly defect (in gel-staining for complex I activity) Molecular analysis of the mutant to identify the disrupted gene You said it already!! Chamy is cool! Beat Michigan!
