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Outline Part II: Derived Mitochondria Endosymbiont hypothesis & the tree(s) of life Hydrogenosomes Anaerobic mitochondria Mitosomes Iron-Sulfer Clusters.

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Presentation on theme: "Outline Part II: Derived Mitochondria Endosymbiont hypothesis & the tree(s) of life Hydrogenosomes Anaerobic mitochondria Mitosomes Iron-Sulfer Clusters."— Presentation transcript:

1 Outline Part II: Derived Mitochondria Endosymbiont hypothesis & the tree(s) of life Hydrogenosomes Anaerobic mitochondria Mitosomes Iron-Sulfer Clusters Protein import Adenine Transporters Summary of diverse mitos Who cares? Big picture

2 Endosymbiont hypothesis revisited

3 (Nature, 1998) Rickettsia is the etiological agent of typhus

4 Old eukaryotic tree of life proposing when endosymbiosis took place Rooted rRNA tree Anaerobes without canonical mitos

5 Derived mitochondria MLO= mitochondria like organelles Harbored by anaerobes and/or parasites

6 Hydrogenosome of Trichomonas vaginalis Anaerobic parasite of vaginal tract Common venereal disease Model organism for hydrogenosomes Bound by double membranes No DNA No cristae No Krebs Cycle No electron transport chain No oxidatative phosphorylation

7 Why hydrogenosome? Ferredoxin (electron acceptor- instead of ubiquonine) Pyruvate:ferridoxin Oxidoreductase (PFO) Produce ATP by substrate level phosphorylation Chemical phosphorylation instead of generation of ATP by proton motive force generated by OxPhos Oxidation of pyruvate and malate to H 2, CO 2 and acetate to make ATP Participation of 2 remnant complex I (1 st big complex of respiratory chain) subunits for malate catabolism (Hrdy et al., 2004, Nature) Hydrogenase Substrate level phosphorylation Dehydrogenase

8 Anaerobic mitochondria Anaerobic ciliate Nyctotherus ovalis Lives in hindgut of cockroaches (!!!) double membrane organelle cristae organellar DNA Has ΔΨm has shown by Rhodamine 123 and other vital dyes Subunits of complex I in both organellar and nuclear genomes Subunits of ccomplex II in nuclear genome Electron transport? cV? Like hydrogenosome, releases H 2, CO 2, and acetate Missing link between mito and hyrogenosomes? (Boxama et al., 2005, Nature)

9 Mitosomes Found in the following parasites: Entamoeba histolytica Anaerobic parasite infecting intestinal tract Giardia intestinalis Anaerobic parasite infecting intestinal tract Microsporidia Intracellular parasite Fungi

10 Mitosomes Double membrane bound organelles No DNA No cristae No ATP synthesis So what do they do? Fe-S assembly! Immunogold electron microscopy labeling protein IscU

11 Iron-Sulfur Clusters Important co-factors for ~ 100 proteins in typical eukaryotic cell Important co-factors in catalysis of redox reactions Electron transport/transfer Thiolation [4Fe4S] [2Fe2S]

12 Iron-Sulfur containing proteins Lill and Muhlenhoff, 2008, Annu Rev Biochem

13 Iron-sulfur cluster assembly ferredoxin electron doner Nfs-Isd11 complex cysteine desulfurase S doner Nfs ≈ bacterial IscS frataxin Fe 2+ doner Scaffold protein * * Lill and Muhlenhoff, 2008, Annu Rev Biochem *

14 Iron-sulfer assembly proteins in mitosomes Microsporidia localized with specific antibody (Goldberg et al., 2008, Nature) Giardia localized with specific antobody Trichomonas localized with C- terminal tag (Tovar et al., 2003, Nature) (Sutak et al., 2004, Nature)

15 Iron-Sulfur Synthesis The only essential function of mitochondria Yeast without mtDNA can grow in fermentable media as ρ mutants However, interference with Fe-S assembly is lethal in fermentable media since it affects too many other cellular events Presence in mitosomes and hydrogenosomes of Fe-S assembly proteins supports this notion

16 Conservation of protein import C-terminal tagged G. intestinalis mitosomal proteins expressed in T. vaginalis Conclusion: they share protein import machinery (Dolezal, 2005, PNAS)

17 Protein import Several Protein complexes required for mt protein import Translocase of Outer membrane Export and assembly machinery of inner membrane Mitochondrial IMS Assembly Machinery -Redox mediated import - intraprotein disulfide bridge formation Sorting And Assembly Machinery Carrier Translocase Of Inner Membrane Presequence translocase of inner membrane & Presequence translocase-associated motor

18 Pre-sequence Signal peptides Usually N-terminal Postively charged (basic residues His, Lys and Arg) interspersed with hydrophobic residues Lengths vary between systems These properties are the basis of programs used to predict mt signal peptides, e.g. MITOPROT and SignalP on Internet NC

19 Protein import Translocase of outer membrane Basic Features: Presequence and "carrier" (internal signal) initially bind 2 different TOM subunits, but are transferred to the same translocation machinery TOM20 (binding presequence signal) has domain of negative charge No ATP required (Chacinska et al., 2009, Cell)

20 Protein import Presequence translocase of inner membrane Basic Features: ΔΨm needed for initial presequence signal peptide penetration into mt matric ATP hydrolysis required for pulling rest of protein into matrix Several heat shock proteins 70 (HSP 70) is believed to work in conjunction HSP70 consumes ATP HSP

21 Adenine Nucleotide Transporters ANT ATP/ADP antiporter in mitos exchange b/w cyto and matrix Estimated that human ortholog traffics kg ATP per day! Gets ATP out of mito and into cytoplasm where it can be used putative role apoptosis as part of mtPTP maintain ΔΨm in some ρ mutants Characterization of ANT in the mitosome of the microsporidian E. cuniculi this one actually takes ATP from cyto and exchanges it for ADP from mito Novel ATP/ADP transporter in mitosome of Entamoeba also reported (Tsaousis et al., 2008, Nature)

22 Summary of derived mitochondria MLOs= mitochondria like organelles Canonical Mitochondria Anaerobic Mitochondria (Hydrogenosome) HydrogenosomeMitosome Cristae DNA* “Powerhouse” Respiratory Chain ΔΨm Fe-S machinery TOM/TIM Protein Import ANT → ATP out/ ADP in *not always=petite mutants Cristae DNA Some electron transport complexes “Powerhouse” ΔΨm TOM/TIM Protein Import (most likely) ANT → ATP out/ ADP in (most likely) “Powerhouse” Remnant cI of respiratory chain (T.v.) Fe-S machinery TOM/TIM Protein Import ANT → ATP out/ ADP in (most likely) Fe-S machinery TOM/TIM Protein Import ANT → ATP out/ ADP (microsporidia) No identifiable ANT in Giardia but believed that ATP has to get in somehow Novel ANT in Entamoeba ALL ARE BOUND BY DOUBLE MEMBRANES

23 Summary of derived mitochondria MLOs= mitochondria like organelles MLOs all over tree of life. Evidence of single endosymbiont acquisition

24 Mitochondrial variety even in mammals? ρ 0 mutants in Chinese hamster fibroblast cells Significant amount of energy requirements satisfied by glycolysis Limiting glucose only modestly increases respiration Different cell types with different energy requirements Muscle and nerve cells have high energy needs Susceptible to mutations affecting nuclear and mito encoded mito proteins such as Freidreich’s ataxia Cardiac muscleAdrenal cortex Sarcomeres (From Scheffler, Mitochondria)

25 That's nice, but who cares?

26 Who cares about mitochondrial diversity anyway? This guy does: Vamsi Mootha at Harvard Medical School and Massachusetts General Hospital Exploits innate differences in mitochondrial physiology from several cell types to gain insight into human mitochondrial (and pathologies caused by their dysfunction). Case study: using proteomics, bioinformatics, computational strategies (and verified some experimentally) to create a Mitocarta of human mito proteins

27 Complex I of the respiratory chain Largest of respiratory complexes by far 45 known subunits in human mitos Also requires assembly factors for its biogenesis

28 Orthologues of human Mitocarta proteins have telling pattern of occurrence Presence of Mitocarta genes in 500 seq genomes Origin of Mitocarta genes Basic energy Import carriers Energy OxPhos Phylogenetic origin of Mitocarta proteins compared to whole mouse

29 Identification of proteins involved in complex I biogenesis Using bioinformatics, looked for ABSENCE of 19 complex I candidate genes in the genomes of organisms without this complex lost 4 times in evolution: 2x in yeast 1x in apicomplexans (very reduced mito but still with DNA) 1x in the mitosome containing clade Assumption #1: 19 candidates present in bacteria to assemble its complex I Assumption #2: present in complex I containing eukaryotes where needed and lost in those without

30 Validation of one candidate by RNAi affect on complex I and a clinical case Effect of RNAi on complex I Western with complex I subunit Ab Real time qPCR of targeted candidate mRNA Complex I activity Mutation in C8orf38 ORF causes a defect in complex I activity, resulting in pathologies (ataxia, decreased strength, eventual cardiac arrest) in 2 infant patients

31 Big Picture In textbooks, mitochondria have been presented as “powerhouses” of the cell based on studies on yeast and metazoans However, these organelles are far more diverse than that! Plenty of other roles including: Apoptosis signaling Fe-S cluster assembly Ion homeostasis This role as a powerhouse does not apply for all mitochondria Also, it seems that DNA is also dispensable under the right conditions: Fermentable media Anaerobic Parasitism And if mtDNA is so easy to lose, why is it maintained in canonical mitos? So far, no amitochondrial organisms reported (Archezoa all have mitosomes) Double membraned organelles ultimately essential? Diversity of mitochondria can be elegantly exploited to reveal molecular mechanisms underlying mitochondrial functions

32 What will be on the test? Answers: HH Q1: B HH Q2: D HH Q3: True HH Q4: Mud Lick, Kentucky, USA HH Q5: W HH Q6: (Trick question, leave blank)


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