Outline

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

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Endosymbiont hypothesis revisited
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Endosymbiont hypothesis revisited
(Nature, 1998)
Rickettsia is the etiological agent of typhus
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Old eukaryotic tree of life proposing when
endosymbiosis took place
Anaerobes without canonical mitos
Rooted rRNA tree
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Derived mitochondria MLO mitochondria like
organelles

• Harbored by anaerobes and/or parasites

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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

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Why hydrogenosome?
Dehydrogenase
(Hrdy et al., 2004, Nature)
Ferredoxin (electron acceptor-instead of
ubiquonine)
Hydrogenase
Pyruvateferridoxin Oxidoreductase (PFO)
Substrate level phosphorylation

• 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 H2, CO2 and
  acetate to make ATP
• Participation of 2 remnant complex I (1st big
  complex of respiratory chain) subunits for malate
  catabolism

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Anaerobic mitochondria

• Anaerobic ciliate Nyctotherus ovalis
• Lives in hindgut of cockroaches (!!!)

• double membrane organelle
• cristae
• organellar DNA

• Has ??m 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 H2, CO2, and
  acetate
• Missing link between mito and hyrogenosomes?

(Boxama et al., 2005, Nature)
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Mitosomes
Found in the following parasites

• Giardia intestinalis
• Anaerobic parasite infecting intestinal tract

• Entamoeba histolytica
• Anaerobic parasite infecting intestinal tract

• Microsporidia
• Intracellular parasite
• Fungi

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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
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Iron-Sulfur Clusters
Important co-factors for 100 proteins in
typical eukaryotic cell
4Fe4S
2Fe2S

• Important co-factors in catalysis of redox
  reactions
• Electron transport/transfer
• Thiolation

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Iron-Sulfur containing proteins
Lill and Muhlenhoff, 2008, Annu Rev Biochem
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Iron-sulfur cluster assembly

• frataxin
• Fe2 doner

Scaffold protein

• Nfs-Isd11 complex
• cysteine desulfurase
• S doner
• Nfs bacterial IscS

• ferredoxin
• electron doner

Lill and Muhlenhoff, 2008, Annu Rev Biochem
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Iron-sulfer assembly proteins in mitosomes

• Giardia
• localized with specific antobody

(Tovar et al., 2003, Nature)

• Trichomonas
• localized with C-terminal tag

• Microsporidia
• localized with specific antibody

(Sutak et al., 2004, Nature)
(Goldberg et al., 2008, Nature)
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Iron-Sulfur Synthesis The only essential function
of mitochondria

• Yeast without mtDNA can grow in fermentable
  media as ? 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

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Conservation of protein import
C-terminal tagged G. intestinalis mitosomal
proteins expressed in T. vaginalis
Conclusion they share protein import machinery
(Dolezal, 2005, PNAS)
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Protein import Several Protein complexes required
for mt protein import
Translocase of Outer membrane

• Mitochondrial
• IMS
• Assembly
• Machinery
• Redox mediated import
• intraprotein disulfide bridge formation

Sorting And Assembly Machinery
Export and assembly machinery of inner membrane
Carrier Translocase Of Inner Membrane
Presequence translocase of inner membrane
Presequence translocase-associated motor
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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

N
C
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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)
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Protein import Presequence translocase of inner
membrane

• Basic Features
• ??m 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
(Chacinska et al., 2009, Cell)
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Adenine Nucleotide Transporters

• ANT
• ATP/ADP antiporter in mitos
• exchange b/w cyto and matrix
• Estimated that human ortholog traffics 50-60 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 ??m in some ? mutants

(Tsaousis et al., 2008, Nature)

• 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

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Summary of derived mitochondria MLOs
mitochondria like organelles
Anaerobic Mitochondria (Hydrogenosome)
Canonical Mitochondria
Hydrogenosome
Mitosome

• Cristae
• DNA
• Powerhouse
• Respiratory Chain
• ??m
• Fe-S machinery
• TOM/TIM Protein Import
• ANT ? ATP out/ ADP in
• not alwayspetite mutants

• Cristae
• DNA
• Some electron transport complexes
• Powerhouse
• ??m
• 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
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Summary of derived mitochondria MLOs
mitochondria like organelles
MLOs all over tree of life. Evidence of single
endosymbiont acquisition
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Mitochondrial variety even in mammals?

• ?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 Freidreichs
  ataxia

Sarcomeres
Cardiac muscle
Adrenal cortex
(From Scheffler, Mitochondria)
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That's nice, but who cares?
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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
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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

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Orthologues of human Mitocarta proteins have
telling pattern of occurrence
Presence of Mitocarta genes in 500 seq genomes
Origin of Mitocarta genes
Phylogenetic origin of Mitocarta proteins
compared to whole mouse
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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

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

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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)