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
Endosymbiont hypothesis revisited
Endosymbiont hypothesis revisited
(Nature, 1998)
Rickettsia is the etiological agent of typhus
Old eukaryotic tree of life proposing when endosymbiosis took place
Anaerobes
without
canonical
mitos
Rooted rRNA tree
Derived mitochondria
MLO= mitochondria like organelles
• Harbored by
anaerobes and/or
parasites
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
Why hydrogenosome?
Dehydrogenase
(Hrdy et al., 2004, Nature)
Ferredoxin
(electron
acceptorinstead of
ubiquonine)
Pyruvate:ferridoxin
Oxidoreductase (PFO)
Hydrogenase
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
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 H2, CO2, and acetate
• Missing link between mito and hyrogenosomes?
(Boxama et al., 2005, Nature)
Mitosomes
Found in the following parasites:
Entamoeba histolytica
• Anaerobic parasite infecting
intestinal tract
Microsporidia
• Intracellular parasite
• Fungi
Giardia intestinalis
• Anaerobic parasite infecting
intestinal tract
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
Iron-Sulfur Clusters
Important co-factors for ~ 100 proteins in typical eukaryotic cell
[2Fe2S]
[4Fe4S]
Important co-factors in catalysis of redox reactions
• Electron transport/transfer
• Thiolation
Iron-Sulfur containing proteins
Lill and Muhlenhoff, 2008, Annu Rev Biochem
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
Iron-sulfer assembly proteins in mitosomes
Giardia
• localized with specific antobody
(Tovar et al., 2003, Nature)
Trichomonas
• localized with Cterminal tag
Microsporidia
• localized with specific antibody
(Goldberg et al., 2008, Nature)
(Sutak et al., 2004, Nature)
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
Conservation of protein import
C-terminal tagged G. intestinalis mitosomal proteins expressed in T. vaginalis
Conclusion: they share protein import machinery
(Dolezal, 2005, PNAS)
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
Export and
assembly
machinery
of inner
membrane
Sorting
And
Assembly
Machinery
Carrier Translocase
Of Inner Membrane
Presequence translocase of inner membrane &
Presequence translocase-associated motor
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
C
N
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)
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
(Chacinska et al., 2009, Cell)
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
(Tsaousis et al., 2008, Nature)
• 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
Summary of derived mitochondria
MLOs= mitochondria like organelles
Anaerobic
Mitochondria
(Hydrogenosome)
Canonical
Mitochondria
• 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)
Hydrogenosome
• “Powerhouse”
• Remnant cI of respiratory
chain (T.v.)
• Fe-S machinery
• TOM/TIM Protein Import
•ANT → ATP out/ ADP in
(most likely)
ALL ARE BOUND BY DOUBLE MEMBRANES
Mitosome
• 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
Summary of derived mitochondria
MLOs= mitochondria like organelles
MLOs all over tree of life. Evidence of single endosymbiont acquisition
Mitochondrial variety even in mammals?
• ρ0 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 muscle
(From Scheffler, Mitochondria)
Adrenal cortex
Sarcomeres
That's nice, but who cares?
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
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
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
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
Validation of one candidate by RNAi affect on complex I and a clinical case
Complex I activity
Real time qPCR of
targeted candidate
mRNA
Western with complex
I subunit Ab
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
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
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)