Mitochondria
Prof. Daniel C. Hoessli, Geneva, Switzerland,
Dr. Waqar Hameed, PCMD
March 2013
Figure 14-4 Essential Cell Biology (© Garland Science 2010)
Figure 14-3 Essential Cell Biology (© Garland Science 2010)
Mitochondria: where do
they come from ?
Mitochondria probably originate from archeobacteria that were engulfed by a phagocytic
cell.
The two have lived in symbiosis ever since.
Then what has the bacterium brought in that is
beneficial to the host cell ? The capacity to
transport electrons and make a proton gradient
across the membrane
How do mitochondria and host cells
share resources ?
Mitochondria have their own DNA and
express their genes to produce proteins
active in the electron transport chain.
However, most of the proteins they need are
encoded in the nucleus of the cell. They
need to import most of their proteins to
function.
Mitochondrial division and segregation
Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al
Mitochondrial genome: evolutional influence
Overview of mitochondrial gene expression
Mitochondrial genome
http://zhangzhiyi.weibin.me/2012/10/31/mitochondrial-and-plastids-dna/
Mitochondrial genome: human Vs yeast
http://www.cbs.dtu.dk/staff/dave/roanoke/genetics44.html
Mitochondrial DNA replication fork: critical
proteins required for DNA replication
Copeland W. C. et al., 2012
Mitochondrial DNA replication
Copeland W. C. et al., 2012
Mitochondrial DNA transcription initiation
machinery and promoters
Mitochondrial transcription: Lessons from mouse
Gene Regulatory Mechanisms Volume 1819, Issues 9?10 2012
961 - 969
http://dx.doi.org/10.1016/j.bbagrm.2011.11.001
Mitochondria have a specific genetic code
Alterations in the Standard Genetic Code in Mitochondria
Codon
Standard
Code:
NuclearEncoded
Proteins
Mitochondria
Mammals
Drosophil
a
Neurospora
Yeasts
Plants
UGA
Stop
Trp
Trp
Trp
Trp
Stop
AGA,
AGG
Arg
Stop
Ser
Arg
Arg
Arg
AUA
Ile
Met
Met
Ile
Met
Ile
AUU
Ile
Met
Met
Met
Met
Ile
UUU,
CUG,
CUA,
CUG
Leu
Leu
Leu
Leu
Thr
Leu
Biogenesis of mitochondrial proteins
Pfanner et al., 2009
Mitochondrial targeting and sorting signals
Pathways of protein import into mitochondria
Two models for unfolding and translocation of
preproteins across the mitochondrial membranes
Neupert W. and Brunner M. (2002)
The translocase of the inner mitochondrial
membrane for presequence-carrying
preproteins
Import of a hydrophobic carrier protein into
the inner mitochondrial membrane
The mitochondrion
Is the site of the citric acid cycle, where the substrates
NADH and FADH2 donate electrons to the inner
mitochondrial membrane electron transport chain.
The result of electron transport is to build up a gradient of
H+ protons outside the inner mitochondrial membrane
(IMM)
The accumulated H+ protons traverse the IMM back to
the matrix and in so doing, activate the membrane ATP
synthase and generate 80% of the cellular ATP
Oxidative phosphorylations
Oxidative phosphoryations are a chemiosmotic process whereby high energy
electrons of NADH are converted into high
energy phosphate bonds of ATP
Oxidative phosphorylations do not need O2 to
produce ATP.
O2 is actually combined with the electrons at
the end of the transport chain to produce
water.
Figure 14-8 Essential Cell Biology (© Garland Science 2010)
The majority of ATP molecules are
produced in the inner mitochondrial
membranes
Mitochondrial membranes contain highly
specialized proteins that:
Transport electrons from one protein to
another,
Thus providing energy to pump H+ protons
from one side of the membrane to the other,
and
Allow the ATP synthase transmembrane
protein to produce ATP

The key bioenergetic molecules
The electron and proton transporters: NADH
FADH2 and the final products: ATP
Figure 14-5 Essential Cell Biology (© Garland Science 2010)
Figure 14-7 Essential Cell Biology (© Garland Science 2010)
Figure 14-11 Essential Cell Biology (© Garland Science 2010)
Figure 14-20 Essential Cell Biology (© Garland Science 2010)
Figure 14-14 Essential Cell Biology (© Garland Science 2010)
Figure 14-9 Essential Cell Biology (© Garland Science 2010)
Ubiquinone
Cytochrome c
Horse Cytochrome c
Stevens. Metallomics, 3:319 (2011)
Figure 14-6 Essential Cell Biology (© Garland Science 2010)
The cytochome oxidase: the 3rd complex in the electron transport chain
Figure 14-22 Essential Cell Biology (© Garland Science 2010)
The ATP synthase machine
Figure 14-12 Essential Cell Biology (© Garland Science 2010)
The last (and most important steps) of cellular
energy production occur in mitochondria
The mitochondrion



Maintains cellular homeostasis of
energy levels (ATP), Ca and of
reactive oxygen species (ROS).
The electron transport chain produces
most of the ROS present in the cell
Cytochrome c is capable of both
sensing the ATP-ADP levels and
decreasing the level of ROS
The other side of mitochondria:
apoptosis regulation
Mitochondria not only sustain life by producing
ATP, they also control the health of the cell and
may induce it to die in the controlled manner of
apoptosis, without causing inflammatory
damages. Apoptosis is therefore called:
programmed cell death
Apoptosis
Through the release of Cytochrome c,
the mitochondrion controls (induces or
prevents) most decisions to undergo
programmed cell death by the cell: this
implies both mostly the intrinsic and to
a lesser extent the extrinsic pathways
of apoptosis
Modalities of cell death
necrosis
apoptosis
phagocytosis
Nature reviews Mol.Cell Biol. 9; 2008
The Cytochrome c
Is evolutionarily a conserved, nuclear-encoded and
highly charged (pI 9.6) mitochondrial protein of 104 aa.
Contains a heme group covalently bound to cysteines 14
and 17 via thioether bonds. The heme iron is in a
hexacoordinate configuration with histidine 18 and
methionine 80 and lies in a very hydrophobic
environment
Is a multifunctional enzyme involved in: electron transfer,
apoptosome formation, cardiolipin peroxidation and
which contains 4 phosphorylation sites (Y97, Y48, T28
and S/T 47)
The Cytochrome c
Contains 4 phosphorylatable residues (Y97, Y48,
T28 and S/T47) that can be modified following
yet to be defined extracellular signals.
Contains an ATP binding site (Glu69, Asn 70,
Lys88 and Lys72, Lys86, Lys87) and may slow
down ATP production when the site is occupied.
A phospholipid-binding site interacts with
cardiolipin, the membrane lipid that attaches
Cytc to the membrane, and specific Lys (39, 25
and 7) are involved in contacting Apaf-1.
Nature reviews Mol.Cell Biol. 9; 2008
The many functions of cytochrome c
Hütteman et al. Mitochondrion 11, 369 (2011
Critical amino acid residues in Cytochrome c
Hütteman et al. Mitochondrion 11, 369 (2011
Hüttemann et al. 2011. Mitochondrion vol 11
Unifying hypothesis for
mitochondrial function
Phosphorylation of Cytc (Y48 and Y97) ensures
controlled ATP production, without excessive ROS
generation. Dephosphorylation of Cytc will cause:
Increased oxidative phosphorylations, with increased
membrane mitochondrial potential (Δψm) and increased
ROS production
 Release of Cytc from the IMM (inner mitochondrial
membrane) following oxidation of cardiolipin.
 A dephosphorylated Cytc is necessary for apoptosome
formation and activate caspases.

Which are the cytoplasmic proteins
that impact on mitochondria and
control apoptosis ?
The Bcl-2 family of proteins, which may be
pro- or anti-apoptotic
The Ras small GTPases, which may enhance
or decrease the action of Bcl-2 proteins
Bcl-2 family of proteins
The major role of Bcl-2 family of proteins is to
control the mitochondrial outer membrane
permeability (MOMP).
MOMP controls the release into the cytoplasm of
proteins contained in the mitochondrial
intermembrane space, including cytochrome c.
Cytochrome c, when in the cytoplasm, interacts
with Apaf-1 and leads to the assembly of the
apoptosome to carry out apoptosis
Developmental Cell 21, 2011
Groups of Bcl-2 proteins
The anti-apoptotic proteins (Bcl-2, Bcl-XL; Bcl-w; Mcl-1
and A1/Bfl-1) display the BH1, BH2, BH3 and BH4
domains, and a transmembrane domain ™.
The pro-apoptotic proteins (Bax; Bak and Bok/Mtd) also
display BH1 to BH4 domains and TM domain.
The BH3-only proteins (Bid, Bim/Bod, Bad, Bmf,
Bik/Nbk, Blk, Noxa, Puma/Bbc3 and Hrk/DP5) lack TM
domains and thus directly interact with anti- or proapoptotic Bcl-2 family proteins
Bad (BH3-only protein) sequesters Bcl-2 (an anti-apoptotic
protein) in cells responding to apoptotic stimuli.
When the Akt kinase phosphorylates Bad, Bcl-2 is released and
its anti-apoptotic potential restored.
Interaction of pro-apoptotic Bax with anti-apoptotic Bcl-xL: Bax-Bcl-xL complexes shuttle
from the cytoplasm to the OMM, thus preventing accumulation of Bax in the OMM, oligomerization of Bax and permeabilization of the OMM (MOMP), and consequently release
of cytochrome c
Dev. Cell 2011, 21:92-101
The BH3-only proteins
The BH3-only proteins interact with anti-apoptotic proteins (i.e.
Bad with Bcl-2 or Bcl-xL) and allow Bax-Bak oligomerization in
the mitochondial membrane, followed by cytochrome C release
and apoptosis.
The BH3-only proteins have little in common among
themselves, aside the BH3 domain.
They act as sensors for cellular stress, cell damage, infection,
growth factor deprivation, and any other signal that causes
apoptosis.
Activation of BH3-only proteins occurs by a variety of means
such as transcriptional up-regulation, limited proteolysis or
dephosphorylation.
Mol. Cell. 36; 2009
Other cytoplasmic small GTPases can
influence Bcl-2 proteins
Ras proteins constitute a very large family of
small GTPases that can influence the
function of Bcl-2 proteins in either
promoting or inhibiting their action.
Intracellular switches
Mitochondrial dynamics
Highly connected mitochondria support a
higher level of ATP production. For instance,
starved cells tend to increase the size of their
mitochondrial networks and cells undergoing
apoptosis fragment their mitochondrial
networks.
Fusion and fission of mitochondria is carried
out by mitofusins (fusion) and Drp1 (fission),
proteins, which are dynamin-related
GTPases.
Mol. Cell. 36; 2009
Do mitochondria function
differently in cancer cells ?
The metabolic phenotype of cancer cells is
the consequence of a remodeling of
mitochondria, resulting in:
1) suppressed oxidative phosphorylations,
2) enhanced glycolysis
3) suppressed apoptosis
Mitochondria in cancer cells
In the initial stages of tumor growth, cancer cells
live in relative hypoxic conditions, which favors
glycolytic degradation of glucose for energy
production. This indeed promotes an acidic
cytoplasmic pH (lactate accumulation) which
suppresses apoptosis, but also facilitates
extracellular matrix breakdown, cell motility and
invasion.
Mitochondria in cancer cells
It was considered earlier that the „glycolytic“
phenotype of cancer cells was the consequence
(due to damaged mitochondria), rather than the
cause of cancer.
It is now believed that the bioenergetic cancer
defect of mitochondria is not permanent, but
reversible, and could be targeted in cancer
treatment.
How to modify the metabolic
phenotype of cancer cell
mitochondria ?
The enzyme pyruvate dehydrogenase (PDH):
uses the pyruvate made by glucose degradation
outside the mitochondrion to make acetyl CoA, which
will be utilized in the Krebs cycle in the mitochondrion.
This key enzyme, which can be activated with the simple
molecule dichloracetate to treat children with
congenital lactic acidosis, has been tested in cancer
cells to target mitochondrial metabolism
The enzyme pyruvate
dehydrogenase (PDH)
The enzyme pyruvate
dehydrogenase (PDH
.
Remodeling of cancer cell mitochondria
with DCA
DCA activates PDH and:
Shifts mitochondrial metabolism from aerobic
glycolysis to glucose oxidation,
Decreases the mitochondrial membrane potential
and opens the MTP (mitochondrial transition
pores), releasing Cytochrome c and apoptosisinducing factors (AIF).
Increases mitochondrial production of H2O2,
Decreases intracellular K concentration by
upregulating plasma membrane K+ channels
Releases inhibition on caspases by lowering K+
concentrations.
Consequences of DCA treatment (I)
By activating PDH, DCA causes increased:
acetylCoA influx in the Krebs cycle,
delivery of NADH to the ETC,
H2O2 production, causing damage to complex I,
with reduced H+ efflux, lower Δψm, pore opening in the
mitochondrial membrane and release of Cytochrome c
and AIF.
Cytochrome c and H2O2 open the K+ (Kv1.5) plasma
membrane channels, decrease cytoplasmic levels of
K+ and
ACTIVATE CASPASES AND FORM APOPTOSOMES
Consequences of DCA treatment (II)
Cytochrome c and H2O2 both open the Kv1.5
channels, hyperpolarizing the cells and inhibiting
the voltage-dependent Ca++ entry.
Decreased Ca++ desactivates the NFAT
transcription factor, removes it from the nucleus,
and leads to uncontrolled Kv1.5 expression and
further K+ efflux and cytoplasmic decrease, thus
FURTHER ENHANCING CASPASE
ACTIVATION
Nutrient sensors are influenced by NAD+/NADH and
AMP/ATP ratios, as well as AcetylCoA levels
Cell 148, 2012
Life and death of a
mitochondrion and its
intracellular interactions
Cell 148, 2012
The cellular environment and contacts of the mitochondrion
Cell 148, 2012
How mitochondria may elicit responses
from the whole organism
Cell 148, 2012
Summary
Malfunctioning mitochondria are apoptosis inducers,
essentially through the release of cytochrome c.
The hypoactivity of mitochondria in cancer cells may
be corrected by forcing acetylCoA into the Krebs cycle
and providing excess fuel to the electron transport
chain (pyruvate dehydrogenase activation by DCA).
Apoptosis is thus induced, mainly by Cytochrome c
release and caspase activation by intracellular [K]
decrease.
The key mitochondrial protein in linking the
bioenergetic function of mitochondria with apoptosis in
Cytochrome c