Discipline course -1

Semester -1 Botany
Paper – Plant Cell Biology
Lesson- Mitochondria
Lesson Developer: Dr Renu Kathpalia
College /Department: Kirori Mal College
University of Delhi

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 Mitochondria

Table of Contents

Chapter: Mitochondria

• Introduction

• Historical Background
• Size and number
• Morphology
o The outer membrane
o The innermembrane
o The inter membrane space
o Matrix

• Mitochondrial DNA
• Mitochondrial Biogenesis
• Marker Enzymes
• Functions of mitochondria
 Cellular respiration
• Glycolysis
• Kerb’s cycle
• Oxidative phosphorylation
• Electron transport chain
• Chemiosmotic coupling
• Role in apoptosis
• Cell specific functions

• Endosymbiotic origin of mitochondria

• Import of proteins into mitochondria
• Summary
• Exercise/ Practice
• Glossary
• References/ Bibliography/ Further Reading

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Introduction
In eukaryotic cells most of the generation of metabolic energy occurs in the organelle called
mitochondria (singular: mitochondrion) often referred to as the power house of the cell.
The energy is derived by the breakdown of carbohydrates, amino acids and fatty acids and
is used in the formation of energy rich molecules the ATP (often referred to as the energy
currency of the cell) by the process of oxidative phosphorylation.
Mitochondria are found in the cytoplasm of nearly all eukaryotic cells and occupy a
substantial portion of the cytoplasm. They are large enough to be resolved in the light
microscope but are generally not visible as they lack contrast. Special stains are used to
make them visible, for e.g. Janus Green B.
Each cell contains hundreds to thousands of mitochondria (e.g., the liver cell contains 1000-
2000 mitochondria occupying one fifth of the cell volume). Mitochonria are double
membrane organelles. Their number and size varies in metabolically different cells. The
mitochondria are highly plastic and constantly change their shape and position. In some
cells, however, they remain in a fixed position and provide ATP, e.g., the muscle cells and
around the flagellum of a sperm.

Figure: The Electron micrograph of normal adult muscle showing mitochondria (arrows).

Source:http://missinglink.ucsf.edu/lm/ids_104_musclenerve_path/student_musclen

erve/normal2.html

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Mitochondria have their own circular DNA and synthesize some of their proteins. Thus, they
are said to be “semi-autonomous” organelles. Most of the proteins required by the
mitochondria, however, are encoded by the nuclear genes and are imported from the
cytosol. The organelle is believed to have originated by the process of endosymbiosis.

Historical Background

1894- Richard Altmann, discovered the cell organelle and called it as "bioblasts”.

1898- Carl Benda coined the term "mitochondria". The word mitochondrion is derived from

the Greek word mitos- thread, and chondrion- granule.

1900 – L. Michaelis (of enzyme kinetics) found that mitochondria in living cells could be
specifically stained green by the dye ‘Janus Green B’. Because the dye must be oxidized to
give the green colour, Michaelis proposed that mitochondria are cellular oxidizing organelles.

1904- Friedrich Meves for the first time observed mitochondria in plants (Nymphaea alba).

1908- Friedrich Meves and Claudius Regaud suggested that proteins and lipids are present

in mitochondria.

1912- B. F. Kingsbury related this organelle with cell respiration.

1913- O.H.Warburg extracted particles from guinea-pig liver and conformed that enzymes
catalyzing oxidative reactions are present in the particles identified as mitochondria, and
thus, linked them to respiration.

1925- David Keilin discovered cytochromes in mitochondria.

1937 – Based on work of many scientists such as Szent-Gyorgyi, Martius, Knoop and others
and his own work, Hans Krebs presented the complete tricarboxylic acid cycle and was
awarded a Nobel prize in 1953.

1939- It was demonstrated that in minced muscle cells one molecule of oxygen can lead to

formation of two ATP.

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1941- F.A.Lipmann gave the Unifying concept of ATP as the energy currency or the primary
and universal carrier of chemical energy in cells. energy rich phosphate bond in cellular
metabolism. He shared the Nobel prize with Krebs in 1953.

1946- A.Claude isolated mitochondria from other cell fraction and did biochemical analysis.

He also isolated cytochrome oxidase and other enzymes responsible for the respiratory
chain.

1956 – George Palade and Fritjof Sjostrand published high resolution electron micrographs
showing the presence of two mitochondrial membranes and the cristae formed by the folded
inner mitochondrial membrane.

1957- Philip Siekevitz named the mitochondria as "the powerhouse of the cell", because it

generates adenosine triphosphate (ATP).

1960- Efraim Racker gave evidence for presence of particles involved in coupling ATP
synthesis to electron transport and called them coupling factors or F1 particles.

1961- Peter D. Mitchell proposed the chemi-osmotic mechanism to explain biosynthesis of

ATP and was awarded a Nobel prize in 1978.

1964 – H. Fernandez-Moran – presented electron micrographs of negatively stained inner

mitochondrial membrane containing sub-mitochondrial particles which he named as
“elementary particles”.

1967- Ribosomes were isolated from mitochondria.

1968- The mapping of mitochondrial genes was done.

1976- The complete genetic and physical map of yeast mitochondria was developed.

1978- Peter Mitchell was awarded noble prize for proposing chemiosmotic mechanism of

oxidative phosphorylation.

Size and number

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The size of mitochondria varies from 0.5 to 1.0 μm in diameter and 1 to 4 μm in length. As
mentioned earlier these organelles tend to change their shapes frequently and are usually
seen as elongated cylinders. These also occur as highly branched, interconnected tubular
network. The number of mitochondria varies considerably. These are present where ATP is
needed the most for example the metabolically active tissues. In many unicellular
organisms only single mitochondrion is present. Mitochondria can fuse with one another or
divide in two. The balance between fission and fusion determines the number, size and
degree of interconnection. The number of mitochondria can be as high as several thousand
e.g., in human liver cells 1000-2000 mitochondria/cell are present. The exception is human
mature red blood cells, which lacks mitochondria. The RBCs of vertebrates lose their
mitochondria during differentiation; it is a rare example of a vertebrate cell generating all of
its ATP by glycolysis alone. In sperm cell the mitochondria is located in the central region,
just behind the nucleus. The movement of sperm is energized by ATP produced in
mitochondria. In plants, they are the primary supplier of ATP in non-photosynthetic tissues
as well as source of energy in photosynthetic tissues during dark. Therefore the
mitochondria is present in both chemotropic and phototropic tissues in animal as well as
plant cells.

Morphology
Mitochondria are double membranous organelle. The outer membrane is smooth and
continuous but the inner membrane is folded into finger like projections called cristae that
project into the matrix. The two chemically and physiologically different membranes in
mitochondria create two separate mitochondrial compartments- the matrix and the
intermembrane space.

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Figure: Electron micrograph of mitochondria

Source:http://i681.photobucket.com/albums/vv177/akucic_biology/mitocho
ndria.jpg

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Figure: The diagrammatic representation of the mitochondria showing a continuous outer

membrane and folded inner membrane with large number of F0-F1particles. Presence of
mitochondrial DNA and ribosomes can also be seen in the matrix.

Source: http://kids.britannica.com/comptons/art-179599/Mitochondria-produce-energy-
that-the-cell-needs-to-function

Embed Video: <object width="480" height="380"

data="http://www.dnatube.com/nuevo/player.swf" type="application/x-
shockwave-flash"><param name="movie"
value="http://www.dnatube.com/nuevo/player.swf"><param name="flashvars"
value="config=http://www.dnatube.com/nuevo/econfig.php?key=0610b44d9b9f
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value="true"></object>

Video: Tomogram of mitochondria

Source: http://www.dnatube.com/video/103/3D-structute-of-mitochondrion

The Outer Membrane

The outer membrane is a simple phospholipid bilayer, containing 50% lipid and 50%
proteins by weight. And mixture of enzymes involved in degradation of tryptophan and the
elongation of fatty acids.The outer membrane is a simple phospholipid bilayer, containing

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50% lipid and 50% proteins by weight. And mixture of enzymes involved in degradation of
tryptophan and the elongation of fatty acids.

The outer mitochondrial membrane is homologous to an outer membrane present in as part

of the cell wall of certain bacterial cells. In contrast to the inner membrane the outer
mitochondrial membrane is highly permeable as it contains special protein called porins that
form aqueous channels which allows free diffusion of molecules of 5000 Daltons or less.The
proteins of outer membrane also contains proteins include a number of enzymes involved in
mitochondrial lipid synthesis, degradation of tryptophan, elongation of fatty acids, in
division and fusion of mitochondria etc., and many other proteins such as the receptor
proteins that recognize the import signal and also the enzymes that are involved in the
division and fusion of the mitochondria.

The Inner Membrane

As mentioned earlier the inner membrane is impermeable to most ions and small charged
molecules and thus forms a functional barrier to the free passage of molecules between the
cytosol and the matrix. The inner membrane contains more than 100 different polypeptides;
it has high protein/lipid ratio. It is rich in unusual phospholipids named cardiolipins (which
have four fatty acids rather than two), which is characteristic of bacterial membrane.
Presence of the phospholipid cardiolipin makes the inner membrane impermeable to ions
though oxygen, carbon dioxide and water can move freely through this layer. This property
enables the membrane to maintain the proton gradient that drives the oxidative
phosphorylation. The inner membrane is thus the principal site of ATP synthesis. Another
structural feature that enables ATP synthesis is that the inner membrane is folded into
finger like projections called cristae (singular-crista). The folding of the inner membrane
increases the total surface area. The number and shape of the cristae is highly variable.
The greater the demand of ATP, more the number of cristae.

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Source: http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/images/mito.gif

The inner membrane is highly complex, containing complexes of the electron transport
chain, the ATP synthase and transport proteins. The proteins present in the inner membrane
can be divided into three principal types:

• Those that are part of the electron transport chain and carry out oxidation reaction
• ATP synthase the enzyme that is involved in synthesis of ATP
• Transport proteins that are involved in the transport of molecules like fatty acids and
pyruvate between the cytosol and mitochondria.

The main functions of the inner mitochondrial membrane proteins are:

• Oxidative phosphorylation
• ATP synthesis
• Regulation of protein transport
• Protein import
• Mitochondria fusion and fission

The intermembrane space

The outer chamber or intermembrane space is 40-70 Ao in width and filled with watery fluid.
This space can be increased by placing the isolated mitochondria in a sucrose solution. The
sucrose can penetrate only in this chamber but not in the inner chamber. Since, the outer

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membrane is permeable and the constitution of the inner chamber resembles that of the
cytosol. It has an important role in the primary function of mitochondria, which is oxidative
phosphorylation. The intermembrane space contains the enzymes that use the ATP to
phosphorylate other nucleotides.

Matrix

The matrix contains insoluble inorganic salts having binding sites for divalent cations (Mg2+
and Ca2+) in the form of dense granules (300-500Ao), ribosomes, tRNAs, several copies of
mitochondrial DNA and enzymes required for the expression of mitochondrial genes. In
addition, the matrix also contains the enzymes required for the oxidation of pyruvate and
fatty acids and for the citric acid cycle. The matrix components can easily diffuse to inner
membrane or inner chamber because of the folds of the cristae.

Mitochondrial DNA

Mitochondrial DNA .swf

Video: Mitochondrial genome

Source: http://www.dnalc.org/view/16647-Animation-30-Higher-cells-incorporate-an-
ancient-chromosome-.html

The mitochondria contain their own genetic material which is distinct from the nuclear genome.
The mitochondrial DNA (mtDNA) and protein synthesizing machinery resembles the prokaryotic
genome supporting the endosymbiotic theory that postulated that organelles like chloroplast
and mitochondria originated from bacteria that eventually established a symbiotic relationship
with the host cell. Mitochondrial DNA originated from aerobic bacteria and during evolution
most of the genes were either lost or transferred to the nucleus of host cell. The mitochondrial
proteins are thus encoded by two different genetic systems-

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• the nuclear genes that encode the mitochondrial proteins that are imported from the
cytosol and

• the mitochondrial genes.

Mitochondria (and chloroplast) are therefore referred to as semi- autonomous organelle for
this reason.

There are multiple copies of mitochondrial DNA contained in the matrix of these organelles
and are distributed in several nucleoids that are believed to be attached to the inner
mitochondrial membrane. Mitochondrial DNA varies in size from less than 6000 bp in
Plasmodium falciparum to more than 300,000 bp in some land plants. The human
mitochondrial genome has been completely sequenced and is known to be 16,569 base pair
long .

Video: Mitochondria have their own DNA

Source: Douglas Wallace a molecular geneticist talks about Mitochondrial DNA

http://content.dnalc.org/content/c15/15179/wallace01_02.mp4

The characteristic features of the mitochondrial genomes in comparison with the nuclear and
chloroplast genome:

• Dense packing of genes

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• Relaxed codon usage –Only 22 tRNAs are present in the mitochondria unlike the 30 or
more tRNAs that are present in the cytosol. The normal codon –anticodon pairing rules
are relaxed and each codon can be recognized by more than one tRNAs.

• Variation in genetic role- Four of the 64 codons specify amino acid sequences from those
of the same codon in other genomes.

Human mitochondrial DNA has 37 genes of which 13 of these genes are involved in enzymes for
oxidative phosphorylation forming components of NADH dehydrogenase, cytochrome b,
cytochrome c oxidase and ATP synthase. The remaining genes are involved in making 22
different molecules of transport RNA (tRNAs) and 2 ribosomal ribonucleic acids (rRNAs).

Figure: Human mitochondrial DNA mapped for some of the important genes.

Source: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/mtDNA2.gif

Mitochondrial genes are inherited in non mendelian fashion

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Source: http://naturalishistoria.files.wordpress.com/2013/02/nuclear-mtdna-
diagram.jpg (CC)

Unlike the nuclear genes the mitochondrial genes are not inherited according to
Mendelian rules. In higher organisms the cytoplasm is contributed by the female parent
and therefore the inheritance of the organelles and their DNA is dependent on the
female. Such a pattern is called as the cytoplasmic/ maternal/uniparental inheritance
pattern.

Mitochondrial Biogenesis

Mitochondria are dynamic organelles frequently dividing, fusing and changing in number and
shape. The process of formation of new mitochondria in the cell is known as mitochondrial
biogenesis. This process is activated by different signals during cellular stress or in
response to environmental stimuli. Mitochondrial biogenesis involves all the processes
required in mitochondrial maintenance, growth, division and segregation during cell cycle.
Although mitochondrion has its own genome but more than 95% of mitochondrial proteins
are synthesized by nucleus genome. The nuclear encoded proteins are transported into the
mitochondria by specialized transport processes.

Figure: 95% of the mitochondrial proteins and the tRNAs are encoded by the nuclear
genes.

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Source:http://commonweb.unifr.ch/biol/pub/zoology/Homepage/Trypanos/projects/trnaorig
in.jpg

As the cell divides the number of the mitochondria and its DNA content doubles. The
mitochondria divide by fission and are then segregated to the daughter cells.

Figure: Mitochondria divide by fission.

Source:http://www.visualphotos.com/image/1x8466336/stages_of_mitochondrial_division_t
em

Marker Enzymes
Enzyme specific for an organelle, cell or cellular component is called marker enzyme.
Since these are not present anywhere else, their detection is an indication of the presence
of the source e.g., acid phosphatase is marker enzyme for lysosome and succinate
dehydrogenase for mitochondria. Marker enzymes act as biomarkers to characterize a cell
type. They are also used in the isolation of the cell components. In mitochondria each
component has its own marker enzyme.
Table. Marker enzymes of mitochondrial components
Source: Author
Name of the enzyme Location in mitochondria

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Rotenone-insensitive NADH cytochrome c outer mitochondrial membrane

reductase

Kynurenine hydroxylase outer mitochondrial membrane

Monoamine oxidase outer mitochondrial membrane

Succinate cytochrome c reductase inner mitochondrial membrane

Rotenone-sensitive NADH cytochrome c inner mitochondrial membrane

reductase

Adenylate kinase intermembrane space(outer chamber)

NAD+ malate dehydrogenase mitochondrial matrix(inner chamber)

mitochondrial matrix
NAD+ glutamate dehydrogenase

mitochondrial matrix
Citrate synthase

Functions of Mitochondria

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Video: <iframe width="420" height="315"

src="//www.youtube.com/embed/KXsxJNXaT7w" frameborder="0"
allowfullscreen></iframe>

Source: http://www.youtube.com/watch?v=KXsxJNXaT7w

Cellular respiration

The foremost important role of mitochondria is cellular respiration involving the oxidative
breakdown of glucose and fatty acids. Mitochondria can use both pyruvate (which comes
from the glucose and other sugars by glycolysis) and fatty acids (from fats). These
molecules are transported into the mitochondrial matrix and converted to acetyl CoA. The
acetyl CoA is then oxidized by the enzymes of the mitochondrial matrix to CO2 (Krebs cycle)
along with the generation of high energy electrons (released from the NADH and FADH2).
These electrons are transported to the inner mitochondrial membrane and enter into the
electron transport chain.

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Source: http://www.theoneclickgroup.co.uk/images/Myhill%20-
%20Fig%201%20Energy%20Metabolism,%20Cell%20Mitochonrion.png

The cellular respiration consists of three main steps:

• Glycolysis
• Krebs cycle
• Oxidative phosphorylation

Glycolysis

The first step of oxidation of carbohydrates – glycolysis occurs in the cytosol; it is the
process of conversion of glucose into pyurvate. When glucose (6 carbon compound) enters
into the cell, it is broken down into two molecules of pyurvic acid (3 carbon compound)
generating 2ATP molecule in the cytoplasm in the absence of oxygen. Only a small amount
of energy is released through glycolysis, most of the energy is stored in pyruvate and
NADH.

Pyruvate is then transported into the mitochondrial matrix and decarboxylated to form a
two carbon acetyl group. The acetyl group combines with coenzyme A to form acetyl CoA.
This reaction is catalyzed by a large multienzyme complex known as pyruvate
dehydrogenase.

Krebs cycle/Citric acid cycle/TCA cycle

This newly formed acetyl-CoA binds to oxaloacetic acid (4-carbon molecule) to form citric
acid. The citric acid undergoes two cycles of decarboxylation, phosphorylation and using one
glucose molecule generates 2 ATP, 6 NADH+H+, 2 FADH2 and 4 CO2. This cyclic pathway is
called the tricarboxylic acid (TCA) cycle. This is also referred as Kerb’s cycle after the
British biochemist Hans Kerbs, who elucidated the pathway in the 1930s.

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Figure: Citric acid cycle

Source:http://upload.wikimedia.org/wikipedia/commons/thumb/0/0b/Citric_acid_cycle_with
_aconitate_2.svg/754px-Citric_acid_cycle_with_aconitate_2.svg.png

Oxidative phosphorylation

Oxidative phosphorylation refers to the chemiosmotic process that converts the oxidation
energy into ATP. The initial energy released from burning of carbohydrates and fats etc., are
stored initially in the form of high energy electrons in NADH and FADH2. The NADH2+ and
FADH2 molecules give up their electrons to electron transport complexes located in the inner
mitochondrial membrane, which in turn move protons from inner compartment to outer
compartment. This proton gradient creates free energy potential which activates turbine like
ATPase pump to generate ATP (oxidative phosphorylation). The final acceptor of electrons is
oxygen that in turn reacts with hydrogen and forms water.

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Step I:

Figure: The diagram showing location and connection of electron transport chain and citric
acid cycle in the mitochondria.

Source:http://en.wikipedia.org/wiki/Oxidative_phosphorylation

Electron transport chain

Video: Electron transport chain

Source: http://vcell.ndsu.nodak.edu/animations/etc/movie-flash.htm

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Video: ETC and ATP synthesis.swf

Source: Author

The electron transport chain is located in the mitochondrial membrane where electron
carrier proteins and proton pumps are present.

The electron transport chain contains five types of carriers; flavoproteins, cytochromes,
copper atoms, ubiquinone and iron-sulfur proteins. These electron carriers are present in
inner mitochondrial membrane in the form of four complexes, named as complexes I, II, III
and IV. Two component of the electron transport chain, Ubiquinone and cytochrome c, are
not part of any of these four complexes.

A series of electron moves through electron transport chain. These reactions are coupled
with conformational changes in electron carrier that move proton outwards across the inner
mitochondrial membrane. At the end low energy electron are transferred to the terminal
electron acceptor, molecular oxygen (O2), which becomes reduced to water.

Chemiosmotic coupling

The coupling of the mechanism of electron transport chain and ATP synthesis is referred to
as chemi-osmotic coupling. The outward movement of proton creates a potential difference
across the mitochondrial inner membrane. The proton moves down the electrochemical
gradient, through ATP-synthesizing complex- ATP synthase. This was first proposed by
Peter Mitchell in 1961, and this is known as chemiosmotic mechanism. Mitochondria utilizes
gradient across inner membrane to synthesize ATP. This process of ATP formation driven by
electron movement generated by substrate oxidation is known as oxidative
phosphorylation.

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Source:http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/images/f1fo.gif

ATP synthase

On the matrix side of the inner membrane stalked particles called inner-membrane spheres
are present. These spheres are ATP-synthesizing enzyme - ATP synthase. Mammalian liver
mitochondria have 15,000 copies of the ATP synthase. Each mushroom shaped protein
complex is composed of two main components;

• Head or sphere of diameter of 80-90Ao; It contain a protein complex called F1. It

contains five subunits- in the stoichiometry 3 α:3 β:1 γ: 1 δ:1 ε.
• Stalk of 30-35Ao wide and 45-50Ao long, embedded in the inner membrane. This
multisubunit complex is called as the Fo portion. It has three subunits a,b,c
(Stoichiometry 1:2:9-12). The c subunit is hydrophobic with a transmembrane helix
turn helix structure.

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Figure: Diagrammatic representation and molecular model of ATP synthase

Source: http://ap.lanexdev.com/user_images/images/ATPase.jpg,
http://en.wikipedia.org/wiki/File:Atp_synthase.PNG

For further details visit: http://www.atpsynthase.info/

The enzyme ATP synthase is considered to be the nature’s smallest rotary motors that
provides the chemical energy. ATP synthase is a multisubunit protein complex that has two
parts F0 and F1. The F0 is embedded in the membrane whereas the F1 containing the catalytic
site. The F0 and F1 together carry out the ATP synthesis as protons are carried along the
electrochemical gradient. Paul Boyer proposed the ‘rotary binding change’ mechanism
explaining the fact that the ATP synthesis occurs spontaneously from tightly bound ADP and
inorganic phosphate. The energy released from proton movement across the
electrochemical gradient is however used to release the ATP. This is followed by binding of
ADP and phosphate at the catalytic site.

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Figure: a. Diagrammatic representation of the molecular mechanism involved in ATP

synthesis at the catalytic site of ATP synthase.

Step 1- the γ-subunit rotates 1200 and causes conformational changes in the adjacent sites
changing the affinity between the products and their substrates (L- loose; T-tight; O-open).

Step 2- ATP synthesis occurs spontaneously due the tight binding of ADP and phosphate at
the catalytic site. The energy released due the proton movement across the gradient causes
the release of ATP. This followed by the binding of the ADP and phosphate and the rotation
of the γ-subunit. The cycle thus continues.

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 Mitochondria

b. View of the F0F1 particle from the side. As mentioned earlier the ATP synthase is a
multiprotein complex with several subunits. The proton enters the a subunit of the F0
particle and the rotation of the c subunit carries it further.

Source: http://www.nature.com/nature/journal/v427/n6973/full/427407b.html

Paul D. Boyer and John E. Walker were awarded the nobel prize in 1997 for their work in
discovering the enzymatic mechanism of ATP synthesis and Jens C. Skou for the discovery
of ion transporting enzyme Na+ K+ ATPase.

Video: Mechanism of ATP synthesis by ATP synthase

Source: http://www.dnatube.com/video/104/ATP-synthase-structure-and-mechanism

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Video: ATP Synthase uses the proton gradient to synthesize ATP

Source: http://vcell.ndsu.nodak.edu/animations/atpgradient/movie-flash.htm

Role in apoptosis

In addition to supplying energy, mitochondria play role in regulating self-destruction of cells

known as apoptosis.

Apoptosis is principally regulated by a group of proteins called caspases (cysteine

proteases) which exist as inactive proenzymes in the cells. The mitochondria are the site
where not only the signals for the activation of these caspases originate but also the site
where these proteins interact. The mitochondrial protein cytochrome c acts as an activator
of initiator caspase-9 after its release from the mitochondria.

For further details :http://www.annualreviews.org/doi/full/10.1146/annurev-genet-102108-

134850?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed

Cell Specific functions

The other functions of mitochondria are related to the type of cell in which they are found.
Mitochondria are important for production of cholesterol and heme. The biosynthesis of
steroid hormones requires oxidative enzymes present in mitochondria and endoplasmic
reticulum. In mitochondria the cholesterol is converted to precursors which are required for
synthesis of all kinds of steroids. Mitochondria are also involved in cell signaling, cellular
differentiation, cell death, as well as the control of the cell cycle and cell growth.

Mitochondrial Disorders
Diseases from Abnormal Mitochondrial function
Mitochondrial DNA is always under attack by oxygen radicals released in electron
transport processes. In addition, it is not protected by DNA repair systems as in case
of nuclear DNA. Thus, mtDNA experience 10 times more mutations as compared to
nuclear DNA. The abnormal mtDNA contributes to a number of neurological
disorders; most prevalent among all is Parkinson’s disease (PD).The patients having
PD are deficient in complex I of the mitochondrial respiratory chain. This is probably

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due to increase rate of mutations in mt DNA of these cells as compared to normal

cells. Most of the disorders lead to degeneration of muscle and brain tissue, both of
these require exceptionally large amount of ATP. Premature- aging phenotypes in
mice are also caused by increased mutations in mt DNA.
Mutations of the polypeptide encoding mitochondrial genes results in disorders that
affect the muscles and the brain probably because these tissues have a high
requirement of energy.
For details visit:
http://www.umdf.org/site/c.8qKOJ0MvF7LUG/b.7934627/k.3711/What_is_Mitochond
rial_Disease.htm

Endosymbiotic origin of mitochondria

Mitochondria are considered to have originated from the group of bacteria- the
proteobacteria (most likely from Rickettsiales) through endosymbiosis. The primitive
heterotrophic eukaryotic cell got symbiotically associated with autotrophic bacterium and
gradually became dependent on the host cell.

The various evidences in favor of the endosymbiotic theory are:

• The mitochondria and bacteria are similar in size, 1-10 μm.

• Mitochondria and chloroplast divide like bacteria through binary fission.

• The mitochondria have their own DNA, RNA and ribosomes. The DNA is different
from eukaryotes and similar to that of bacteria in size and shape (both have circular
shape). The ribosome is 70s in mitochondria and bacteria.

• DNA sequence analysis suggests that many of the mitochondrial genes originated
from the bacterial endosymbiont.

• The F1 portion of ATP synthase is similar in both bacteria and mitochondria.

• The inner mitochondrial membrane is similar in lipid composition to the bacterial

membrane.

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 Mitochondria

• The mitochondria have double phospholipid bilayers. The bacteria have one
phospholipid bilayer, but at the time of entry via endocytosis, they are bound by a
vesicle which forms the second layer.

• Mitochondria and purple-aerobic bacteria use oxygen in the formation of ATP. They
both have Krebs cycle and oxidative phosphorylation.

• N-formylmethionine is the initiating amino acid in mitochondria as well as in bacteria.

• Many of the enzymes as well as transport systems are similar in both.

• The existence of amoeba, a eukaryote that lacks mitochondria.

Figure: The steps involved in endosymbiotic origin of mitochondria

Source:http://www.fossilmuseum.net/Evolution/Endosymbiosis.htm

Video: http://www.dnalc.org/view/15978-Mitochondrial-DNA-a-closer-look.html (CC)

Import of proteins into mitochondria

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 Mitochondria

Source: http://vcell.ndsu.nodak.edu/animations/mito-pt/index.htm
Mitochondria are semiautonomous organelles that make only a few proteins ( approximately
1%) using its own machinery. However more than 1000 mitochondrial proteins are encoded
by the nuclear genome. The nuclear proteins synthesized in cytosol on the free ribosome
are transported into mitochondria. This translocation involves large number of multisubunit
complexes present in the membranes of mitochondria. These complexes function to mediate
protein translocation across mitochondrial membranes. These include:
• the TOM complex- Translocase of outer membrane that transfer proteins across the
outer mitochondrial membrane; It consists of three proteins viz., TOM70, TOM20
and TOM22. TOM70 is the major receptor for precursor proteins that contain internal
targeting information; in addition TOM70 is docking point for cytosolic chaperones
Hsp90 and Hsp70 and is directly involved in the import pathway. The main function
of TOM20 is to bind precursor proteins with specific presequences.
• the TIM complex that includes the TIM22 and TIM23 -Translocase of inner membrane
that transfer proteins across the inner membrane.
These complexes contain (a)components that act as receptors for the mitochondrial proteins
and (b) others that act as translocation channels for the proteins to pass through.
There are four sites to which the proteins are transferred:
A. Protein imported into outer membrane
B. Protein imported into inner mitochondrial membrane
C. Protein imported into intermembrane space/outer chamber
D. Protein exported to inner membrane
E. Protein imported into mitochondrial matrix

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 Mitochondria

Figure: The import and export of proteins in mitochondria. There are four sites in
mitochondria to which the proteins are imported (A-D) and one (E) where it is exported. IM-
inner membrane; OM-outer membrane; IMS-intermembrane space.
Source:http://www.sciencedirect.com/science/article/pii/S0960982203002392

Nuclear encoded proteins first bind via their presequences (a stretch positively charged
amino acids at one end and hydrophobic residues at the other end) to the translocase of the
outer membrane- TOM complex. Translocation through the TOM complex is followed by the
transfer and movement through the second complex- TIM23 complex of the inner
membrane into the matrix. The β barrel proteins of the outer membrane are transferred
through the TIM complex and then carried by the mobile chaperones TIM9 and TIM10 to the
second translocator of the outer membrane - the SAM complex that helps them to fold
properly.
Proteins are transported into the mitochondria in their unfolded state assisted by
chaperone proteins belonging to the HSP70 family. These chaperones prevent the
proteins from aggregating and folding up spontaneously before interacting with the TOM
complex.
The protein components of the mitochondrial membranes are also imported through the
TOM complex. However, the outer membrane proteins, which have a hydrophobic
transmembrane sequence, exit the channel laterally.
As mentioned earlier the TIM23 complex mediates the transfer of the soluble proteins into
the mitochondrial matrix. These proteins associate with chaperones mtHsp 70 in the matrix
that mediate their entry into the matrix. Presequences are cleaved by the enzyme peptidase

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 Mitochondria

(MPP) present in the mitochondrial matrix. On entering the matrix these proteins assume
their native conformation by associating with chaperones Hsp60.
For the proteins destined for the inner membrane the TIM22 complex binds to these
proteins that have an internal targeting sequence and inserts them into the inner
membrane.
Proteins synthesized within the mitochondria destined for the inner mitochondrial membrane
are inserted into the membrane by the inner membrane translocator the OXA complex
(export complex). It also helps to insert the proteins into the inner membrane that are
initially transported into the matrix by the other complexes.

Figure: The four translocation complexes in the different components of mitochondria;

Three for import-TOM, TIM 23 and TIM22 and one for export. The proteins with either an
amino-terminal pre-sequence or internal targeting sequence binds to specific receptor(R).
The imported protein in the mitochondrial matrix are immediately bonded by mitochondrial
chaperone-ATP dependent heat shock protein (Hsp70) and folded back.
OM- outer membrane; IM- inner membrane; IMS- intermembrane space
Source:http://www.sciencedirect.com/science/article/pii/S0960982203002392

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 Mitochondria

The energy for translocation of proteins into mitochondria is derived from two sources. The
first is the membrane potential difference (Δψ) across the mitochondrial membranes. The
second source of energy is provided by matrix ATP. The successful import requires the ATP-
dependent action of the molecular chaperone mtHsp70. The mtHsp70 is important for the
folding of an amino-terminal ATPase domain and a peptide-binding domain, which in turn
provides the pulling force on protein being imported.

Video: Structure of export complex of mitochondrial membrane2.swf

Source: Author

Summary
Mitochondria are double membrane-bound organelles, found in almost all eukaryotic cells. It
varies in size and shape, with an average diameter of about 0.5 nm and a length ranging 1-
4 micrometer. The mitochondrial membranes have phospholipid bilayer embedded with
proteins. The outer membrane is continuous, whereas the inner membrane is folded into
fingerlike projections called cristae. These folds enhance the rate of cellular respiration. The
mitochondria has distinct components; outer membrane, inter membrane space, inner
membrane and matrix. Each of these performs different functions. Mitochondria are
semiautonomous in nature as the matrix consists of circular DNA, which codes for some of
the proteins. However, most of the proteins are coded by nuclear genome. Similar to
bacteria mitochondria divides by fission.

Mitochondria are the main site for energy production required by the cell. They are
responsible for the breakdown of carbohydrates and fatty acids. The energy released is
converted to ATP, by the process called oxidative phosphorylation.

Most of the mitochondrial proteins are encoded by the nucleus and are imported into the
mitochondria. These proteins are recognized by their specific signal sequences are imported
into the mitochondria by protein-lined channels.

With reference to its origin it is widely accepted that mitochondria originated as a result of
symbiotic association between bacteria and primitive eukaryotic cell.

Exercises

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 Mitochondria

1. Where do the reactions of the citric acid cycle and oxidative phosphorylation occur in
a mitochondrion?
2. What are the folds on the inner mitochondrial membrane known as?
3. Multiple choice questions
i. What kind of DNA is present in mitochondria
a) Closed circular
b) Nicked circular
c) Linear
d) Open circular
ii. Mitochondria and chloroplasts share several common features
a) Semiautonomous growth and reproduction.
b) Both do not belong to the endomembrane system.
c) Presence of small amount of DNA
d) all of the above
e) none of the above
iii. Human mitochondrial DNA has approximately__________base pair long
a) 1600
b) 16,000
c) 160,000
d) 1,600,000
iv. The mutation rate in mt DNA is
a) lower than in nuclear DNA.
b) higher than in nuclear DNA.
c) same as in nuclear DNA.
d) non-existent
v.Mitochondrial and chloroplast DNA are inherited
a) the maternal side.
b) the paternal side.
c) both parents.
d) symbiotic bacteria.
vi.Mitochondria are involved in which of the following process:
a) Fatty Acid Synthesis
b) DNA Synthesis
c) Fatty Acid Oxidation
d) Protein Synthesis

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 Mitochondria

vii.Which of the following is not the characteristics of mitochondria, they

a) need special stain to be seen
b) have double membranes
c) can divide
d) contain DNA
e) contain some hydrolytic enzymes
viii.Which of the following is not the function of mitochondria.
a) heat production in the brown fat
b) krebs cycle
c) drug detoxification
d) concentration of calcium
ix.In case of outer mitochondrial membrane rupture which of the following will not
function
a) Chemiosmosis
b) Glycolysis
c) Electron transport chain
d) Citric acid cycle
x. Which of the following process is/are required for the complete glucose oxidation?
a) Glycolysis
b) Pyruvate oxidation
c) Krebs cycle
d) All of the above
xi.An autotroph can
a) extract energy from organic sources
b) derive energy from other organisms
c) convert light energy into chemical energy
d) perform both a and c processes
xii. ATP production in Glycolysis takes place by
a) production of glyceraldehydes-phosphate
b) substrate level phosphorylation
c) the reduction of NAD+ to NADH
d) None of the above
xiii. The electrons from NADH and FADH2 can be
a) directly transferred to ATP synthase
b) transferred to inner membrane of the mitochondria

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 Mitochondria

c) matrix of the mitochondria

d) intermembrane space of the mitochondria
xiv. The role of NAD+ in the process of generation of energy is as
a) an electron carrier
b) final electron acceptor
c) an enzyme
d) a nucleotide source
xv. Which of the following are not generated during glycolysis
a) NADH
b) CO2
c) ATP
d) Pyruvate
4. What are the three ways to generate ATP from food, and which organisms use each
pathway?
5. Write the overall equation for cellular respiration.
6. How cellular respiration and breathing related?
7. Why in plants rate of photosynthesis is more than respiration?
8. Name the different components of mitochondria?
9. Name the site and reaction which takes place in different part of mitochondria.
10.With the help of suitable evidences prove that mitochondria originated through
endosymbiosis.
11. Mitochondria and chloroplast are the organelles for ATP generation. What are the
structural and functional similarities in both of these organelles?
12. Fill in the blanks
a) The process of oxidation of organic compounds to extract energy from chemical
bonds is known as___________.
b) _________ is generated by cellular respiration.
c) The electron transport chain creates a ________ gradient in the mitochondria.
d) Many proteins in mitochondria are formed in the _______ .
e) The inner membrane of mitochondria is folded to form __________.
13. Give one term for the followings:
a) The theory proposed for origin of chloroplast and mitochondria.
b) The organisms capable of harvesting the energy from sunlight.
c) The loss of an electron by an atom .
d) The enzyme involve in ATP synthesis.

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 Mitochondria

e) The process of ATP generation by electron transport from oxidation of glucose to

oxygen.
14.How the ultrastructure of mitochondria is related to its function?
15.Name two mitochondrial diseases.
16. Name the site where the following process takes place in the mitochondria
a) Electron transport
b) Krebs cycle
c) Oxidative phosphorylation
17. Who coined the term mitochondria?
18. What are marker enzymes? Name the marker enzymes of different component of
mitochondria.
19. What are mitoplast?
20.Deacribe the structure and function of Fo-F1 particles
21.Name the mitochondria present in the muscle cells .
22.Write down the structural and chemical differences in two membranes of
mitochondria.
23. Write short notes on the following
a) Oxidative phosphorylation
b) Fo-F1 particles
c) Semiautonoums nature of mitochondria
d) Mitochondrial genome
e) Biogenesis of mitochondria
f) Endosymbiotic theory
24. Differentiate between the following
a) Oxidative and photophosphorylation
b) Outer and inner membrane of mitochondria
c) Autotroph and hetrotroph
d) Mitochondrial inheritance and Mendelian inheritance
25. With the help of suitable diagrams illustrate the ultrastructure of mitochondria.
26.Describe the process of import of mitochondrial proteins.
27. How mitochondria are involved in Apoptosis?
28. What is the significance of change in number of cristae in a mitochondria
29. What is the role of hydrogen pumps in electron transport chain?
30. How mitochondria are involved in steroid hormone production?
31.How do mitochondria replicate?

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 Mitochondria

32. Describe the role of TIM TOM complex

33. Define the following:
a) Chemiosmotic theory
b) Chaperone
c) Phosphorylation
d) Semiautonomus
e) Apoptosis
34. Expand the following
a) ATP
b) TIM
c) TOM
d) NADH
e) FADH
f) Hsp
g) PD
h) ETC
35.Describe the structure and mechanism of action of ATP synthease.
36. Match the followings pair:
1. ATP synthease a) matrix
2. mtDNA b) Inner mitochondrial membrane
3. Porin c) Outer chamber
4. Adenylate kinase d) outer membrane

Glossary
Adenosine triphosphate (ATP): Adenosine with three energy rich phosphate bond; it is
the main energy storage compound of almost all the cells.
Aerobic respiration: It is an exergonic process involves the oxidation of glucose to carbon
dioxide and water using oxygen as electron acceptor; the energy released is conserved as
ATP.
Apoptosis: An orderly cell death in which the cell responds to signals lead to
characteristic cell changes and death.ATP synthase: Alternative name of ATPase; the
F0 and F1 complex found in mitochondrial inner membrane.
Chemiosmotic mechanism: The movement of electrons through electron-transport chain
resulting in proton gradient across the inner mitochondrial membrane, thylakoid or the

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 Mitochondria

bacterial membrane, linking oxidation of substrates to the phosphorylation of ADP, i.e., ATP
formation.
Cytochromes: An electron carrier consisting of a protein bound to heme group.
Electron transport or respiratory chain: The movement electron through membrane
embedded carriers that accept high-energy electrons and lowers the energy state of the
electron as it moves through the chain; this result into ATP synthesis or other storage
molecules.
Glycolysis: The first step in catabolism of glucose occurs in cytoplasm and does not require
oxygen; it results in the formation of 2 ATP molecules and pyruvate.
Matrix: The aqueous phase located within the interior of the organelle.
Organelles: The membranous or membrane bounded, functionally and structurally
diversely organized structures in the eukaryotic cells.

References
1. Campbell, N.A. and Reece, J. B. (2008). Biology 8th edition, Pearson Benjamin
Cummings, San Francisco.

2. Raven, P.H et al (2006). Biology 7th edition Tata McGrawHill Publications, New Delhi

3. Sheeler, P and Bianchi, D.E. (2006). Cell and Molecular Biology, 3rd edition, John Wiley
& sons NY

4. Karp, G. 2010. Cell and Molecular Biology: Concepts and Experiments. 6th Edition. John
Wiley & Sons. Inc.

5. De Robertis, E.D.P. and De Robertis, E.M.F. 2006. Cell and Molecular Biology. 8th edition.
Lippincott Williams and Wilkins, Philadelphia.

6. Cooper, G.M. and Hausman, R.E. 2009. The Cell: A Molecular Approach. 5th edition. ASM
Press & Sunderland, Washington, D.C.; Sinauer Associates, MA.

7. Becker, W.M., Kleinsmith, L.J., Hardin. J. and Bertoni, G. P. 2009. The World of the Cell.
7th edition. Pearson Benjamin Cummings Publishing, San Francisco.

Web links

1. http://www.nature.com/scitable/topicpage/mitochondria-14053590

2. http://www.johnkyrk.com/mitochondrion.html

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 Mitochondria

3. http://www.dnalc.org/search?q=mitochondria

4. http://www.sciencedirect.com/science/article/pii/S0960982203002392

5. http://www.pnas.org/content/102/35/12356.full

6. http://www.life.illinois.edu/crofts/bioph354/lect10.html

7. http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/oxphos.ht
m#animat2

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