Structure and function of
mitochondria and peroxisomes
Láng, Orsolya MD, PhD
Dept. Genetics, Cell & Immunobiology, Semmelweis University
Lecture EPh 2015
www.dgci.sote.hu
Endosymbiotic theory – Similar Origin
Similarities:
 origin
 biogenesis
 metabolic activity: beta-oxidation
By Confocal M
Mitochondrion
By TEM
By SEM
History
1894 - Richard Altmann established them as cell organelles and called them
"bioblasts"
1898 - The term "mitochondria" was coined by Carl Benda
1900 - Leonor Michaelis discovered Janus Green can be used as a supravital stain
for mitochondria
1913 particles from extracts of guinea-pig liver were linked to respiration by Otto
Heinrich Warburg, which he called "grana".
1948 - Albert Lester Lehninger described the oxidative phosphorylation
1952 - The first „official portrait” was taken
by high resolution micrographs
1957 - The popular term "powerhouse of the cell"
was coined by Philip Siekevitz
Mitochondrion
Size
Width 0.2-3.0 m
length 7-10 m,
but dynamically
changeable
Inner membrane of Mch
crista
tubular
fingerprint-like
berry-like
Localisation
Striated duct cells
Sperm cell
Number of mitochondria
Number/cell
RBC, anaerobe cells of parasites – 0
Constant
Sperm cell – 24
Dynamically changeable
Leukocytes ~300
Hepatocytes ~2000
Increased number in hyperthyroid patients
Chaos-Chaos ameba - 500.000 !
Fission and fusion
Drp1 (outer and inner membrane fission).
Mitofusin protein (outer membrane fusion),
Fis1 ( works as receptor of Drp1)
OPA1 (inner membrane fusion),
 Drp1-dynamin-related protein 1, Drp1
 Fis1 - Mitochondrial fission 1 protein
Opa1 - Optic Atrophy 1
Dynamic mitochondria
Composition I.
compartmentalisation
Outer membrane
• poor in proteins
• characteristic protein: porin
(b-sheet– trimers form channels)
• permeability up to 5000 dalton
•fatty acids, triptophane and adrenaline
metabolizing enzymes are also localized
in the outer membrane
Outer membrane proteins in Mitochondria
and their function
channel
translocators
apoptosis
fission
What do you know about Tom40 ?
OM- Porin
Big flow of molecules across the membrane
Porin protein: transmembrane protein;
known from outer membranes of bacteria,
mitochondria, and chloroplasts
Characterised by number of antiparallel βstrands and by the shear number
3 porins forms a chanel; <5000 Da can go
through
bigger molecules are transported with active
transport across the mitochondrial transporters
Composition II.
Inner membrane
 Increased surface
 70% proteins:
 e- - transporter chaini
 ATP synthesis
 transporters
 other point impermeable –
20% cardiolipin
Proteins involved in oxidative phosphorylation
http://www.bio.davidson.edu/genomics/2004/Wilson/yeast%20protein.htm
ATP synthase – molecular motor
Matrix/IC
Stator: a,b,d
Enchore the
structure
F1 ATP-ase
chatalytic site
Rotor: Ɛ
Spin clockwise
when H+ enter
IMS / EC
Transmembrane proton carriers subunit
https://www.youtube.com/watch?v=GM9buhWJjlA
Terms of Chemiosmotic theory
Peter Dennis Mitchell
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Mch. Respiratory chain – moves electrons
- pumps H+ into
intermembrane space
Mch. ATP synthase works also as a H+ pump.
H+ in
ATP synthesis
Reversible mechanism:
ATP cleavage
H+ out
Several carrier molecules for metabolites, ions – in the
inner membrane of Mch.
Other point of the inner membrane of Mch. is
impermeable for H+ and OH-.
Composition III.
Matrix
• Pyruvate dehydrogenase complex
• Enzymes of citric acid cycle
• Enzymes of ß-oxydation of fatty acids
• Enzymes of amino acid oxydation
• 5-10 copies of mtDNA (circular)
• Enzymes of mtDNA replication and transcription
•Ribosomes (70S)
• ATP, ADP, Pi
• Mg2+, Ca2+, K+
Function of Mitochondria
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ATP synthesis
Regulation of Ca2+ levels in the cell (cation granula)
Lipid homeostasis (lipid oxidation, steroid synthesis)
Nucleotide metabolism
Amino acid metabolism
FE-S synthesis (Hem)
Ubiquinone synthesis
Cofactor synthesis
Apoptosis
Aging
Heat production
Cellular respiration
ATP synthesis
cytosol
Upon one mol
glucose oxidation, 36
mol ATPs are formed
in eukaryotes
mitochondrium
Szent-Györgyi – Kreb’s cycle
Formation of Acetyl-CoA
Oxidative phosphorylation
Oxygen absent – NAD regeneration by fermentation
Heat production- thermogenesis
It is activated whenever the organism is in
need of extra heat:
febrile state - centrally controlled via
hypothalamus
feeding - low in protein diet, leptindependent hypothalamic control
Thermogenin =
uncoupling
protein 1
(UCP1)
The human mitochondrial genome
MT-DNA is circular, double-stranded structures consists of 16’569 base pairs
carrying the information for 37 genes.
http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-18/18_25.jpg
mt-DNA
• ring shape, 5 –10 copies/Mch.
• 13 Mt genes are coding proteins
• there are no introns
• few regulator genes
• no histons
• replication, transcription, translation
• 22 tRNA, 2 rRNA
Difference in protein synthesis:
 70S ribobome
 protein synthesis starts w/ fMet
 antibiotic sensitivity
Semiautonomous organelle
98 %
• growth and
proliferation of
mitochondria are
controlled by both
nuclear genome and
it’s own genome.
Apx. 1000 proteins are distributed between the outer
membrane, intermembrane space, inner membrane and matrix
space
Selective transport of
proteins to Organells
Major membrane components
TOM
complex
TIM 23
complex
TIM 22
complex
SAM
complex
OXA
complex
Direct import of unfolded protein into
matrix
2
4
1
3
Further requirements
 Chaperons – both cytosolic and mitochondrial HSP70
 IM Membrane potential
 Energy- ATP hydrolysis
Integration of unfolded protein into OM
Beta - signal in C terminus
Chaperons bids to the protein in IMS
 SAM complexes insert the protein inOM
What kind of protein can be inserted in OM?
Integration of protein into IM I.
 N-terminus signal sequence
 Hydrophobic sequence
 TIM23 stops translocation
 N-terminus signal sequence
 Hydrophobic sequence – 2nd
signal
 OXA complex folds it
 Mitochondrial proteins as well
Name one protein!
Integration of protein into IM II.
 Metabolite transporters have internal signal
sequence – loop in TOM
 Chaperons in IMS
 TIM22 is specialized for insertion of multipass IM
proteins
Which pathway can be used for IMS
proteins ?
http://www.biochemie.uni-freiburg.de/ag/pfanner/research
https://www.qiagen.com/geneglobe/static/images/pathways
/mitochondrial%20protein%20import%20pathways.jpg
Mathernal inheritance
2015. February
U.K. Parliament approves controversial
three-parent mitochondrial gene therapy
Mutation rate of mtDNA based familyTree
-week repair mechanism
- high mutation rate 100 times faster than in the nucleus
in 1980s Allan
Wilson tested the
mtDNA of 137
people from
different parts of
the world.
Everyone alive today came from a single woman who lived in Africa about 200,000
years ago: Mitochondrial Eve.
https://abagond.wordpress.com/2010/01/08/mitochondrial-dna/
Mitochondrial disorders and dysfunctions
!
Secondray
Primary events
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Mitochondrial disorders
can be caused by mutations:
in mitochondrial DNA (mtDNA)
or in nuclear genes that code for
mitochondrial components.
Can be acquired mitochondrial
dysfunction due to adverse
effects of drugs, infections, or
other environmental causes
Most sensitive cells are:
Neurons
Muscell cells
http://www.icmr.nic.in/ijmr/2015/janaury/0103.pdf
http://www.icmr.nic.in/ijmr/2015/janaury/0103.pdf
Inherited mitochondrial disorders
Mt-DNA
•Leber's Hereditary Optic
Neuropathy (Complex I)
Nuclear DNA – mt-proteins
•Congenital muscular dystrophy
Both eyes are affected
Reason: nervus opticus
(optical nerve) and retina cells
dye because
Mechanism : cause defects in
several NADH-ubiquinone
oxidoreductase chains, therefore
impair glutamate transport and
increase reactive oxygen species
level
Mitochondria with paracrystalline
Pharmacological aspects of Mtch
Strategies for mitochondrial pharmacology:
 to make molecules selectively accumulate within mitochondria.
 to use molecules that bind targets within mitochondria
 to modulate processes outside mitochondria that ultimately alter mitochondrial function
http://www.cell.com/trends/pharmacological-sciences/fulltext/S01656147%2812%2900042-9
Proofs of bacterial origin
1. Circular mt DNA (Non-Mendelian inheritence)
Animal mt: smallest genetic system known
Translation of 13 polypeptide
2. Size of mitoribosomes (70 S)
3. Formylmethionine initiator amino acid
4. Antibiotic sensitivity
5.Presence of porin in Gram negative bacteria
6. Similarities of the electron transport chain and ATP synthase
7. Division of mitochondria
By Confocal M
Peroxisome
By TEM
By SEM
Structure
 0,3-1,5 µm
 single membrane bound organelle
 Oxidative enzymes :
Catalase
Ureate oxidase crystalloid – not in human
 Selective import
 No genome - No Transcription, No translation
Composition I.
Peroxisomal membrane proteins (PMP):
- peroxins (genes: PEX)
- other PMP e.g. „half „ ABC transporters)
Composition II.
Peroxisomal matrix:
Enzymes of oxidative processes: Superoxid dismutase, catalase, peroxidase,
Enzymes of metabolic pathways: fatty acid oxidation, bile acid synthesis, enzymes of
purin metabolism
Glyoxysome
• Specialized peroxisome for
Glyoxylate cycle – photorespiration in plant
cels
Function of peroxisome
Neutralisation of O2 radicals
Oxidative processes:
Synthesis and degradation of hydrogen peroxide
Oxidation of long-chain fatty acids (fatty acids with 24 to 26 carbons)
 Fatty acid oxidation ( oxidation)
 Purin metabolism
 D amino acid oxidation
Retinoid metabolism
Detoxification:
Catalases uses some parts of it to detoxify alcohols in liver cells
Synthesis:
 Synthesis of cholesterol and bile acids in the liver
Synthesis of certain phospholipids(plasmalogen) in neurons
Summary of lipid metabolism in Peroxisome
Biogenesis of Peroxisome
1.
2.
Sheme of matrix protein import
Requirements : PTS signal: Ser-Lys-Leu(SKL) at C terminus of the protein
most of the 24 pex gene products
1. binding of enzymes (red
circles) by the import
receptors (PEX5);
2 transport of receptor–
enzyme complexes to the
peroxisome surface;
3 docking of with
peroxisomal membrane
proteins, (PEX14 ,PEX13);
4 dissociation
5 receptor recycling,
http://www.nature.com/nrm/journal/v3/n5/fig_tab/nrm807_F1.html
PEX
molecules
Import of matrix protein: catalase enzyme
PTS1 signal
PTS1R =
Pex5
Catalase is
transported in
tetramer form
not in an unfolded
form
Main forms of peroxisomal disorders
PSEDs
PBDs
(peroxisome biogenesis disorders)
(peroxisomal single enzyme disorders)
PEX gene mutations
Defective –Import
Empty peroxisomes
Hypomyelination
enzymopathy
X linked Adrenoleukodystrophy
Symptomes:
Hypotonia
Hepatomegaly
Most serius for is Zellweger syndrome
X-linked Adrenoleukodystrophy
 Mutation of ABCD1 transporter
 accumulation of very long chain fatty acids in tissues
throughout the body,
 many different phenotypes
 most severely affected tissues are the myelin in the central
nervous system, the adrenal cortex and the Leydig cells
Teratment:
Diet - restricting the intake of very-long chain fatty
acids (VLCFA)
Lorenzo's oil : mixture of unsaturated fatty acids
(glycerol trioleate and glyceryl trierucate in a 4:1 ratio),
known as inhibits elongation of saturated fatty acids in
the body.
Future - Gene therapy
By LM
Chloroplast
By TEM
By SEM
Chloroplast
Highly structured, membrane rich organelle:
1. double-membrane envelope
2. stroma: large soluble interior
3. thylakoid membrane system - granum
4. intrathylakoid space or lumen
Functions
1. many important biochemical (anabolic) pathways, e.g.,
photosynthesis
starch synthesis
fatty acid synthesis
amino acids synthesis
pigment synthesis
nucleotide synthesis
nucleic acids and protein synthesis
sulfur and nitrogen assimilation
2. own genetic machinery*
* Indicates that pathway involves a chloroplast encoded
gene in at least some organisms
Protein transport I.
http://www.nature.com/nrm/journal/v12/n1/fig_tab/nrm3027_F1.html
Protein transport II.
Chloroplast DNA (cpDNA)
“relaxed” cpDNA molecule from lettuce
General features:
1.
double-stranded,
circular molecule
2.
no histones, but have
bound proteins (e.g.,
Hu), organized into
nucleoids
3.
G-C content typically
less than nuclear DNA
4.
multiple copies (~30100) per plastid (i.e.,
all cp genes are multicopy)
5.
can be 10-20% of the
total DNA in leaves
From Kolodner & Tewari
Reproduction
• all plant and eukaryotic algal
cells have plastids
• chloroplasts form by division;
semi-autonomous
• Involves proteins (Fts) similar
to those that mediate cell
division in bacteria
From Miyagishima et al.
Photorespiration
occurs when the CO2 levels
inside a leaf become low
Rubisco starts to combine O2
with RuBP instead of CO2.