Structure and function of mitochondria and peroxisomes

www.dgci.sote.huLecture EPh 2015

Láng, Orsolya MD, PhDDept. Genetics, Cell & Immunobiology, Semmelweis University

Endosymbiotic theory – Similar Origin

Similarities: origin biogenesis metabolic activity: beta-oxidation

Mitochondrion

By TEM By SEM

By Confocal M

1894 - Richard Altmann established them as cell organelles and called them "bioblasts"1898 - The term "mitochondria" was coined by Carl Benda1900 - 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

History

Mitochondrion

Size

Width 0.2-3.0 mlength 7-10 m,

but dynamically changeable

Inner membrane of Mch

crista

tubular

berry-likefingerprint-like

Localisation

Striated duct cells

Sperm cell

Number of mitochondria

Number/cellRBC, anaerobe cells of parasites – 0

ConstantSperm cell – 24

Dynamically changeableLeukocytes ~300Hepatocytes ~2000Increased number in hyperthyroid patients

Chaos-Chaos ameba - 500.000 !

Fission and fusion

Drp1 (outer and inner membrane fission).

Fis1 ( works as receptor of Drp1)

Mitofusin protein (outer membrane fusion),

OPA1 (inner membrane fusion),

Drp1-dynamin-related protein 1, Drp1 Fis1 - Mitochondrial fission 1 proteinOpa1 - 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

apoptosisfission

channeltranslocators

and their function

What do you know about Tom40 ?

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 β-strands 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

OM- Porin

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

https://www.youtube.com/watch?v=GM9buhWJjlA

ATP synthase – molecular motor

Transmembrane proton carriers subunit

F1 ATP-asechatalytic site

Stator: a,b,dEnchore the structure

Rotor: ƐSpin clockwise when H+ enter

Matrix/IC

IMS / EC

Terms of Chemiosmotic theory

• Mch. Respiratory chain – moves electrons - pumps H+ into

intermembrane space• Mch. ATP synthase works also as a H+ pump.

• Reversible mechanism:

• 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-.

H+ in ATP synthesis

ATP cleavageH+ out

Peter Dennis Mitchell

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+

Composition III.

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

Function of Mitochondria

Cellular respiration

Upon one mol glucose oxidation, 36 mol ATPs are formed

in eukaryotes

mitochondrium

cytosolATP synthesis

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 hypothalamusfeeding - low in protein diet, leptin-dependent 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

• growth and proliferation of mitochondria are controlled by both nuclear genome and it’s own genome.

Semiautonomous organelle

Apx. 1000 proteins are distributed between the outer membrane, intermembrane space, inner membrane and matrix space

98 %

Selective transport of proteins to Organells

Major membrane components

TIM 23 complex

TIM 22 complex

OXA complex

SAMcomplex

TOMcomplex

Direct import of unfolded protein into matrix

1

2

3

4

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 wellName 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

https://abagond.wordpress.com/2010/01/08/mitochondrial-dna/

-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.

Mitochondrial disorders and dysfunctions

Primary events

Secondray!

!

!

!

Mitochondrial disorders can be caused by mutations: in mitochondrial DNA (mtDNA) or in nuclear genes that code for mitochondrial components.

Most sensitive cells are:NeuronsMuscell cells

Can be acquired mitochondrial dysfunction due to adverse effects of drugs, infections, or other environmental causes

http://www.icmr.nic.in/ijmr/2015/janaury/0103.pdf

http://www.icmr.nic.in/ijmr/2015/janaury/0103.pdf

Mt-DNA

• Leber's Hereditary Optic Neuropathy (Complex I)

Nuclear DNA – mt-proteins

• Congenital muscular dystrophy

Mitochondria with paracrystalline

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

Inherited mitochondrial disorders

Pharmacological aspects of Mtch

http://www.cell.com/trends/pharmacological-sciences/fulltext/S0165-6147%2812%2900042-9

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

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

Proofs of bacterial origin

Peroxisome

By TEM By SEM

By Confocal M

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

Peroxisomal membrane proteins (PMP):

- peroxins (genes: PEX)

- other PMP e.g. „half „ ABC transporters)

Composition I.

Peroxisomal matrix:

Enzymes of oxidative processes: Superoxid dismutase, catalase, peroxidase,

Enzymes of metabolic pathways: fatty acid oxidation, bile acid synthesis, enzymes of purin metabolism

Composition II.

Glyoxysome• Specialized peroxisome for

Glyoxylate cycle – photorespiration in plant cels

Function of peroxisome

Neutralisation of O2 radicals

Oxidative processes:Synthesis and degradation of hydrogen peroxideOxidation of long-chain fatty acids (fatty acids with 24 to 26 carbons) Fatty acid oxidation ( oxidation) Purin metabolism D amino acid oxidationRetinoid metabolism

Detoxification:Catalases uses some parts of it to detoxify alcohols in liver cells

Synthesis: Synthesis of cholesterol and bile acids in the liverSynthesis of certain phospholipids(plasmalogen) in neurons

Summary of lipid metabolism in Peroxisome

Biogenesis of Peroxisome

1.

2.

Sheme of matrix protein import

http://www.nature.com/nrm/journal/v3/n5/fig_tab/nrm807_F1.html

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,

Requirements : PTS signal: Ser-Lys-Leu(SKL) at C terminus of the protein most of the 24 pex gene products

PEX molecules

PTS1R = Pex5

Catalase is transported in tetramer formnot in an unfolded form

Import of matrix protein: catalase enzyme

PTS1 signal

PBDs

(peroxisome biogenesis disorders)

PSEDs

(peroxisomal single enzyme disorders)

Main forms of peroxisomal disorders

PEX gene mutations

Defective –Import Empty peroxisomesHypomyelination

Symptomes:HypotoniaHepatomegaly

enzymopathy

X linked Adrenoleukodystrophy

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

Chloroplast

By TEM By SEM

By LM

Chloroplast

Highly structured, membrane rich organelle:1. double-membrane envelope2. stroma: large soluble interior3. thylakoid membrane system - granum4. intrathylakoid space or lumen

1. many important biochemical (anabolic) pathways, e.g., photosynthesis

starch synthesis fatty acid synthesisamino acids synthesispigment synthesisnucleotide synthesisnucleic acids and protein synthesissulfur and nitrogen assimilation

2. own genetic machinery* * Indicates that pathway involves a chloroplast encoded

gene in at least some organisms

Functions

http://www.nature.com/nrm/journal/v12/n1/fig_tab/nrm3027_F1.html

Protein transport I.

Protein transport II.

From Kolodner & Tewari

“relaxed” cpDNA molecule from lettuce

Chloroplast DNA (cpDNA)

General features:1. double-stranded,

circular molecule2. no histones, but have

bound proteins (e.g., Hu), organized into nucleoids

3. G-C content typically less than nuclear DNA

4. multiple copies (~30-100) per plastid (i.e., all cp genes are multi-copy)

5. can be 10-20% of the total DNA in leaves

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.

Photorespirationoccurs when the CO2 levels inside a leaf become low

Rubisco starts to combine O2 with RuBP instead of CO2.