BS108 Lecture 3

Mitochondrial and Plastid Genomes

Mitochondria

Mitochondria
Mitochondria are the cells' power sources. Their main function is to convert potential energy from food to ATP
Cells which require and use a lot of energy typically have a lot of mitochondria (muscle cells).

Numbers in the cells

The number of mitochondria found in different types of cells varies widely. At one end of the spectrum, the Trypanosome protozoan has one large mitochondrion; by contrast, human liver cells normally have between one thousand and two thousand each. Mitochondria can occupy up to 25% of cell cytosol.

Mitochondria

They are distinct organelles with two membranes.

Usually they are rod-shaped, however they can be round.

The outer membrane limits the organelle. The inner membrane is thrown into folds or shelves that project inward. These are called "cristae mitochondriales".

Inter-membrane space

The Outer Membrane

The outer membrane contains many transmembrane channel proteins called porins. They form channels through which a variety of molecules and ions move in and out of the mitochondrion. The outer membrane is permeable to molecules which are 5000 daltons or less (including small proteins). So the inter-membrane space and the cytosol contain similar concentration of small solutes and proteins.

The Inner Membrane

The lipid bilayer contains a high proportion of cardiolipin ( a type of phospholipid). There is a higher proportion of proteins embedded in the inner membrane compared to other membranes. Contains a variety of transport proteins for molecules that are metabolized or required by many mitochondrial enzymes.

The Inner Membrane

The inner membrane contains 5 complexes of integral membrane proteins: NADH dehydrogenase succinate dehydrogenase cytochrome c reductase (also known as the cytochrome b-c1 complex) cytochrome c oxidase ATP synthase

Mitochondria divide by fission

Mitochondria replicate like bacteria cells Will divide by fission when grown to a certain size.

Fission and Fusion of mitochondria

A balance of fission and fusion determines the arrangement of mt in different cells.

Production of ATP

Glycolysis, the breakdown of glucose to form pyruvate, takes place in the cytoplasm outside mitochondria. The Kreb's cycle, breaking down acetyl coenzyme A into carbon dioxide and NADH, takes place in the matrix of the mitochondrion.

Some ATP is generated directly during the Kreb's cycle, but most of the ATP produced in tissue respiration is generated by the electron transfer chain which takes place across the membranes of the mitochondrion. The inner surface of cristae contains the enzymes required to make ATP from ADP and phosphate.

Plastids

Plastids develop from proplastids which are small organelles found in the plant cell. Several kinds of plastid e.g. chloroplasts, chromoplasts and amyloplasts develop from the proplastid and each have a different function. During plant cell differentiation, the undifferentiated proplastid found in meristematic cells of the shoot and the root can differentiate into a number of specialized plastid types. e.g. Green chloroplasts are found in mature mesophyll cells, colourless amyloplasts are present in roots and tubers and coloured chromoplasts are found in fruit The most common plastid found in the plant cell is the chloroplast. There can be up to several hundred chloroplasts in a cell.

Chloroplasts

The main function of the chloroplast is to harness the energy from sunlight and use it to convert carbon dioxide and water to sugars that can used or stored by the plant. This process is known as photosynthesis. There are close to 50 chloroplasts in a typical plant cell.

The structure of Chloroplast

The chloroplast is made up of 3 types of membrane: A smooth outer membrane which is freely permeable to molecules. A smooth inner membrane which contains many transporters: integral membrane proteins that regulate the passage in an out of the chloroplast of

small molecules like sugars proteins synthesized in the cytoplasm of the cell but used within the chloroplast

A system of thylakoid membranes

Chloroplast

The Thylakoid Membranes

The thylakoid membranes enclose a lumen: a system of vesicles (that may all be interconnected). At various places within the chloroplast these are stacked in arrays called grana (resembling a stack of coins). Four types of protein assemblies are embedded in the thylakoid membranes: Photosystem I which includes chlorophyll and carotenoid molecules Photosystem II which also contains chlorophyll and carotenoid molecules Cytochromes b and f ATP synthase
These carry out the so-called light reactions of photosynthesisphotosynthetic electron transfer.

The thylakoid membranes are surrounded by a fluid stroma. The stroma contains:

all the enzymes, e.g., RUBISCO, needed to carry out the "dark" reactions of photosynthesis; that is, the conversion of CO2 into organic molecules like glucose. A special set of ribosomes, RNAs and the chloroplast DNA.

Main Polypeptide Transport Complexes of the Outer and Inner Membranes of Mitochondria and Chloroplasts

TOM: Translocase of the outer mitochondrial membrane TIM: Translocase of the inner mitochondrial membrane TOC: Transclocase of the outer chloroplast membrane TIC: Translocase of the inner chloroplast membrane

Experimental Demonstration That Polypeptides Span Both Mitochondrial Membranes During Import
Cell-free import system was incubated on ice. At lower temperature, the translocation of the of the polypeptide stalls The transit sequence at the N-terminus is cleaved by the transit peptidase located in the mitochondrial matrix. Proteolytic enzymes added to the outside of the mitochondrion will leave the C-terminal part of the polypeptide. Analyze peptides.

Proteins transiently span inner & outer membranes

Protein import by mitochondria

Comparison between mitochondrion and chloroplast

Chloroplast is generally larger Chloroplast has another set of membrane, the thylakoid membrane. The inner mitochondrial membrane is folded into cristae. Mitochondria import most of their lipids; chloroplasts make most of theirs.

Both Mitochondria and chloroplasts have their own genome.

Mitochondria genome size : 6000 bp to 300,000 bp Chloroplast genome size: 70,000 bp to 200,000 bp

Out of phase replication

In most cells, the replication of the organelle DNA is not limited to the S phase of the cell cycle, when the nuclear DNA replicates, but occurs throughout the cell cycleout of phase with cell division. Under constant conditions, the process is regulated to ensure that the total number of organelle DNA molecules doubles in every cell cycle.

Mitochondrial DNA (mt DNA)

Mitochondria have their own DNA and ribosomes. They can make some of their own proteins. Still require nuclear DNA for other proteins.

Mitochondrial Genomes

All of the mitochondrial genomes are circular. Even though there is a large size discrepancy between different species, especially between plants and animals. Human mt DNA encodes 13 proteins, the 22-fold larger Arabidopsis mt DNA encodes only 32 proteins. Thus the extra DNA that is found in the larger genomes does not appear to be required. Studies have shown that much of this DNA is in fact repeated sequences.

Mitochondrial genomes vary in sizes

Species Human Drosophila Yeast Turnip Corn

Size (kb) 16 18 75 218 570

Relative amounts of mitochondria DNA in some cells and tissues

Organism Tissue or cell type DNA mt per cell % mt molecules DNA/total per mt cellular DNA

Rat Yeast* Frog

liver vegetative egg

5 - 10 2 - 50 5 - 10

1000 1 - 50 107

1 15 99

* The large variation in the number and size of mt per cell in yeasts is due to mt fusion and fission

Mitochondrial Genome

The human mitochondrion contains about 5 circular mtDNA. Each is about 16,569 bp Genes they code for: 2 ribosomal RNA (rRNA) molecules 22 transfer RNA (tRNA) molecules (shown in the figure as yellow bars; two of them labeled) 13 polypeptides The 13 polypeptides participate in building several protein complexes embedded in the inner mitochondrial membrane. 7 subunits that make up the mitochondrial NADH dehydrogenase 3 subunits of cytochrome c oxidase 2 subunits of ATP synthase cytochrome b

Mitochondrial Genome

Human mt genome

Special Features of Human Mitochondrial Genome

Dense gene packing. Nearly every nt seems to be part of a coding sequence, either for a protein or tRNAs or rRNAs. Little or no regulatory elements.

Special Features

Relaxed codon usage. 30 or more tRNAs specify amino acids in the cytosol or chloroplasts, only 22 tRNAs are required for mitochondria.

Variant genetic code. 4 of the 64 codons have different meanings from those of the same codons in other genomes.

Mitochondrial Genetic Codes

Codon Universal Mammals code Mt code STOP Trp Yeasts Mt code Trp Plants Mt code Stop

UGA

AUA

Ile

Met

Met

Ile

CUA

Leu

Leu

Trp

Leu

Mitochondrial Genetic Codes

Codon Universal Mammals code Mt code Arg STOP Yeasts Mt code Arg Plants Mt code Arg

AGA

AGG

Arg

STOP

Arg

Arg

Mitochondria inheritance

In mammals, the mitochondria are from the mothers. Sperms carry about 100 mitochondria while the oocytes contain about 100,000 mitochondria. As the cells divide, more and more of the paternal mt are diluted out.

Mutations from mitochondrial genome

Mutations from the mt genome can cause diseases.

Some example of diseases:

Leber's hereditary optic neuropathy (degeneration of the optic nerve, accompanied by increasing blindness): caused by mutation to the gene encoding subunit 4 of the NADHCoQ reductase. "ragged muscle fibers" associated with jerky movements is caused by mutation of mitochondrial lysine tRNA. Kaerns-Sayre syndrome: eye defects, abnormal heartbeat, Central nervous system degeneration. Several large deletions in mtDNA.

Non Mendelian Inheritance

Affected Mother

Affected Daughters

Affected Son

Affected Grandchildren

Healthy Grandchildren

What happened to old mt?

Mitochondrial numbers are controlled by autophagy. This is a process by which lysosomes are involved in controlling cell constituents. The process begins by wrapping endoplasmic reticulum membranes around the mitochondrion. Then, vesicles come from the Golgi complex and join with the autophagic vacuole.

Plastids have their own DNA

All plastids studied to date contain their own DNA, which is actually a reduced "genome" derived from a cyanobacterial ancestor that was captured early in the evolution of the eukaryotic cell. Land plant chloroplast genomes typically contain around 110-120 unique genes. Some algae have retained a large chloroplast genome with more than 200 genes, while the plastid genomes from nonphotosynthetic organisms may retain only a few dozen genes.

Chloroplast genome

Chloroplast Genome

Chloroplast DNAs are circular molecules of 70,000 200,000 bp. About 120 genes are encoded. About 60 genes are involved in RNA transcription and translation, including genes for rRNAs, tRNAs and RNA polymerase subunits. About 20 genes encode subunits of chloroplast photosynthetic electron-transfer complexes and the F0F1 ATPase complexes. About 40 genes encode proteins of unknown functions.

Pollen does not carry chloroplast DNA

One striking feature of chloroplast is that its genome is maternally inherited in most plants (in 2/3 of higher plants). This means that the pollen grains (the male reproductive cells) only contribute the genetic information contained in the nucleus but not in the chloroplast to the zygote.

Variegation in plants

In some plants, pollen chloroplasts enter the zygote, making chloroplast inheritance biparental. A mixture of defective and normal chloroplasts in the zygote may sort out by mitotic segregation during plant growth and development to produce variegated leaves.

Variegated leaves

In the white patches, the plant cells have inherited a defective chloroplast.

Where did mitochondria and chloroplasts come from?

The endosymbiosis theory postulates that

The mitochondria of eukaryotes evolved from aerobic bacteria (probably related to the rickettsias) living within their host cell. The chloroplasts of eukaryotes evolved from endosymbiotic cyanobacteria (autotrophic prokaryotes).

Suggested evolution pathway for the origin of mitochondria

Rickettsias

Rickettsias. These bacteria are too small to be clearly seen under the light microscope. Almost all are obligate intracellular parasites -can only grow and reproduce while within the living cells of their host. Rickettsia prowazekii causes typhus fever when it is transmitted to humans by lice. Because of the similarities of their genomes, rickettsias may be the closest relatives to the ancestors of mitochondria.

Cyanobacteria (blue-green algae)

photosynthetic prokaryotes use chlorophyll a (as do plants)

use water as the source of electrons to reduce CO2 to carbohydrate

Evidence that mt and chloroplast come from symbiotic prokaryotes

Both mitochondria and chloroplasts can arise only from preexisting mitochondria and chloroplasts. They cannot be formed in a cell that lacks them because nuclear genes encode only some of the proteins of which they are made. Both mitochondria and chloroplasts have their own genome and it resembles that of prokaryotes not that of the nuclear genome.

Evidence (contd)

Both genomes consist of a single circular molecule of DNA. There are no histones associated with the DNA. Both mitochondria and chloroplasts have their own protein-synthesizing machinery, and it more closely resembles that of prokaryotes than that found in the cytoplasm of eukaryotes.

Evidence (contd)

The first amino acid of their transcripts is always formyl-Met as it is in bacteria (not methionine [Met] that is the first amino acid in eukaryotic proteins). A number of antibiotics (e.g., streptomycin) that act by blocking protein synthesis in bacteria also block protein synthesis within mitochondria and chloroplasts. They do not interfere with protein synthesis in the cytoplasm of the eukaryotes.

Evidence (contd)

Conversely, inhibitors (e.g., diphtheria toxin) of protein synthesis by eukaryotic ribosomes do not have any effect on bacterial protein synthesis nor on protein synthesis within mitochondria and chloroplasts.

The antibiotic rifampicin, which inhibits the RNA polymerase of bacteria, also inhibits the RNA polymerase within mitochondria. It has no such effect on the RNA polymerase within the eukaryotic nucleus.

Chloroplast genetic engineering

Foreign gene is introduced into the chloroplast genome. Very high level of transgene expression is observed.

Pollen doesnt contain chloroplast (only in some plants). Therefore the risk of transgene escape will be rare if the gene of interest is inserted into the chloroplast genome.

Applications of chloroplast genetic engineering

Glyphosate resistance Glyphosate is a potent broad spectrum herbicide that is effective in the control of most grasses and broadleaf weeds.

This chemical affects the aromatic amino acid biosynthetic pathway in plants and microorganisms and is, therefore, not toxic to animals.

Daniell et al. (1998) successfully transformed a gene conferring glyphosate resistance into the tobacco chloroplast genome.
The transgenic tobacco plants were able to survive on ten times the normally lethal concentration of glyphosate. The gene is contained within the chloroplast and will not be transmitted via pollen to other plants.

Applications of Chloroplast genetic engineering

Insect resistance via Bt toxin

Bacillus thuringensis (Bt) toxins are harmful to insects when ingested. Kota et al. (1999) observed hyper-expression of Bt toxins in transformed tobacco chloroplasts that resulted in a high insect mortality rate.
The advantage of this application is that the toxins are located in green leaf tissue which is the most likely part of the plant to be consumed by insects.

Where do modern man come from?

Two main hypotheses

Multiregional view: modern humans evolved from earlier hominids in different parts of the world.
Out of Africa view: modern humans evolved in Africa before colonizing the world. The two main hypotheses agree that Homo erectus evolved in Africa and spread to the rest of the world around 1 - 2 million years ago; it is regarding our more recent history where they disagree.

Origins of the modern man

Multi-regional evolution

suggests that modern humans evolved from archaic forms (such as Neanderthal and Homo erectus) concurrently in different regions of the world supported by physical evidence, such as the continuation of morphological characteristics between archaic and modern humans now a minority standpoint

Origins of modern man

Recent African origin

proposes that modern humans evolved once in Africa between 100 - 200 thousand years ago modern humans subsequently colonized the rest of the world without genetic mixing with archaic forms supported by the majority of genetic evidence

The evolutionary trees

Mitochondrial DNA -- maternal DNA -- is used to construct evolutionary trees. This is done by measuring the substitution rate of the mitochondrial genomes.

Why is mitochondria DNA used?

Mitochondria have their own genome.

They are present in large numbers in each cell, so fewer samples is required.
They have a higher rate of substitution (mutations where one nucleotide is replaced with another) than nuclear DNA making it easier to resolve differences between closely related individuals. They are inherited only from the mother, which allows tracing of a direct genetic line.

They don't recombine. The process of recombination in nuclear DNA (except the Y chromosome) mixes sections of DNA from the mother and the father creating a garbled genetic history.

How old is the modern man?

A new study shows that modern humans appeared 171,500 years ago in Africa.

Taiwanese Tribes and Polynesians

An analysis of mitochondrial DNA from nine Taiwanese tribes has revealed a genetic link between aboriginal Taiwanese and Polynesian populations, to the exclusion of mainland Asians. Genetic techniques involving mitochondrial DNA (mtDNA) have been used to compare the genetic profiles of Polynesians with people from mainland China, Southeast Asia, and Taiwan. Mitochondria reside in the cell cytoplasm and contain separate DNA that is inherited only from the mother. This means that while a persons nuclear DNA comes from a large number of ancestors, mtDNA can be traced back to a single ancestor.

mtDNA can be used to trace ancestors

In theory, every person should have a copy of mtDNA identical to this original ancestor. In practice, this is not the case because random errors occur in the replication process. Different populations will experience mutations at different locations in their mtDNA, and these will be passed on to future generations. The result is that some groups of people will end up with mtDNA that is very different from another group. By comparing how much mtDNA different populations have in common, an ancestral relationship can be determined and dated.

Additional roles of the mitochondria

Mitochondria play an important role in the regulation of cell death. anti-apoptotic members of the Bcl-2 family of proteins, such as Bcl-2 and Bcl-XL, are located in the outer mitochondrial membrane and act to promote cell survival. Many of the pro-apoptotic members of the Bcl-2 family, such as Bad and Bax also mediate their effects though the mitochondria, either by interacting with Bcl-2 and BclXL, or through direct interactions with the mitochondrial membrane.

Bcl-2 Family

The bcl-2 proteins are a family of proteins involved in the response to apoptosis. Some of these proteins (such as bcl-2 and bcl-XL) are antiapoptotic, while others (such as Bad or Bax) are pro-apoptotic. When there is an excess of pro-apoptotic proteins the cells are more sensitive to apoptosis, when there is an excess of anti-apoptotic proteins the cells will tend to be less sensitive.

Bcl-2 Family

The pro-apoptotic bcl-2 proteins are often found in the cytosol where the act as sensors of cellular damage or stress. Following cellular stress they relocate to the surface of the mitochondria where the anti-apoptotic proteins are located. This interaction between pro- and anti-apoptotic proteins disrupts the normal function of the anti-apoptotic bcl-2 proteins and can lead to the formation of pores in the mitochondria and the release of cytochrome C and other pro-apoptotic molecules from the intermembrane space. This in turn leads to the formation of the apoptosome and the activation of the caspase cascade.

Caspase activation

Release of cytochrome C from mitochondria can lead to the activation of caspase 9, and then of caspase 3. This effect is mediated through the formation of an apoptosome, a multi-protein complex consisting of cytochrome C, Apaf-1, pro-caspase 9 and ATP.

Note: There are other mechanisms to activate the caspase cascades.