Transcript Document

Molecular Biology 325 2006
Molecular biology of mitochondria
Mitochondria are the main site of ATP synthesis in
eukaryote cells and as such are vital for the health
and survival of the cell
They are also one of the sites at which apoptosis is
mediated
These lectures will explore the molecular genetics of
mitochondria, how they are made, the structure of their
genome, how they evolved , and how mitochondrial
gene expression is controlled.
Mitochondrial molecular genetics 1
• focus on mitochondria: brief overview of their
function and structure
• mtDNA structure and replication:
- animals
- yeast
- plants
• inheritance of mitochondria
- petite mutants of yeast
• biogenesis of mitochondria by fission
MITOCHONDRIA
• essential for cell life
- ATP synthesis
- many metabolic intermediates
• essential for cell death
- unprogrammed death: necrosis
( eg, due to loss of energy status)
- programmed cell death
(apoptosis - controlled cell destruction)
Mitochondrial structure
• Two membranes
• Inner membrane invaginated
• Numbers of mitochondria per cell
vary but usually 100s/cell
Matrix contains the TCA
cycle (and other) soluble
enzymes
Inner membrane
contains metabolite
transporters and the
electron transport chain
Overview of
aerobic
respiration
Outline of Tricarboxylic Acid Cycle
3-Carbons
One pyruvate molecule is
completely oxidised to CO2
CO2
4-Carbons
6-Carbons
NADH
NADH + CO2
FADH
NADH
+ CO2
The NADH and FADH
produced are oxidised
by the respiratory
electron transport chain
Four large, multi-subunit
protein complexes
- complex I is a NADHubiquinone reductase
- complex II is succinate
dehydrogenase (part of the
TCA cycle)
- complex III is the
ubiquinone -cytochrome c
reductase
- complex IV is cytochrome
oxidase
The respiratory electron transport chain
Mitochondria have their own DNA and Ribosomes
Mitochondria have some of their own DNA, ribosomes, and can make many
of their own proteins. The DNA is circular and lies in the matrix in structures
called "nucleoids". Each nucleoid may contain 4-5 copies of the
mitochondrial DNA (mtDNA).
mitochondrial
DNA
Mitochondria also have their own ribosomes and tRNA:
• 22 tRNAs
• rRNAs (16S and 12S)
The ribosomes can actually be visualized in some mitochondria. In these
figures, they are seen in the matrix as small dark bodies. DNA can also be
visualized in mitochondria. The DNA is circular and resembles that of a
bacterium in its basic structure.
This micrograph shows the DNA and ribosomes in a close-up view. Note
that the circular structure of the DNA is not evident. It is noted by an
arrow. There are two sets of ribosomes seen, each is circled
To visualize the structure of mitochondrial DNA, we have to extract the
DNA and float it on a water surface. Then, it can be picked up by a plastic
coated grid, and examined in the electron microscope. Mitochondrial
circular DNA is shown in the figure.
Mitochondrial Inheritance
Yeast has been used extensively to study
mitochondrial inheritance.
There is a Yeast strain, called "Petite" that have
structurally abnormal mitochondria that are incapable
of oxidative phosphorylation. These mitochondria
have lost some or all of their DNA.
Genetic crosses between petite and wt strains
showed that inheritance of this trait did not segregate
with any of the nuclear chromosomes.
Mitochondrial Inheritance
Mitochondrial inheritance from yeast is biparental,
and both parent cells contribute to the daughter
cells when the haploid cells fuse. After meiosis and
mitosis, there is random distribution of
mitochondria to daughter cells. If the fusion is with
yeast that are petite and yeast that are not, a
certain percentage of the daughter cells will be
"petite".
Mitochondrial Inheritance in Yeast
Mitochondrial Inheritance
This led to the suggestion that some
genetic element existed in the cytoplasm
and was inherited in a different manner
from nuclear genes. This is called “nonMendelian inheritance” or “cytoplasmic
inheritance”.
In yeast and animals, this indicated inheritance
of mitochondrial genes: in plants it also
includes inheritance of chloroplast genes
Mitochondrial replication
Mitochondrial replication
cell division: random distribution
of mitos between daughter cells
mitochondrial
replication
Mitochondria replicate much like bacterial cells.
When they get too large, they undergo fission.
This involves a furrowing of the inner and then
the outer membrane as if someone was
pinching the mitochondrion. Then the two
daughter mitochondria split. Of course, the
mitochondria must first replicate their DNA. An
electron micrograph depicting the furrowing
process is shown in these figures.
Sometimes new mitochondria are synthesized in centres that are rich
in proteins and polyribosomes needed for their synthesis. The electron
micrograph in the following figure shows such a centre. It appears that
the cluster of mitochondria are sitting in a matrix of proteins and other
materials needed for their production.
Certain mitochondrial
proteins are needed
before the
mitochondria can
divide.
giant
mitochondrion
This has been shown in a
study by Sorgo and Yaffe,
J Cell Bio. 126: 1361-1373,
1994. They showed the
result of the removal of an
outer membrane protein
from mitochondria called
MDM10. This figure shows
the results. The
mitochondria are able to
take in components and
produce membranes and
matrix enzymes. However,
fission is not allowed and
the result is a giant
mitochondrion.
Organisation of the
mitochondrial chromosome
Human mtDNA
• small, double stranded
circular chromosome
• 16,569 bp in total
• no non-coding DNA
• no introns
• polycistronic replication
which is initiated from
the D (displacement)- loop
region
• followed by splicing of
transcript to form
messages.
Yeast
mitochondrial
chromosome
yeast mtDNA
human mtDNA
Human DNA
• 16,569 bp;
• no non-coding DNA
• no introns
• polycistronic replication followed by splicing to form messages.
Yeast mtDNA
• 68-75 kb, similar in structure to bacterial genome
• contains introns and non-regions between genes.
• Same proteins made as in animals
• genes transcribed separately
Despite having their own genome, most mitochondrial
proteins are encoded in the nucleus, made in the cytosol and
imported into the mitochondria
Synthesis of mitochondrial proteins
In all organisms, only a few of the proteins of the
mitochondrion are encoded by mtDNA, but the precise
number varies between organisms
• Subunits 1, 2, and 3 of cytochrome oxidase
• Subunits 6, 8, 9 of the Fo ATPase
• Apocytochrome b subunit of complexIII
• Seven NADH-CoQ reductase subunits (except in yeast)
The nucleus encodes the remaining proteins which are
made in the cytosol and imported into the mitochondrion.
Most of the lipid is imported.
Plant mtDNA
• chromosome size is much bigger but varies dramatically between
species (200-2000 kb)
• arranged as different size circles, sometimes with plasmids.
• The plant mtDNA contains chloroplast sequences, indicating
exchange of genetic information between organelles in plants.
• Much of the plant mtDNA is non-coding, but coding regions are
larger than animals and fungi.
• Number of proteins synthesised not known definitely but more
than in animals and yeast (probably about 50)
Plant mitochondria have specialised functions
• in leaves they participate in photorespiration
• sites of vitamin synthesis (vit C, folic acid, biotin)
maize mitochondrial
genome
In plants, respiration and photosynthesis operate
simultaneously in the light
NIGHT
DAY
Chloroplasts are the
site of photosynthesis
and belong to the
plastid family of
organelles - they
develop from
proplastids in the
light
proplastid
thylakoid
stacks
amyloplast (in storage organs)
Rice mitochondrial
and chloroplast
genomes
Plant
mitochondria
contain
chloroplast genes
- suggesting that
genetic transfer
occurs between
the two organelles
Rib osome
RuBisCO
Cyto chrome b 6/ f
Photosystem I
Stroma
Chlo roplas t
Photosystem II
ATP synthase
Lu men
Rib osome
Matrix
Mitochond ria
Compl ex I
Compl ex II
Inte rmemb rane Space
Complex III
Compl ex IV
Compl ex V
Mitochondrial DNA of animals and fungi uses a different genetic code
than the “universal” code
RNA processing in mitochondria
Plant mitochondria “edit” their RNA transcripts. This
was first noticed when comparing cDNA sequences
with genomic DNA sequences.
The most common change is to replace C with U,
although in some instances other changes can occur.
Matrix enzymes are thought to be responsible for this,
but the reason for the editing is not known.
Most of the DNA in plant mitochondria is non-coding,
only some of which is transcribed. RNA editing
occurs even in non-coding regions such as introns.
Evolution of mitochondria
Mitochondria are generally thought to have
evolved endosymbiotically when an
anaerobic prokaryotic cell engulfed an
aerobic bacterium and formed a stable
symbiosis. Loss of most of the aerobe’s
genome to the nucleus of the host allowed
the latter to control the former.
This is supported by gene sequence analysis
which shows remarkable homology between
bacteria and mitochondrial genes.