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Endosymbiosis and the
Origin of Eukaryotes:
Are mitochondria really just
bacterial symbionts?
Timothy G. Standish, Ph. D.
©1999 Timothy G. Standish
Outline
Mitochondria - A very brief overview
Endosymbiosis - Theory and evidence
Archaezoa - Eukaryotes lacking mitochondria
Gene expression - Mitochondrial proteins
coded in the nucleus
Mitochondrial genetic codes
Gene transport - Mitochondria to nucleus
Conclusions
©1999 Timothy G. Standish
Mitochondria
Mitochondria are organelles found in most
eukaryotic organisms.
The site of Krebs cycle and electron transport
energy producing processes during aerobic
respiration
Are inherited only from the mother during sexual
reproduction in mammals and probably all other
vertebrates.
Because of their mode of inheritance genetic
material found in mitochondria appears to be useful
in determining the maternal lineage of organisms.
©1999 Timothy G. Standish
Mitochondria
Outer membrane
Matrix
Inner membrane
mtDNA
Inter membrane space
©1999 Timothy G. Standish
Extranuclear DNA
Mitochondria and chloroplasts have their own DNA
This extranuclear DNA exhibits non-Mendelian inheritance
Recombination is known between some mt and ctDNAs
Extranuclear DNA may also be called cytoplasmic DNA
Generally mtDNA and ctDNA is circular and contains
genes for multimeric proteins, some portion of which are
also coded for in the nucleus
Extranuclear DNA has a rate of mutation that is independent
of nuclear DNA
Generally, but not always, all the RNAs needed for
transcription and translation are found in mtDNA and
ctDNA, but only some of the protein genes
©1999 Timothy G. Standish
mtDNA
Mitochondrial DNA is generally small in animal cells,
about 1.65 kb
In other organisms sizes can be more than an order of
magnitude larger
Plant mtDNA is highly variable in size and content with
the large Arabidopsis mtDNA being 200 kb.
The largest known number of mtDNA protein genes is 97
in the protozoan Riclinomonas mtDNA of 69 kb.
“Most of the genetic information for mitochondrial
biogenesis and function resides in the nuclear genome,
with import into the organelle of nuclear DNA-specified
proteins and in some cases small RNAs.” (Gray et
al.,1999)
©1999 Timothy G. Standish
Endosymbiosis
©1999 Timothy G. Standish
Origin of Eukaryotes
Two popular theories presupposing naturalism seek to
explain the origin of membrane-bound organelles:
1 Endosymbiosis to explain the origin of mitochondria and
chloroplasts (popularized by Lynn Margulis in 1981)
2 Invagination of the plasma membrane to form the
endomembrane system
©1999 Timothy G. Standish
Origin of Eukaryotes
Two popular theories presupposing naturalism seek to
explain the origin of membrane-bound organelles:
1 Endosymbiosis to explain the origin of mitochondria and
chloroplasts (popularized by Lynn Margulis in 1981)
2 Invagination of the plasma membrane to form the
endomembrane system
Mitochondria
©1999 Timothy G. Standish
Origin of Eukaryotes
Two popular theories presupposing naturalism seek to
explain the origin of membrane-bound organelles:
1 Endosymbiosis to explain the origin of mitochondria and
chloroplasts (popularized by Lynn Margulis in 1981)
2 Invagination of the plasma membrane to form the
endomembrane system
Endoplasmic
Mitochondria
Reticulum
Nucleus
Chloroplast
Golgi
Body
©1999 Timothy G. Standish
Origin of Eukaryotes
Two popular theories presupposing naturalism seek to
explain the origin of membrane-bound organelles:
1 Endosymbiosis to explain the origin of mitochondria and
chloroplasts (popularized by Lynn Margulis in 1981)
2 Invagination of the plasma membrane to form the
endomembrane system
Endoplasmic Reticulum
Mitochondria
Nucleus
Chloroplast
Golgi Body
©1999 Timothy G. Standish
How Mitochondria Resemble Bacteria
Most general biology texts list ways in which
mitochondria resemble bacteria. Campbell et al.
(1999) list the following:
Mitochondria resemble bacteria in size and morphology.
They are bounded by a double membrane: the outer thought
to be derived from the engulfing vesicle and the inner from
bacterial plasma membrane.
Some enzymes and inner membrane transport systems
resemble prokaryotic plasma membrane systems.
Mitochondrial division resembles bacterial binary fission
They contain a small circular loop of genetic material
(DNA). Bacterial DNA is also a circular loop.
They produce a small number of proteins using their own
ribosomes which look like bacterial ribosomes.
Their ribosomeal RNA resembles eubacterial rRNA.
©1999 Timothy G. Standish
How Mitochondria Don’t
Resemble Bacteria
Mitochondria are not always the size or morphology of
bacteria:
– In some Trypanosomes (i.e., Trypanosoma brucei)
mitochondria undergo spectacular changes in morphology that
do not resemble bacteria during different life cycle stages
(Vickermann, 1971)
– Variation in morphology is common in protistans,
“Considerable variation in shape and size of the organelle can
occur.” (Lloyd, 1974 p 1)
Mitochondrial division and distribution of mitochondria
to daughter cells is tightly controlled by even the simplest
eukaryotic cells
©1999 Timothy G. Standish
How Mitochondria Don’t
Resemble Bacteria
Circular mtDNA replication via D loops is different from
replication of bacterial DNA (Lewin, 1997 p 441).
mtDNA is much smaller than bacterial chromosomes.
Mitochondrial DNA may be linear; examples include:
Plasmodium, C. reinhardtii, Ochromonas, Tetrahymena,
Jakoba (Gray et al., 1999).
Mitochondrial genes may have introns which eubacterial
genes typically lack (these introns are different from
nuclear introns so they cannot have come from that
source) (Lewin, 1997 p 721, 888).
The genetic code in many mitochondria is slightly
different from bacteria (Lewin, 1997).
©1999 Timothy G. Standish
Archaezoa
©1999 Timothy G. Standish
Giardia - A “Missing Link”?
The eukaryotic parasite Giardia has been suggested as a
“missing link” between eukaryotes and prokaryotes
because it lacks mitochondria (Friend, 1966; Adam,
1991) thus serving as an example of membrane
invagination but not endosymbiosis
Giardia also appears to lack smooth endoplasmic
reticulum, peroxisomes and nucleoli (Adam, 1991) so
these must have either been lost or never evolved
©1999 Timothy G. Standish
A Poor “Missing Link”
As a “missing link” Giardia is not a strong argument due
to its parasitic life cycle which lacks an independent
replicating stage outside of its vertebrate host
– Transmission is via cysts excreted in feces followed by
ingestion
– As an obligate parasite, to reproduce, Giardia needs other more
derived (advanced?) eukaryotes
Some other free-living Archaezoan may be a better
candidate
©1999 Timothy G. Standish
Origin of Giardia
Giardia and other eukaryotes lacking mitochondria and
plastids (Metamonada, Microsporidia, and Parabasalia )
have been grouped by some as “Archaezoa” (CavalierSmith, 1983; Campbell et al., 1999 p 524-6)
This name reflects the belief that these protozoa split from
the group which gained mitochondria prior to that event.
The discovery of a mitochondrial heat shock protein
(HSP60) in Giardia lamblia (Soltys and Gupta, 1994) has
called this interpretation into question.
Other proteins thought to be unique to mitochondria,
HSP70 (Germot et al., 1996), chaperonin 60 (HSP60)
(Roger et al., 1996; Horner et al., 1996) and HSP10 (Bui
et al., 1996) have shown up in Giardia’s fellow
©1999 Timothy G. Standish
Origin of Archaezoa
The authors who reported the presence of mitochondrial
genes in amitochondrial eukaryotes all reinterpreted
prevailing theory in saying that mitochondria must have
been present then lost after they had transferred some of
their genetic information to the nucleus.
The hydrogenosome, a structure involved in carbohydrate
metabolism found in some Archaezoans (Muller, 1992), is
now thought to represent a mitochondria that has lost its
genetic information completely and along with that loss,
the ability to do the Krebs cycle (Palmer, 1997).
Alternative explanations include transfer of genetic
material from other eukaryotes and the denovo production
of hydrogenosomes by primitive eukaryotes.
©1999 Timothy G. Standish
Origin of Archaezoa:
Mitochondrial Acquisition
©1999 Timothy G. Standish
Origin of Archaezoa:
Gene Transfer and Loss
mtGenes
Lost
genetic
material
©1999 Timothy G. Standish
Origin of Archaezoa:
Option 1 - Mitochondrial Eukaryote Production
©1999 Timothy G. Standish
Origin of Archaezoa:
Option 2 - Mitochondrial DNA Loss/
Hydrogenosome production
Hydrogenosome
©1999 Timothy G. Standish
Origin of Archaezoa:
Option 2A - Mitochondria/Hydrogenosome Loss
©1999 Timothy G. Standish
Gene Transport
©1999 Timothy G. Standish
“All in all then, the host nucleus
seems to be a tremendous
magnet, both for organellar
genes and for endosymbiotic
nuclear genes.”
Palmer, 1997
©1999 Timothy G. Standish
Steps in Mitochondrial Acquisition:
The Serial Endosymbiosis Theory
Fusion of Rickettsia with either a
nucleus containing Archaezoan or
an archaebacterium
Rickettsia
Host Cell
Primitive
eukaryote
DNA
reduction/transfer
to nucleus
Ancestral eukaryote
(assuming a nucleus)
©1999 Timothy G. Standish
Steps in Mitochondrial Acquisition:
The Hydrogen Hypothesis
Fusion of proteobacterium with an
archaebacterium
Hydrogen
producing
proteobacterium
Hydrogen
requiring
archaebacterium
DNA
reduction/transfer
nucleus production
Ancestral eukaryote
With nucleus containing both
archaebacterium and
proteobacterium genes
©1999 Timothy G. Standish
Phylogeny
Bacteria
Microsporidia,
and Parabasalia
Metamonada Eukaryota
Bacteria
mtDNA Hydrogenosome/
loss mitochondria
loss
mtDNA
loss
Gene transfer
Cell fusion
Origin of Life
©1999 Timothy G. Standish
Timing of Gene Transfer
Because gene transfer occurred in eukaryotes lacking
mitochondria, and these are the lowest branching
eukaryotes known:
Gene transfer must have happened very early in the
history of eukaryotes.
The length of time for at least some gene transfer
following acquisition of mitochondria is greatly
shortened.
No plausible mechanism for movement of genes from
the mitochondria to the nucleus exists although
intraspecies transfer of genes is sometimes invoked to
explain the origin of other individual nuclear genes.
©1999 Timothy G. Standish
Gene
Expression
©1999 Timothy G. Standish
Cytoplasmic Production of
Mitochondrial Proteins
Mitochondria produce only a small subset of the proteins
used in the Krebs cycle and electron transport. The
balance come from the nucleus
As mitochondrial genomes vary spectacularly between
different groups of organisms, some of which may be
fairly closely related, if all came from a common
ancestor, different genes coding for mitochondrial
proteins must have been passed between the nucleus and
mitochondria multiple times
©1999 Timothy G. Standish
The Unlikely Movement of Genes
Between Mitochondria and the Nucleus
Movement of genes between the mitochondria
and nucleus seems unlikely for at least two
reasons:
1 Mitochondria do not always share the same
genetic code with the cell they are in
2 Mechanisms for transportation of proteins
coded in the nucleus into mitochondria seem to
preclude easy movement of genes from
mitochondria to the nucleus
©1999 Timothy G. Standish
Protein Production
Mitochondria and Chloroplasts
Cytoplasm
Nucleus
G
AAAAAA
Export
Mitochondrion
Chloroplast
©1999 Timothy G. Standish
Protein Production
Mitochondria and Chloroplasts
Cytoplasm
Nucleus
Mitochondrion
Chloroplast
©1999 Timothy G. Standish
Protein Production
Mitochondria
Outer membrane
Inner membrane
Matrix
Inter membrane space
©1999 Timothy G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
ATP
ATP
P +ADP
Matrix
MLSLRQSIRFFKPATRTLCSSRYLL
P +ADP
Outer membrane
Inner membrane
Inter
membrane
space
©1999 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Peptidease
cleaves off
the leader
Inner membrane
Matrix
Inter
membrane
space
©1999 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©1999 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©1999 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Hsp60
Hsp60
Matrix
Chaperones
Inner membrane
Inter
membrane
space
©1999 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Mature protein
Inter
membrane
space
©1999 Timothy
G. Standish
M
L
S
L Polar
R
I
Q
S
NonR
polar
F
First 12 residues are sufficient for
transport to the mitochondria
F
K
A
P
R
T
L Polar
C
R
P
S
S
Y
L
Neutral Non-polar
Polar
Basic
Acidic
MLSLRQSIRFFKPATRTLCSSRYLL
Recognized by peptidase?
T
Yeast Cytochrome C
Oxidase Subunit IV Leader
This leader does not resemble other
eukaryotic leader sequences, or other
mtProtein leader sequences.
Probably forms an a helix
This would localize specific classes of
amino acids in specific parts of the helix
There are about 3.6 amino acids per turn
of the helix with a rise of 0.54 nm per turn
©1999 Timothy G. Standish
Yeast Cytochrome C1 Leader
Charged leader sequence signals
for transport to mitochondria
First cut
MFSNLSKRWAQRTLSKTLKGSKSAAGTATSYFEKLVTAGVAAAGITASTLLYANSLTAGA-------------Uncharged second leader sequence signals for transport
across inner membrane into the intermembrane space
Second cut
Cytochrome c functions in electron transport and is
thus associated with the inner membrane on the
intermembrane space side
Cytochrome c1 holds an iron containing heme
group and is part of the B-C1 (III) complex
C1 accepts electrons from the Reiske protein and
passes them to cytochrome c
Neutral Non-polar
Polar
Basic
Acidic
©1999 Timothy G. Standish
Protein Production
Mitochondria
Outer membrane
Inner membrane
Matrix
Inter membrane space
©1999 Timothy G. Standish
Protein Production
Mitochondria
ATP
Leader sequence
binding receptor
P +ADP
Outer membrane
ATP
P +ADP
Peptidease
cleaves off
the leader
Matrix
Inner membrane
Inter
membrane
space
©1999 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©1999 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©1999 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©1999 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©1999 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Peptidease
cleaves off the
second leader
Matrix
Inter
membrane
space
©1999 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©1999 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Matrix
Inter
membrane
space
©1999 Timothy
G. Standish
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Mature protein
Matrix
Inter
membrane
space
©1999 Timothy
G. Standish
Building a Minimally Functional
Nuclear Mitochondrial Gene
Given that a fragment of DNA travels from the
mitochondria to the nucleus and is inserted into the
nuclear DNA
Nuclear DNA
Control Sequence Signal Sequence
Mitochondrial Gene
Control Sequence
Additional hurdles may
include:
Signal Sequence
Resolution of problems resulting from
differences
Mitochondrial
Gene
between mitochondrial and nuclear introns
Resolution of problems resulting from differences
between mitochondiral and nuclear genetic codes
©1999 Timothy G. Standish
Additional Requirements
In addition to addition of appropriate control and
leader sequences to mitochondrial genes, the
following would be needed:
Recognition and transport mechanisms in the
cytoplasm
Leader sequence binding receptors
Peptidases that recognize leader sequences and
remove them
©1999 Timothy G. Standish
No Plausible Mechanism Exists
If genes were to move from the mitochondria to the nucleus
they would have to somehow pick up the leader sequences
necessary to signal for transport before they could be
functional
While leader sequences seem to have meaningful portions
on them, according to Lewin (1997, p 251) sequence
homology between different sequences is not evident, thus
there could be no standard sequence that was tacked on as
genes were moved from mitochondria to nucleus
Alternatively, if genes for mitochondrial proteins existed in
the nucleus prior to loss of genes in the mitochondria, the
problem remains, where did the signal sequences come
from? And where did the mechanism to move proteins with
signal sequences on them come from?
©1999 Timothy G. Standish
Mitochondrial
Genetic Codes
©1999 Timothy G. Standish
Variation In Codon Meaning
Lack of variation in codon meanings across almost all phyla is
taken as an indicator that initial assignment must have occurred
early during evolution and all organisms must have descended
from just one individual with the current codon assignments
Exceptions to the universal code are known in a few single-celled
eukaryotes, mitochondria and at least one prokaryote
Most exceptions are modifications of the stop codons UAA, UAG
and UGA
Organism
Codon/s
Tetrahymena thermophila UAA UAG
A ciliate
Paramecium
UAA UAG
A ciliate
Common Meaning Modified Meaning
Stop
glutamine
Stop
glutamine
Euplotes octacarinatus
UGA
Stop
cysteine
Mycoplasma capricolum
UGA
Stop
tryptophan
Candida
CUG
serine
leucine
A ciliate
A bacteria
A yeast
Neutral Non-polar, Polar
©1999 Timothy G. Standish
AUA=Met
CUN=Thr
Universal
Code
AAA=Asn
AUA=Ile
AAA=Asn
Vertebrates
Insects
Molluscs
Echinoderms
Nematodes
Platyhelmiths
Yeast/
Molds
Plants
Cytoplasm/
Nucleus
Variation in Mitochondrial
Codon Assignment
UGA/G=Stop
NOTE - This would mean
AUA changed from Ile to
Met, then changed back to
AUA=Met
Ile in the Echinoderms
AGA/G=Ser
AAA must have changed from Lys to
Asn twice
UGA=Trp
UGA must have changed to Trp then back to stop
Differences in mtDNA lower the number of tRNAs needed
©1999 Timothy G. Standish
Problems Resulting From
Differences in Genetic Codes
Changing the genetic code, even of the most
simple genome is very difficult.
Because differences exist in the mitochondrial
genomes of groups following changes in the
mitochondrial genetic code, mitochondrial genes
coding differently must have been transported to
the nucleus.
These mitochondrial genes must have been edited
to remove any problems caused by differences in
the respective genetic codes.
©1999 Timothy G. Standish
No Modern Examples
Unfortunately for Margulis and S.E.T. [the serial
endosymbiotic theory], no modern examples of prokaryotic
endocytosis or endosymbioses exist . . . She discusses any
number of prokaryotes endosymbiotic in eukaryotes and
uses Bdellovibrio as a model for prokaryotic endocytosis.
Bdellovibrios are predatory (or parasitoid) bacteria that feed
on E. coli by penetrating the cell wall of the latter and then
removing nutrient molecules from E. coli while attached to
the outer surface of its plasma membrane. Although it is
perfectly obvious that this is not an example of one
prokaryote being engulfed by another Margulis continually
implies that it is.
P.J. Whitfield, review of “Symbiosis in Cell Evolution,” Biological
Journal of the Linnean Society 18 [1982]:77-78; p 78)
©1999 Timothy G. Standish
Conclusions
Presence of mitochondrial genes in nuclear DNA reduces
the window of time available for mitochondrial
acquisition in eukaryotes.
Understanding the structure of mitochondrial genes in
the nucleus and how they are expressed makes the
transfer of genes from protomitochondria to the nucleus
appear complex.
Differences between mitochondrial genetic codes and
nuclear genetic codes adds to the complexity of gene
transfer between mitochondria and nucleus.
As molecular data accumulates, the endosymbiotic origin
of mitochondria appears less probable.
©1999 Timothy G. Standish
©1999 Timothy G. Standish