Powerpoint Endosymbiosis

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

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.

Mitochondria
Outer membrane
Inner membrane
Matrix mtDNA
Inter membrane space

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
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)

Endosymbiosis

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

system
Mitochondria

system
Endoplasmic
Mitochondria Reticulum
Nucleus
Golgi
Chloroplast Body
system
Endoplasmic Reticulum
Mitochondria
Nucleus
Golgi Body
Chloroplast
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.

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
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).
Archaezoa

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

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

Origin of Giardia
 Giardia and other eukaryotes lacking mitochondria and
plastids (Metamonada, Microsporidia, and Parabasalia ) have
been grouped by some as “Archaezoa” (Cavalier-Smith, 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 Archaezoans
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.
Origin of Archaezoa:
Mitochondrial Acquisition

Gene Transfer and Loss
mtGenes
Lost
genetic
material
Option 1 - Mitochondrial Eukaryote Production

Option 2 - Mitochondrial DNA Loss/
Hydrogenosome production
Hydrogenosome

Option 2A - Mitochondria/Hydrogenosome Loss

Gene Transport

“All in all then, the host nucleus
seems to be a tremendous
magnet, both for organellar
genes and for endosymbiotic
nuclear genes.”
Palmer, 1997

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)
Steps in Mitochondrial Acquisition:
The Hydrogen Hypothesis
Fusion of proteobacterium with an

archaebacterium
Hydrogen
producing Hydrogen
proteobacterium requiring
archaebacterium
DNA
reduction/transfer
nucleus production
Ancestral eukaryote
With nucleus containing both
archaebacterium and
proteobacterium genes

Phylogeny
Microsporidia,
Bacteria and Parabasalia Metamonada Eukaryota Bacteria
mtDNA Hydrogenosome/
loss mitochondria
loss
mtDNA
loss
Gene transfer
Cell fusion
Origin of Life
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.
Gene
Expression

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

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
Protein Production
Mitochondria and Chloroplasts
Cytoplasm
Nucleus G AAAAAA
Export
Mitochondrion Chloroplast

Protein Production
Mitochondria and Chloroplasts
Cytoplasm
Nucleus
Mitochondrion Chloroplast

Protein Production
Mitochondria
Outer membrane
Inner membrane
Matrix

Protein Production
Mitochondria
Leader sequence
ATP binding receptor
Outer membrane
P +ADP
ATP MLSLRQSIRFFKPATRTLCSSRYLL
P +ADP
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
M
LS
Peptidease LR
cleaves off QS
the leader
RFI
FK
PA
Inner membrane
TR
TL
Inter
CS
membrane
S RY
Matrix space
LL

Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
ML
SL RQ
SIR
FFK Inner membrane
PA
T RT Inter
LC
SSR membrane
YL
Matrix L space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Hsp60
Hsp60
Inner membrane
Inter
Chaperones membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix Mature protein
space
M
L Yeast Cytochrome C
S
L Polar
R
Oxidase Subunit IV Leader
Q Neutral Non-polar
S First 12 residues are sufficient for Polar
I Non- transport to the mitochondria
Basic
polar
R Acidic
F
F MLSLRQSIRFFKPATRTLCSSRYLL
K  This leader does not resemble other eukaryotic
P
A leader sequences, or other mtProtein leader
Recognized by peptidase?
T
R sequences.
T  Probably forms an  helix
L Polar
C  This would localize specific classes of amino
S acids in specific parts of the helix
S
R
Y
 There are about 3.6 amino acids per turn of the
helix with a rise of 0.54 nm per turn
L
P ©1999 Timothy G. Standish
Yeast Cytochrome C1 Leader
Charged leader sequence signals
for transport to mitochondria First cut
MFSNLSKRWAQRTLSKTLKGSKSAAGTATSYFE-
KLVTAGVAAAGITASTLLYANSLTAGA--------------
Second cut
Uncharged second leader sequence signals for transport
across inner membrane into the intermembrane space Neutral Non-polar
 Cytochrome c functions in electron transport and is thus Polar
Basic
associated with the inner membrane on the Acidic
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
Protein Production
Mitochondria
Outer membrane
Inner membrane
Matrix

Protein Production
Mitochondria
Leader sequence
ATP binding receptor
Outer membrane
P +ADP
ATP
P +ADP
Peptidease
cleaves off
the leader Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Peptidease
cleaves off the
second leader
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Inter
membrane
Matrix space
Protein Production
Mitochondria
Leader sequence
binding receptor
Outer membrane
Inner membrane
Mature protein
Inter
membrane
Matrix space
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 between
Mitochondrial Gene
mitochondrial and nuclear introns
 Resolution of problems resulting from differences between
mitochondiral and nuclear genetic codes
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

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?

Mitochondrial
Genetic Codes

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 Common Meaning Modified Meaning
Tetrahymena thermophila UAA UAG Stop glutamine
A ciliate
Paramecium UAA UAG Stop glutamine
A ciliate
Euplotes octacarinatus UGA Stop cysteine
A ciliate
Mycoplasma capricolum UGA Stop tryptophan
A bacteria
Candida CUG serine leucine
A yeast
Neutral Non-polar, Polar©1999 Timothy G. Standish
Variation in Mitochondrial
Codon Assignment
Platyhelmiths
Echinoderms
Vertebrates
Cytoplasm/
Nematodes
Molluscs
Nucleus
Insects
Molds
Yeast/
Plants
AUA=Met AAA=Asn AUA=Ile UGA/G=Stop

CUN=Thr AAA=Asn
 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
Universal UGA must have changed to Trp then back to stop

Code  Differences in mtDNA lower the number of tRNAs needed
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.
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)

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.

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Endosymbiosis and the

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

Inter membrane space

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

Fusion of proteobacterium with an

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

Inter membrane space

©1999 Timothy G. Standish

©1999 Timothy G. Standish

Inter membrane space

©1999 Timothy G. Standish

Control Sequence Signal Sequence Mitochondrial Gene

©1999 Timothy G. Standish

©1999 Timothy G. Standish

©1999 Timothy G. Standish

AUA=Met AAA=Asn AUA=Ile UGA/G=Stop

©1999 Timothy G. Standish

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