endosymbiosis and the origin of eukaryotes: are mitochondria really just bacterial symbionts?

©1999 Timothy G. Standish

Endosymbiosis and the Endosymbiosis and the Origin of Eukaryotes:Origin of Eukaryotes:Are mitochondria really just Are mitochondria really just

bacterial symbionts?bacterial symbionts?

Timothy G. Standish, Ph. D.


OutlineOutline 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


MitochondriaMitochondria 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.


MitochondriaMitochondria

Matrix

Inter membrane space

Inner membraneOuter membrane

mtDNA


Extranuclear DNAExtranuclear 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


mtDNAmtDNA 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)


EndosymbiosisEndosymbiosis


Origin of EukaryotesOrigin of EukaryotesTwo 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






Nucleus

Endoplasmic Reticulum

Golgi Body

Mitochondria

Chloroplast






Chloroplast

Endoplasmic Reticulum

Nucleus

Golgi Body

Mitochondria


How Mitochondria Resemble BacteriaHow Mitochondria Resemble BacteriaMost 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’tHow Mitochondria Don’tResemble BacteriaResemble 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’tHow Mitochondria Don’tResemble BacteriaResemble 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).


ArchaezoaArchaezoa


GiardiaGiardia - A “Missing Link”? - 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”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 Origin of GiardiaGiardia 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 ArchaezoaOrigin 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:Origin of Archaezoa:Mitochondrial AcquisitionMitochondrial Acquisition


Origin of Archaezoa:Origin of Archaezoa:Gene Transfer and LossGene Transfer and Loss

mtGenes

Lost genetic

material


Origin of Archaezoa:Origin of Archaezoa:Option 1 - Mitochondrial Eukaryote ProductionOption 1 - Mitochondrial Eukaryote Production


Origin of Archaezoa:Origin of Archaezoa:Option 2 - Mitochondrial DNA Loss/Option 2 - Mitochondrial DNA Loss/

Hydrogenosome productionHydrogenosome productionHydrogenosome


Origin of Archaezoa:Origin of Archaezoa:Option 2A - Mitochondria/Hydrogenosome LossOption 2A - Mitochondria/Hydrogenosome Loss


Gene TransportGene 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:Steps in Mitochondrial Acquisition:The Serial Endosymbiosis TheoryThe Serial Endosymbiosis Theory

Fusion of Rickettsia with either a nucleus containing Archaezoan or

an archaebacteriumRickettsia

DNA reduction/transfer to nucleus

Ancestral eukaryote(assuming a nucleus)

Primitive eukaryote

Host Cell


Steps in Mitochondrial Acquisition:Steps in Mitochondrial Acquisition:The Hydrogen HypothesisThe Hydrogen Hypothesis

Fusion of proteobacterium with an archaebacterium

Hydrogen producing proteobacterium

DNA reduction/transfer nucleus production

Hydrogen requiring archaebacterium

Ancestral eukaryoteWith nucleus containing both archaebacterium and proteobacterium genes


Metamonada

Hydrogenosome/mitochondria

loss

Hydrogenosome/mitochondria

loss

Microsporidia, and Parabasalia

mtDNAloss

mtDNAloss

mtDNAloss

mtDNAloss

PhylogenyPhylogeny

Eukaryota BacteriaBacteria

Origin of Life

Gene transferGene transfer

Cell fusionCell fusion


Timing of Gene TransferTiming 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 Gene ExpressionExpression


Cytoplasmic Production of Cytoplasmic Production of Mitochondrial ProteinsMitochondrial 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 The Unlikely Movement of Genes Between Mitochondria and the NucleusBetween 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


Cytoplasm

Nucleus

Protein Production Protein Production Mitochondria and ChloroplastsMitochondria and Chloroplasts

G AAAAAA

Export

ChloroplastMitochondrion


Cytoplasm

Nucleus

ChloroplastMitochondrion

Protein Production Protein Production Mitochondria and ChloroplastsMitochondria and Chloroplasts


Protein Production Protein Production MitochondriaMitochondria

Matrix





Matrix

Inter membrane

space

Inner membrane

Outer membrane

Leader sequence binding receptor

ML

SLR

QS

IRF

FK

PA

TR

TL

CS

SRY

LL

P +ADP

ATP

P +ADP

ATP

©1999 Timothy G. StandishMatrix

Inner membrane

Outer membrane

MLSLR

QSIR

FFKPA

TRTLC

SSRY

LL

Inter membrane

space



Peptidease cleaves off the leader



Matrix

Inner membrane

Outer membrane

MLSLRQSIRFFKPATRTLCSSRYLL

Inter membrane

space




Matrix

Inner membrane

Outer membrane

Inter membrane

space




Matrix

Inner membrane

Outer membrane

Inter membrane

space


Hsp60

Hsp60

Chaperones



Matrix

Inner membrane

Outer membrane

Inter membrane

space


Mature protein


Yeast Cytochrome C Yeast Cytochrome C Oxidase Subunit IV LeaderOxidase Subunit IV Leader

MLSLRQSIRFFKPATRTLCSSRYLL

PolarPolar

PolarPolar

Non-Non-polarpolar

RY

PL

T

CS

R

L

S

T

I

KP

R

F

A

F

M

RQ

L

L

S

S

This leader does not resemble other eukaryotic leader sequences, or other mtProtein leader sequences.

Probably forms an 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

First 12 residues are sufficient for transport to the mitochondria

Neutral Non-polarPolarBasicAcidic

Recognized by peptidase?


Yeast Cytochrome C1 LeaderYeast Cytochrome C1 Leader

MFSNLSKRWAQRTLSKTLKGSKSAAGTATSYFE-KLVTAGVAAAGITASTLLYANSLTAGA--------------

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-polarPolarBasicAcidic

Second cut

First cut

Uncharged second leader sequence signals for transport across inner membrane into the intermembrane space

Charged leader sequence signals for transport to mitochondria



Matrix





Matrix

Inter membrane

space

Inner membrane

Outer membrane


P +ADP

ATP

P +ADP

ATP

Peptidease cleaves off the leader



Matrix

Inter membrane

space

Inner membrane

Outer membrane




Matrix

Inter membrane

space

Inner membrane

Outer membrane


Peptidease cleaves off the second leader



Matrix

Inter membrane

space

Inner membrane

Outer membrane




Matrix

Inter membrane

space

Inner membrane

Outer membrane


Mature protein


Nuclear DNA

Building a Minimally Functional Building a Minimally Functional Nuclear Mitochondrial GeneNuclear Mitochondrial Gene

Given that a fragment of DNA travels from the mitochondria to the nucleus and is inserted into the nuclear DNA

Additional hurdles may include: Resolution of problems resulting from differences between

mitochondrial and nuclear introns Resolution of problems resulting from differences between

mitochondiral and nuclear genetic codes

Mitochondrial Gene

Signal Sequence

Control Sequence

Mitochondrial GeneSignal SequenceControl Sequence


Additional RequirementsAdditional RequirementsIn 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 ExistsNo 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 Mitochondrial Genetic CodesGenetic Codes


Variation In Codon MeaningVariation 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

serine

Stop

Stop

Common Meaning

Stop

CandidaA yeast

Euplotes octacarinatusA ciliate

ParameciumA ciliate

OrganismTetrahymena thermophila

A ciliate

leucine

cysteine

glutamine

Modified Meaning

CUG

UGA

UAA UAG

Codon/sUAA UAG

glutamine

StopMycoplasma capricolumA bacteria tryptophanUGA

Neutral Non-polar, Polar


Variation in Mitochondrial Variation in Mitochondrial Codon AssignmentCodon Assignment

UGA/G=Stop

UniversalCode

Cyt

opla

sm/

Nu

cleu

s

Pla

nts

Yea

st/

Mol

ds

Pla

tyh

elm

ith

s

Ech

inod

erm

s

Mol

lusc

s

Inse

cts

Ver

teb

rate

s

UGA=Trp

AGA/G=Ser

AUA=Met

AUA=MetCUN=Thr

AUA=IleAAA=Asn

AAA=AsnN

emat

odes

NOTE - This would mean AUA changed from Ile to Met, then changed back to Ile in the Echinoderms

UGA must have changed to Trp then back to stop Differences in mtDNA lower the number of tRNAs needed

AAA must have changed from Lys to Asn twice


Problems Resulting From Problems Resulting From Differences in Genetic CodesDifferences 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 ExamplesNo Modern ExamplesUnfortunately 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)


ConclusionsConclusions 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.

endosymbiosis and the origin of eukaryotes: are mitochondria really just bacterial symbionts?

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