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

Inner membrane

Matrix

mtDNA

Inter membrane space

1999 Timothy G. Standish

Extranuclear DNA
Mitochondria and chloroplasts have their own DNA This extranuclear DNA exhibits non-Mendalian 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 Extra-nuclear 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 geneome, 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 naturlaism seek to explain the origin of membrane bound organelles:
1 Endosymbiosis to explain the origin of mitochondria and chloroplasts (popularized by Lynn Margulis (Margulis, 1981) 2 Invagination of the plasma membrane to form the endomembrane system

1999 Timothy G. Standish

Origin of Eukaryotes
Two popular theories presupposing naturlaism seek to explain the origin of membrane bound organelles:
1 Endosymbiosis to explain the origin of mitochondria and chloroplasts (popularized by Lynn Margulis (Margulis, 1981) 2 Invagination of the plasma membrane to form the endomembrane system

Mitochondria

1999 Timothy G. Standish

Origin of Eukaryotes
Two popular theories presupposing naturlaism seek to explain the origin of membrane bound organelles:
1 Endosymbiosis to explain the origin of mitochondria and chloroplasts (popularized by Lynn Margulis (Margulis, 1981) 2 Invagination of the plasma membrane to form the endomembrane system Endoplasmic Mitochondria Reticulum Nucleus Golgi Body
1999 Timothy G. Standish

Chloroplast

Origin of Eukaryotes
Two popular theories presupposing naturlaism seek to explain the origin of membrane bound organelles:
1 Endosymbiosis to explain the origin of mitochondria and chloroplasts (popularized by Lynn Margulis (Margulis, 1981) 2 Invagination of the plasma membrane to form the endomembrane system Endoplasmic Reticulum Mitochondria Nucleus Golgi Body
1999 Timothy G. Standish

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.
1999 Timothy G. Standish

How Mitochondria Dont Resemble Bacteria

Mitochondria are not always the size or morphology of bacteria:
In some Trypanosomes (ie 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 p1)

Mitochondrial division and distribution of mitochondria to daughter cells is tightly controlled by even the simplest eukaryotic cells
1999 Timothy G. Standish

How Mitochondria Dont Resemble Bacteria

Circular mtDNA replication via D loops is different from replication of bacterial DNA (Lewin, 1997 p441). 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 p721, 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 Gardia
Gardia and other eukaryotes lacking mitochondira and plastids (Metamonada, Microsporidia, and Parabasalia ) have been grouped by some as Archezoa (CavalierSmith, 1983; Campbell et al., 1999 pp524-6) 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 Gardias fellow Archezoans

1999 Timothy G. Standish

Origin of Archezoa
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 Archezoans (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 Archezoa:
Mitochondrial Aquisition

1999 Timothy G. Standish

Origin of Archezoa:
Gene Transfer and Loss

mtGenes

Lost genetic material

1999 Timothy G. Standish

Origin of Archezoa:
Option 1 - Mitochondrial Eukaryote Production

1999 Timothy G. Standish

Origin of Archezoa:
Option 2 - Mitochondrial DNA Loss/ Hydrogenosome production Hydrogenosome

1999 Timothy G. Standish

Origin of Archezoa:
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 Archazoan 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 mitochondira 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 geneomes 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

ATP P +ADP ATP P +ADP
MLSLRQSIRFFKPATRTLCSSRYLL

Leader sequence binding receptor

Outer membrane

Inner membrane
Inter membrane space 1999 Timothy G. Standish

Matrix

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

Inner membrane
Inter membrane space 1999 Timothy G. Standish

Matrix

Chaperones

Protein Production Mitochondria

Leader sequence binding receptor Outer membrane

Inner membrane

Matrix

Mature protein

Inter membrane space 1999 Timothy G. Standish

L Polar R
I

Yeast Cytochrome C Oxidase Subunit IV Leader

First 12 residues are sufficient for transport to the mitochondria
Neutral Non-polar Polar Basic Acidic

NonR polar

F
K

F
P

MLSLRQSIRFFKPATRTLCSSRYLL
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

A
R

Recognized by peptidase?

L Polar
C

S
Y

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
Neutral Non-polar Polar Basic Acidic

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

1999 Timothy G. Standish

Protein Production Mitochondria

Outer membrane

Inner membrane

Matrix

Inter membrane space

1999 Timothy G. Standish

Protein Production Mitochondria

ATP P +ADP ATP P +ADP
Peptidease cleaves off the leader

Leader sequence binding receptor

Outer membrane

Inner membrane
Inter membrane space 1999 Timothy G. Standish

Matrix

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 Control Sequence Mitochondrial Gene

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, p251) 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

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

Variation In Codon Meaning

Organism
A ciliate
A ciliate

Codon/s

Common Meaning Modified Meaning Stop Stop Stop Stop serine glutamine glutamine cysteine tryptophan leucine
1999 Timothy G. Standish

Tetrahymena thermophila UAA UAG A ciliate Paramecium UAA UAG Euplotes octacarinatus Mycoplasma capricolum
A bacteria A yeast

UGA UGA CUG

Candida

Neutral Non-polar, Polar

Variation in Mitochondrial Codon Assignment

Platyhelmiths
Echinoderms

Cytoplasm/ Nucleus

Nematodes

Molluscs

AUA=Met CUN=Thr

Plants

Yeast/ Molds

AAA=Asn

Universal Code

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

AUA=Ile AAA=Asn

Insects

UGA/G=Stop

Vertebrates

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