Endosymbiosis and the Origin of Eukaryotes Are mitochondria

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 mt. DNA Inter membrane space © 1999 Timothy

Extranuclear DNA � Mitochondria and chloroplasts have their own DNA � This extranuclear DNA exhibits non-Mendalian inheritance � Recombination is known between some mt and ct. DNAs � Extranuclear DNA may also be called cytoplasmic DNA � Generally mt. DNA and ct. DNA 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 mt. DNA and ct. DNA, but only some of the protein genes © 1999 Timothy G. Standish

� Mitochondrial mt. 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 mt. DNA is highly variable in size and content with the large Arabidopsis mt. DNA being 200 kb. � The largest known number of mt. DNA protein genes is 97 in the protozoan Riclinomonas mt. DNA 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 Chloroplast Golgi Body © 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 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 r. RNA. © 1999 Timothy G. Standish

How Mitochondria Don’t 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 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 mt. DNA replication via D loops is different from replication of bacterial DNA (Lewin, 1997 p 441). � mt. DNA 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

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” (Cavalier. Smith, 1983; Campbell et al. , 1999 pp 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 (HSP 60) in Giardia lamblia (Soltys and Gupta, 1994) has called this interpretation into question. � Other proteins thought to be unique to mitochondria, HSP 70 (Germot et al. , 1996), chaperonin 60 (HSP 60) (Roger et al. , 1996; Horner et al. , 1996) and HSP 10 (Bui et al, 1996) have shown up in Gardia’s 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 mt. Genes Lost genetic material © 1999

Origin of Archezoa: Option 1 - Mitochondrial Eukaryote Production © 1999 Timothy G. Standish

Origin of Archezoa: Option 2 - Mitochondrial DNA Loss/ Hydrogenosome production Hydrogenosome © 1999

Origin of Archezoa: Option 2 A - 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

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 mt. DNA Hydrogenosome/ loss mitochondria loss mt. DNA 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

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

Protein Production Mitochondria Leader sequence binding receptor ATP P +ADP Matrix MLSLRQSIRFFKPATRTLCSSRYLL P +ADP

Protein Production Mitochondria Leader sequence binding receptor LS M LR QS IR Peptidease cleaves off the leader Outer membrane FF KP AT RT Inner membrane SS LC RY LL Matrix Inter membrane space © 1999 Timothy G. Standish

Protein Production Mitochondria Leader sequence binding receptor ML SL R Matrix QSI R FFK

Protein Production Mitochondria Leader sequence binding receptor Outer membrane Inner membrane Matrix Inter membrane

Protein Production Mitochondria Leader sequence binding receptor Outer membrane Hsp 60 Matrix Chaperones Inner

Protein Production Mitochondria Leader sequence binding receptor Outer membrane Inner membrane Matrix Mature protein

M L S L Polar R I Q S Non. R polar F First 12 residues are sufficient for transport to the mitochondria F K A T L Polar R P S S Y L � This P R C Neutral Non-polar Polar Basic Acidic MLSLRQSIRFFKPATRTLCSSRYLL Recognized by peptidase? T Yeast Cytochrome C Oxidase Subunit IV Leader leader does not resemble other eukaryotic leader sequences, or other mt. Protein leader sequences. � Probably forms an a helix � This would localize specific classes of amino acids in specific parts of the helix � There 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 C 1 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 c 1 holds an iron containing heme group and is part of the B-C 1 (III) complex C 1 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 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 Peptidease cleaves off

Protein Production Mitochondria Leader sequence binding receptor Outer membrane Inner membrane Mature protein Matrix

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 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 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 mt. DNA lower the number of t. RNAs needed � Universal Code © 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