1 Mitochondrial HSP 70 is encoded by the host nucleus but is of - - PDF document

1 Probing early eukaryotic evolution using phylogenetic methods

The Universal SSU rRNA Tree

Wheelis et al. 1992 PNAS 89: 2930

Archezoa The SSU Ribosomal RNA Tree for Eukaryotes

Mitochondria?

Prokaryotic

• utgroup

Animals Fungi Ciliates + Apicomplexa Stramenopiles Euglenozoa Giardia Trichomonas Plants / green algae Red algae Entamoebae Choanozoa Dictyostelium Physarum Microsporidia Percolozoa

The Archezoa Hypothesis - primitively amitochondriate eukaryotes

(Tom Cavalier-Smith 1983)

Trichomonas Microsporidia Giardia

‘Tree’ courtesy of W. Ford Doolittle

Entamoeba

The Archezoa Hypothesis

• T. Cavalier-Smith (1983)
• The Archezoa hypothesis would fall if:

– Find mitochondrial genes on archezoan genomes – Find that archezoans branch among aerobic species with mitochondria – Find mitochondrion-derived organelles in archezoans

2

Mitochondrial HSP 70 is encoded by the host nucleus but is of endosymbiotic origin

N C

ATPase DOMAIN

PEPTIDE BINDING DOMAIN

Targeting sequences Retention sequences

mitochondrial HSP70 alpha-proteobacteria chloroplasts cyanobacteria Cytosolic HSP70 RER HSP70

• ther proteobacteria

Gram +ve & Archaea

Endosymbiotic

• rigin

Mitochondrial genes in Archezoa

Giardia / Spironucleus Trichomonas Microsporidia Entamoeba Heat shock 70, Chaperonin 60 Heat shock 70, Chaperonin 60 Heat shock 70 Heat shock 70, chaperonin 60

*defined as forming a monophyletic group with mitochondrial homologues in a non-controversial species phylogeny

Proteins of mitochondrial origin* Archezoa

Chaperonin 60 Protein Maximum Likelihood Tree

(PROTML, Roger et al. 1998, PNAS 95: 229)

A case of Eukaryote Eukaryote HGT?

Note 100% Bootstrap support

Long branches may cause problems for phylogenetic analysis

• Felsenstein (1978) made a simple model phylogeny including four

taxa and a mixture of short and long branches

• Methods which assume all sites change at the same rate

(e.g. PROTML) may be particularly sensitive to this problem

A B C D TRUE TREE WRONG TREE A B C D p p q q q

p > q Chaperonin 60 Protein Maximum Likelihood Tree

(PROTML, Roger et al. 1998, PNAS 95: 229) Longest branches

3

• Does the Cpn60 tree topology change:

– If we remove long-branch outgroups – If we remove sites where every species has the same amino acid

A simple experiment:

Cpn-60 Protein ML tree (PROTML) from variable sites with outgroups removed

Giardia Entamoeba Dictyostelium

30 31

Plants

Apicomplexa Euglena & Trypanosoma

Trichomonas Animals & Fungi

The Archezoa Hypothesis

• T. Cavalier-Smith (1983)
• The Archezoa hypothesis would fall if:

– Find mitochondrial genes on archezoan genomes – Find that archezoans branch among aerobic species with mitochondria – Find mitochondrion-derived organelles in archezoans a-tubulin trees place Microsporidia with Fungi

Edlind TD, et al. (1996). Mol Phylog Evol 5 :359-67 Keeling PJ, Doolittle WF. (1996). Mol Biol Evol 13 :1297-305. Microsporidia Fungi Other eukaryotes

65

Microsporidia are primitive and early branching eukaryotes or relatives of fungi?

• Microsporidia branch deep in some trees but not in others

– Deep: SSU rRNA, EF-2 – With fungi: tubulin and HSP70

• Microsporidia contain a mitochondrial HSP70

– They once contained the mitochondrial endosymbiont and thus are not Archezoa sensu Cavalier-Smith

– Hirt et al., 1997

– Or they obtained this gene (and the one for tubulin) from fungi via HGT

– Sogin, 1997 Curr. Op. Genet. Dev. 7: 792-799

4

75 83 88

The tree shown is the maximum likelihood tree

• btained using a

program called PROTML

Maximum likelihood and phylogenetic inference

• The maximum likelihood tree is the one with

the highest probability of giving rise to the data under a particular model

• It is not the probability that it is the “true”

tree

Assumptions underpinning the PROTML model

• Assumes all sites in the molecule can change
• Assumes that all sequences are evolving in

the same way

• EF-2 violates both of these assumptions:

Human CAGGAATCACCTACGATCCTCGGCGGCGTTACGAACCGAA 53%G+C Saccharomyces CAGGAATCACTTACGATCTTAAGCGGCGTTACGAACAGAA 46%G+C Chlorella CAGGAATCACCTACGATCCTCAGCGGCGCTACGAACCGGC Dictyostelium CAGGAAACACTTTCGATCTTGTTCGGAGTAATTCTGCGGC Trypanosoma CAGGAATCACTTTCGATCCTGAGCGGCGATATGAACCGGC Entamoeba CAGGAATCACTTACGATCCTAAGCGGAGTAACGAACAGCC Cryptosporidium CAGGAATCACTTACGATCCTGCGCGGCGTTACGAACCGGC Tritrichomonas CAGGAATCACCTACGATCCTAAGCGGCGTTACGAACCGCC 54%G+C Tritrichomonas CAGGAATCACCTACGATCCTAAGCGGCGTTACGAACCGCC 54%G+C Giardia CAGGAATCACCTACGATCCTTCGCGGCGTTACGAACCGCA 53%G+C Glugea CAGGAAAGACCTACGATGCTGATCGGAGTAATGAACCGGA 45%G+C Sulfolobus CAGGAAACACACAGGAACCTGTGCGGCTGCTTGATACTTC 43%G+C Methanococcus CAGGAAACACTTTCGAAATTTTGCGGCTGCCTGATTGAGA 44%G+C Halobacterium CAGGAAACACCTACGAAACTACGCGGCTGCATGAACGAGA 58%G+C

LogDet/Paralinear distances for EF-2 DNA variable sites codon positions 1+2

Animals Chlorella Trypanosoma Trichomonas Giardia Dictyostelium Entamoeba Saccharomyces Glugea Cryptosporidium Sulfolobus Methanococcus Halobacterium

60 25 76 70

Archaebacteria

• utgroups

Microsporidia + fungi!

A combination of factors (outgroup GC content and site rate heterogeneity) influence the EF-2 DNA tree

20 40 60 80 100

Methanococcus outgroup (low G+C)

20 40 60 80 100

Halobacterium outgroup Higher G+C

20 40 60 80 100 20 40 60 80 100

(Microsporidia, outgroup)

Fraction of constant sites removed

LogDet Bootstrap values ML estimate

20 40 60 80 100 20 40 60 80 100 20 40 60 80 100 20 40 60 80 100

(Microsporidia, outgroup) (Microsporidia, Fungi)

Fraction of constant sites removed

Bootstrap values

A combination of factors (outgroup GC content & site rate heterogeneity) influence the EF-2 DNA tree

Methanococcus outgroup (low G+C) Halobacterium outgroup Higher G+C

5

A combination of factors (outgroup GC content & site rate heterogeneity) influence the EF-2 DNA tree

20 40 60 80 100 20 40 60 80 100 20 40 60 80 100 20 40 60 80 100

(Giardia, Trichomonas, outgroup)

Fraction of constant sites removed

Bootstrap values

Methanococcus outgroup (low G+C) Halobacterium outgroup Higher G+C

Are the former Archezoa really ancient offshoots?

• Probably not:
• Microsporidia are related to fungi (Tubulin, RNA

polymerase,LSU rRNA, HSP70, TATA binding protein, EF-2, EF-1 alpha, SSU rRNA)

• Evidence for Giardia and Trichomonas branching deeper

than other eukaryotes is based on trees made using unrealistic assumptions (often PROTML)

• There is plenty of room for new hypotheses

If former archezoa contain genes from the mitochondrial endosymbiont what happened to the organelle? Microsporidia are obligate intracellular parasites

Life cycle of microsporidia

Tree of Mitochondrial Hsp70

mtHsp70 has diverse roles in mitochondria

Pfanner and Geissler 2001 Nature Reviews 2: 339-344 mtHsp70 also plays a role in assembly of Fe-S clusters

• an essential function for yeast mitochondria (Lill & Kispal, 2000)

mtHsp70

6

Microsporidial HSP70 have no obvious N-terminal targeting signal

Localisation of a mtHsp70 in Trachipleistophora

Western blot using an antibody to Th-mtHsp70 Infected cells Spores Rabbit

Confocal microscopy using an antibody to Th-mtHsp70 The Hsp70 protein is localised to membrane bound or electron dense structures. Traditional fixation methods show a double membrane Microsporidian

Williams et al. 2002 Nature 418: 865-869

Host cell mitochondrion Microsporidian ‘mitochondrial remnant’

Trees are a good way of exploring the history of genes but care is needed!

• Making trees is not easy:

– Among-site rate heterogeneity, “fast clock” species, shared nucleotide or amino acid composition biases – Different data sets may be affected by individual phenomena to different degrees – Biases need not be large if phylogenetic signal is weak

• Phylogenetic analysis is frequently treated as a

black box into which data are fed (often gathered at considerable cost) and out of which “The Tree” springs

• (Hillis, Moritz & Mable 1996, Molecular Systematics)

Phylogenetic analysis requires careful thought