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Bioinformatics of Mitochondria, …a top-down lecture…. Martijn Huynen. Parkinson. Leigh syndrome. Diabetes. Myopathies. Friedreich’s ataxia. Alzheimer. Leber’s syndrome. Central role of mitochondria in metabolism. Calcium signaling. Coenzyme synthesis. Citric acid cycle. Urea cycle.

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slide2

Parkinson

Leigh syndrome

Diabetes

Myopathies

Friedreich’s ataxia

Alzheimer

Leber’s syndrome

Central role of mitochondria in metabolism

Calcium signaling

Coenzyme synthesis

Citric acid cycle

Urea cycle

Heme synthesis

FeS clusters

Apoptosis

Electrical signaling

ATP production

Fatty acids oxidation

Heat generation

original rationales for the endosymbiosis
Original rationales for the endosymbiosis

1: ATP ? (MCF family is strictly eukaryotic)

2: Oxygen sink ? (Andersson & Kurland)

3: H2 ? (Martin & Muller)

O2

ATP

H2

slide5

Free-living, alpha-proteobacterial ancestor

Gene transfer

Gene loss

Gain (Andersson & Kurland)

and retargeting of proteins

Rickettsia

Mitochondria

identifying eukaryotic proteins with an alpha proteobacterial origin based on their phylogeny
Identifying eukaryotic proteins with an alpha-proteobacterial origin based on their phylogeny

A

B

Eukaryotic + alpha-proteobacterial proteinsg in the same branch

Alpha-proteobacterial proteins with the rest of the bacteria and archaea

slide7

GENOMES

SELECTION OF HOMOLOGS,

(Smith&Waterman)

LIST

ALIGNMENTS AND TREE

(Clustalx, Kimura+Dayhoff)

PHYLOME

Detecting eukaryotic genes of alpha-proteobacterial ancestry

GENOME

6 alpha-proteobacteria (22 500 genes)

6 alpha-proteobacteria

9 eukaryotes

56 Bacteria+Archaea

TREE SCANNING

slide8

species

Genome size

Selected

%

Groups

Rickettsia prowazekii

835

196

23,5

173

Rickettsia conorii

1374

235

17,1

192

Caulobacter crescentus

3718

668

17,9

480

Brucella melitensis

3188

578

18,1

403

Rhizobium loti

7260

969

13,3

516

Rhizobium meliloti

6150

821

13,3

Alpha-proteobacterial genes monophyletic with eukaryotic genes

446

630

Non redundant orthologous groups:

estimating false positives and false negatives 630 orthologous groups appears a lower bound
Estimating false positives and false negatives,630 orthologous groups appears a lower bound:
  • False positives: Of the unrelated Deinococcus radiodurans the algorithm selects 1.3 %
  • False negatives: Of the 66 proteins encoded in the mitochondrial genome of Reclinomonas americana the procedure selects 49%
slide10

Saturation in the estimated size of the proto-mitochondrial proteome with an increasing number of sequenced alpha-proteobacterial genomes

slide11

Increasing the number of genomes leads to more accurate results:

less false negatives, less false positives

slide12

Proto-mitochondrial metabolism

  • Catabolism of fatty acids, glycerol and amino acids-Some pathways are not mitochondrial in present day mitochondria

non-mitoch..

mitochondrial

not in yeast/human

slide13

The majority of the proto-mitochondrial proteome is not mitochondrial (anymore)

566

Yeast mitochondrial proteome:

Proto-mitochondrial proteins in S.cerevisiae

Eric Schon,

Methods Cell Biol 2001

(manually curated)

35

303

59

293

10

Huh et al., Nature 2003

(green fluorescent genomics)

527

Proto-mitochondrial proteins in H.sapiens

755

Human mitochondrial proteome:

Eric Schon,

Methods Cell Biol 2001

113

508

slide14

~65% of the alpha-proteobacteria derived set is not mitochondrial.

~16% of the mitochondrial yeast proteins are of alpha-proteobacterial origin.

From endosymbiont to organell, not only loss and gain of proteins but also “retargeting”:

proteins

loss

re-targeting

Ancestor

Modern mitochondria

gain

t

Gabaldon and Huynen, Science 2003

original rationales for the endosymbiosis15
Original rationales for the endosymbiosis
  • Aerobic (…no hydrogenosomal eukaryotes have been published yet….)
  • Catabolizing lipids, glycerol and amino-acids, incomplete TCA
  • Benefit for host wider than either H2, ATP or O2 consumption: iron-sulfur clusters and a variety of metabolic pathways
slide16

From endosymbiont to organell: a turnover of protein in functional classes and an increase in specialization

(middle columns: yeast, right columns: human)

COG functional classification

F: Nucleotide metabolism

G: Carbohydrate metabolism

M: Cell envelope biogenesis

C: Energy conversion

O: Protein turnover, chaperones

J: Translation, Ribosomal structure

Very low throughout: D: Cell division

slide17

Gene loss in the evolution of mitochondria

  • Mitochondrial FtsZ in a chromophyte alga.Beech PL, Nheu T, Schultz T, Herbert S, Lithgow T, Gilson PR, McFadden GI Science 2000
  • A homolog of the bacterial cell division gene ftsZ was isolated from the alga Mallomonas splendens. The nuclear-encoded protein (MsFtsZ-mt) was closely related to FtsZs of the alpha-proteobacteria, possessed a mitochondrial targeting signal, and localized in a pattern consistent with a role in mitochondrial division. Although FtsZs are known to act in the division of chloroplasts, MsFtsZ-mt appears to be a mitochondrial FtsZ and may represent a mitochondrial division protein.
slide18

Kiefel BRGilson PRBeech PL.Diverse eukaryotes have retained mitochondrial homologues of the bacterial division protein FtsZ.

Protist. 2004 Mar;155(1):105-15.Mitochondrial fission requires the division of both the inner and outer mitochondrial membranes. Dynamin-related proteins operate in division of the outer membrane of probably all mitochondria, and also that of chloroplasts--organelles that have a bacterial origin like mitochondria. How the inner mitochondrial membrane divides is less well established. Homologues of the major bacterial division protein, FtsZ, are known to reside inside mitochondria of the chromophyte alga Mallomonas, a red alga, and the slime mould Dictyostelium discoideum, where these proteins are likely to act in division of the organelle. Mitochondrial FtsZ is, however, absent from the genomes of higher eukaryotes (animals, fungi, and plants), even though FtsZs are known to be essential for the division of probably all chloroplasts. To begin to understand why higher eukaryotes have lost mitochondrial FtsZ, we have sampled various diverse protists to determine which groups have retained the gene. Database searches and degenerate PCR uncovered genes for likely mitochondrial FtsZs from the glaucocystophyte Cyanophora paradoxa, the oomycete Phytophthora infestans, two haptophyte algae, and two diatoms--one being Thalassiosira pseudonana, the draft genome of which is now available. From Thalassiosira we also identified two chloroplast FtsZs, one of which appears to be undergoing a C-terminal shortening that may be common to many organellar FtsZs. Our data indicate that many protists still employ the FtsZ-based ancestral mitochondrial division mechanism, and that mitochondrial FtsZ has been lost numerous times in the evolution of eukaryotes.

slide19

Zooming in on one mitochondrial complex, NADH:ubiquinone oxidoreductase (Complex I), and using gene loss for function prediction

  • -Complex I deficiency is a severe hereditary disease (patients < 5 year) without therapy
  • -For 60% of the patients no mutation is found in known CI genes
slide20

Tracing the evolution of Complex I from 14 subunits in the Bacteria to 46 subunits in the Mammals by comparative genome analysis

Fungi: 37

Mammals: 46

Bacteria: 14 subunits

Plants: 30

Algae: 30

slide21

Issues in homology detection: that we do not detect sequence similarity does not mean that proteins are not homologous.

Latest developments in homology detection: profile vs. profile searches

The fungal ComplexI subunit NUVM is homologous to the Bovine subunit NB5M (B15),

This homology can only be detected by profile vs. profile searches

slide22

Beyond Blastology, Cogoly: Phylogenies for orthology prediction

The Complex I assembly protein CI30 has been duplicated in the Fungi.

This can explain the presence of a CIA30-homolog in Complex I-less S.pombe

slide23

Mining the proto-mitochondrion for new Complex I proteins

}

Metazoa

}

Fungi

}

Alpha-proteobacteria

A methyltransferase derived from the alpha-proteobacterial ancestor of the mitochondria has a phylogenetic distribution identical to Complex I proteins, suggesting involvement of this protein in Complex I

Gabaldon and Huynen, Bioinformatics 2005

function prediction of complex i proteins
Function prediction of Complex I proteins

NUEM

Eukaryotes

CIA30

Cyanobacteria

CIA30 is inserted in NueM in Cyanobacteria, suggesting an interaction between CIA30 and NueM in the eukaryotes as well.

slide25

Plants,algae

Mammals

Insects

Nematode

Fungi

Fish

Distribution of Complex I subunits among model species

Experimentally verified

Homolog present in genome, predicted gene

Homolog present in genome, not predicted

Absent from genome

slide26

Reconstructing Complex I evolution by mapping the variation onto a phylogenetic tree. After an initial “surge” in complexity (from 14 to 35 subunits in early eukaryotic evolution) new subunits have been gradually added and incidentally lost.

Complex I loss is not always “complete”, S.cerevisiae and S.pombe have retained 1 and 3 proteins respectively

Six of the eukaryotic Complex I proteins have been “recruited” from the alpha-proteobacteria

slide27

Tinkering in the eukaryotic evolution of Complex I: new subunits have been added “all over” the complex

Gabaldon et al. (2005) J. Mol. Biol.

See also Science (2005) 308, 167

slide28

Deconstructing protein complexes by tracing their evolution:

The phylogenetic distribution of Complex I subunits suggests the presence of submodules and the functions of the individual proteins

In eukaryotes evolution appears less “sub-modular” than in prokaryotes

T. Friedrich’s model

Huynen et al., FEBS lett. 2005

slide29

How about the origin of the peroxisome?

Like mitochondria an organell involved in oxidative metabolism, but without a genome. Multiplication by fission has suggested an endosymbiotic origin.

slide30

Scenarios for the origin of the peroxisome and mitochondria

A) Independent endosymbiotic origins

B) A single origin followed by fission

C) Retargeting of mitochondrial proteins

Time

slide31

The yeast and rat peroxisomal proteomes contain a large fraction of proteins of alpha-proteobacterial origin (18-19%), besides a large fraction of proteins of eukaryotic origin (37-38%)

Alpha-proteobact

unresolved

Yeast (61 proteins)

Rat (50 proteins)

slide32

Most (90%) of the peroxisomal proteins of alpha-proteobacterial origin have paralogs in the mitochondria

slide33

The retargeting of mitochondrial proteins to the peroxisome has continued in recent evolution

}

Peroxisomal

Mitoch.

Signal peptide cleavage site

Within the Cit1/2 protein family all proteins have a mitochondrial location (exp. data and/or predictions), expect Cit2p which is peroxisomal, and has lost the cleavage site (YS)

slide34

Tracing the evolution of the peroxisome: a continuous retargeting of proteins from various origins.

(yellow = eukaryotic, green = alphaprot, red = actinomyc., blue = cyanobact.)

The “ancestral peroxisome” was likely involved in b-oxidation, harboring Catalase to detoxify hydrogen peroxide.

slide35

Scenarios for the origin of the peroxisome and mitochondria

A) Independent endosymbiotic origins

B) A single origin followed by fission

C) Retargeting of mitochondrial proteins

Time

slide36

Studying evolution of organellar proteomes is not only interesting in itself, it also provides us with clues about the functions of proteins