Transcript Using comparative genomics to study mitochondrial proteome

Bioinformatics of Mitochondria,
…a top-down lecture…
Martijn Huynen
Central role of mitochondria in metabolism
Calcium signaling
Coenzyme synthesis
Parkinson
Leigh syndrome
Citric acid cycle
Diabetes
Heme synthesis
Urea cycle
Friedreich’s ataxia
Myopathies
FeS clusters
Electrical signaling
Apoptosis
Alzheimer
ATP production
Fatty acids oxidation
Heat generation
Leber’s syndrome
Endosymbiotic origin of mitochondria
16S Ribosomal RNA
Original rationales for the endosymbiosis
1: ATP ? (MCF family is strictly eukaryotic)
2: Oxygen sink ? (Andersson & Kurland)
3: H2 ? (Martin & Muller)
ATP
H2
O2
Free-living, alpha-proteobacterial ancestor
Gene transfer
Gene loss
Gain (Andersson & Kurland)
and retargeting of proteins
Mitochondria
Rickettsia
Identifying eukaryotic proteins with an alphaproteobacterial origin based on their phylogeny
Eukaryotic + alpha-proteobacterial
proteinsg in the same branch
Alpha-proteobacterial proteins with the
rest of the bacteria and archaea
Detecting eukaryotic genes of alpha-proteobacterial ancestry
GENOME
6 alpha-proteobacteria (22 500 genes)
GENOMES
6 alpha-proteobacteria
9 eukaryotes
56 Bacteria+Archaea
SELECTION OF HOMOLOGS,
(Smith&Waterman)
LIST
ALIGNMENTS AND TREE
(Clustalx, Kimura+Dayhoff)
TREE SCANNING
PHYLOME
Alpha-proteobacterial genes monophyletic with eukaryotic genes
species
Rickettsia prowazekii
Genome
size
835
Selected
%
Groups
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
Non redundant orthologous groups:
446
630
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%
Saturation in the estimated size of the proto-mitochondrial proteome with an
increasing number of sequenced alpha-proteobacterial genomes
ancient proteome estimate (ML-1)
900
800
700
600
500
400
300
200
100
0
1
2
3
4
5
6
7
8
9
alpha-proteobacterial genom es
10
11
Increasing the number of genomes leads to more accurate results:
less false negatives, less false positives
Species
# proteins
Reclinomonas americana
66
Deinococcus radiodurans
3084
Agrobacterium tumefaciens (Cereon)
5392
Agrobacterium tumefaciens (Washington)
5298
Bradyrhizobium japonicum
8257
Brucella melitensis
3186
Brucella suis
3247
Caulobacter crecentus
3718
Magnetococcus magnetotacticum
4280
Rhizobium loti
7259
Rhizobium meliloti
6149
Rick ettsia conorii
1374
Rick ettsia prowazek ii
834
2003
49
1.3
18.1
17.9
13.3
13.3
17.1
23.5
630
NJ-1
72.3
1.1
13.03
13.11
11.18
16.1
15.86
13.23
11.36
13.08
13.67
20.3
25.06
1026
ML-1
69.70
0.03
7.59
11.76
8.25
11.08
9.67
8.85
8.74
8.94
9.17
16.59
19.78
842
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
The majority of the proto-mitochondrial proteome is not mitochondrial (anymore)
Yeast mitochondrial proteome:
566
Proto-mitochondrial proteins in
S.cerevisiae
Eric Schon,
Methods Cell Biol 2001
(manually curated)
35
293
303
59
10
Huh et al., Nature 2003
(green fluorescent genomics)
Human mitochondrial proteome:
Eric Schon,
Methods Cell Biol 2001
527
755
Proto-mitochondrial proteins in
H.sapiens
113
508
From endosymbiont to organell, not only loss
and gain of proteins but also “retargeting”:
proteins
loss
re-targeting
~65% of the alphaproteobacteria derived set is
not mitochondrial.
Ancestor
Modern mitochondria
~16% of the mitochondrial
yeast proteins are of alphaproteobacterial origin.
gain
t
Gabaldon and Huynen, Science 2003
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
From endosymbiont to organell: a turnover of protein in functional
classes and an increase in specialization
(middle columns: yeast, right columns: human)
30%
alphaprot origin
Non-alpha
25%
20%
15%
10%
5%
0%
J
K
L
D
O
M
N
P
T
C
G
E
F
H
I
Q
COG functional classification
C: Energy conversion
O: Protein turnover, chaperones
J: Translation, Ribosomal structure
F: Nucleotide metabolism
G: Carbohydrate metabolism
M: Cell envelope biogenesis
Very low throughout: D: Cell division
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.
Kiefel BR Gilson PR Beech 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.
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
Tracing the evolution of Complex I from 14 subunits in the
Bacteria to 46 subunits in the Mammals by comparative genome
analysis
Fungi: 37
Bacteria: 14 subunits
Mammals: 46
Plants: 30
Algae: 30
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
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
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
Eukaryotes
NUEM
CIA30
Cyanobacteria
CIA30 is inserted in NueM in Cyanobacteria, suggesting an interaction between
CIA30 and NueM in the eukaryotes as well.
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
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
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
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
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.
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
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%)
eukaryotic
Alpha-proteobact
alpha
actinomycetales
eukaryotic
cyanobacteria
alpha
unresolved
unclear
actinomycetales
cyanobacteria
unclear
Yeast (61 proteins)
Rat (50 proteins)
Most (90%) of the peroxisomal proteins of alphaproteobacterial origin have paralogs in the mitochondria
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)
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.
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
Studying evolution of organellar proteomes is not only interesting
in itself, it also provides us with clues about the functions of
proteins