carrie_10_in_801229.pdf.txt

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"><html xmlns="http://www.w3.org/1999/xhtml">
<head>
<title>An in silico analysis of the mitochondrial protein import apparatus of plants</title>
<meta name="Subject" content="BMC Plant Biology 2010, 10:249. doi:10.1186/1471-2229-10-249"/>
<meta name="Keywords" content=" "/>
<meta name="Author" content="Chris Carrie"/>
<meta name="Creator" content="Arbortext Advanced Print Publisher 10.0.1082/W Unicode"/>
<meta name="Producer" content="Acrobat Distiller 9.0.0 (Windows)"/>
<meta name="CreationDate" content=""/>
</head>
<body>
<pre>
Carrie et al. BMC Plant Biology 2010, 10:249
http://www.biomedcentral.com/1471-2229/10/249

RESEARCH ARTICLE

Open Access

An in silico analysis of the mitochondrial
protein import apparatus of plants
Chris Carrie, Monika W Murcha, James Whelan*

Abstract
Background: An in silico analysis of the mitochondrial protein import apparatus from a variety of species; including
Chlamydomonas reinhardtii, Chlorella variabilis, Ectocarpus siliculosus, Cyanidioschyzon merolae, Physcomitrella patens,
Selaginella moellendorffii, Picea glauca, Oryza sativa and Arabidopsis thaliana was undertaken to determine if
components differed within and between plant and non-plant species.
Results: The channel forming subunits of the outer membrane components Tom40 and Sam50 are conserved
between plant groups and other eukaryotes. In contrast, the receptor component(s) in green plants, particularly
Tom20, (C. reinhardtii, C. variabilis, P. patens, S. moellendorffii, P. glauca, O. sativa and A. thaliana) are specific to this
lineage. Red algae contain a Tom22 receptor that is orthologous to yeast Tom22. Furthermore, plant mitochondrial
receptors display differences between various plant lineages. These are evidenced by distinctive motifs in all plant
Metaxins, which are absent in red algae, and the presence of the outer membrane receptor OM64 in Angiosperms
(rice and Arabidopsis), but not in lycophytes (S. moellendorffii) and gymnosperms (P. glauca). Furthermore, although
the intermembrane space receptor Mia40 is conserved across a wide phylogenetic range, its function differs
between lineages. In all plant lineages, Tim17 contains a C-terminal extension, which may act as a receptor
component for the import of nucleic acids into plant mitochondria.
Conclusions: It is proposed that the observed functional divergences are due to the selective pressure to sort
proteins between mitochondria and chloroplasts, resulting in differences in protein receptor components between
plant groups and other organisms. Additionally, diversity of receptor components is observed within the plant
kingdom. Even when receptor components are orthologous across plant and non-plant species, it appears that the
functions of these have expanded or diverged in a lineage specific manner.

Background
The endosymbiotic event giving rise to the origin of
mitochondria is thought to have occurred 1 to 2 billion
years ago [1,2]. Details of the conditions that favoured
this event and the exact identity of the host cell that
engulfed the a-proteobacterial cell are still unclear. It
has been proposed that the endosymbiosis that gave rise
to mitochondria occurred under anaerobic conditions,
followed by early diversification of eukaryotic cells [3].
For plastids, an endosymbiotic event occurred ~1 billion
years ago when a heterocyst forming cyanobacterium
was engulfed [4,5]. Over time the loss and/or transfer of
genes and genomes from the endosymbionts to the host
cell nucleus has resulted in the formation of organelles
* Correspondence: seamus@cyllene.uwa.edu.au
Australian Research Council Centre of Excellence in Plant Energy Biology,
University of Western Australia, 35 Stirling, Crawley 6009, WA, Australia

with limited coding capacity [6-8]. The majority of proteins located in mitochondria and plastids are encoded
by nuclear located genes, translated in the cytosol and
imported into these organelles. Notably, the proteomes
of both mitochondria and chloroplasts are derived from
a variety of sources and are not simply a subset of the
proteins derived from the ancestral endosymbiont [9]. In
the most extreme cases, it is thought that all genes that
were present in the endosymbiont have been lost, resulting in specialized organelles such as hydrogenosomes
and mitosomes [10].
Although mitochondria have a single origin there is
variation observed between different mitochondria present in the major branches of life [10]. Mitochondria in
plants contain many unique features compared to their
fungal or animal counterparts. These include a larger
genome, ranging from 200 Kb to 2000 Kb in size [11],

© 2010 Carrie et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

Carrie et al. BMC Plant Biology 2010, 10:249
http://www.biomedcentral.com/1471-2229/10/249

extensive cis and trans splicing of introns, [12], relatively
slow rates of mutations [13,14], extensive editing of
mRNA [15] and incorporation of foreign DNA [16].
Another notable feature is the presence of a branched
respiratory chain [17]. Although fungi contain alternative NAD(P)H dehydrogenases and an alternative oxidase, these are usually only expressed under conditions
where the cytochrome chain is inhibited [17]. In contrast, plant mitochondria contain components of the
alternative respiratory pathways which exhibit both constitutive and stress induced expression [18]. Furthermore, mitochondria of plants and animals have
diversified in a lineage specific manner to include or
exclude various biochemical pathways, such as the
b-oxidation of fatty acids that occurs in peroxisomes in
plants and mitochondria in animals [19].
In plants, the presence of plastids in cells also adds to
the complexity of protein sorting required to avoid
mis-targeting of proteins to organelles. Plastidic and
mitochondrial targeting signals, referred to as transit
peptides and presequences respectively, are typically
located at the N-terminal end of the protein and are
enriched in positively charged residues such as lysine
and arginine [20]. It is not known how mis-sorting of
proteins is prevented between plastids and mitochondria. A combination of the predicted ability of transit
peptides and presequences to form different secondary
structures, the proposed presence of cytosolic targeting
factors and even targeting of mRNA to the surface of
organelles, may all combine to achieve the observed specificity of protein targeting [21,22]. There is a mechanistic difference between recognition of targeting signals by
preprotein receptor proteins in plastids and mitochondria, the former involving a GTP/GDP cycle while no
energy requirement is observed for receptor binding in
mitochondria. This mechanism among others may contribute to the specificity of targeting signal recognition
at the surface of each organelle [23,24].
Our knowledge of the mitochondrial protein import
apparatus in plants, both experimental and predicted, is
largely derived from studies in Arabidopsis, and to a lesser extent from Solanum tuberosum (potato). Purification of the translocase of the outer membrane (TOM)
complex from both Arabidopsis and potato revealed
that Tom40 and Tom7 are orthologous with those from
yeast, while Tom20 is not orthologous to yeast or mammalian proteins [25-27]. The other import receptors
characterized in yeast (and mammals), Tom70 and
Tom22, appear to be absent [28,29]. It has been shown
that plant Tom9 is the most likely equivalent to yeast
Tom22, but lacks the cytosolic receptor domain [30].
The mitochondrial processing peptidase has been purified from potato and shown to be integrated into the
cytochrome bc1 complex [31,32]. This is also the case in

Page 2 of 15

lower plants examined both in the elkhorn fern Platycerium bifurcatum and the field horsetail Equisetum
arvense [33]. Biochemical purification of the presequence degradation peptidase (PreP) has shown that it
is a dual targeted protein and that it is a zinc metalloprotease [34]. Biochemical studies have shown that
small intermembrane space proteins also mediate mitochondrial carrier protein import in potato mitochondria.
In addition, the plant TIM17:23 complex differs to that
in yeast in that the Tim17 in Arabidopsis contains a Cterminal extension that must be removed before it can
complement a tim17 mutant in yeast [35,36].
However, there are limited studies on the nature of
the mitochondrial protein import apparatus from other
plants, ranging from single celled algae to monocots.
Thus, in order to gain a better overview of the protein
import apparatus in plants, compared to fungal and animal counterparts, an in silico analysis of these components was carried out. This was based on the fact that
complete genome sequences now exist for the single
celled green algae, Chlamydomonas reinhardtii (Chlorophyte) [37] and Chlorella variabilis, an intracellular single celled green algae photosynthetic symbiont in
Paramecium bursaria [38], a moss, Physcomitrella
patens (Bryophyte) [39], Selaginella moellendorffii, an
ancient vascular plant [40], and higher plants Oryza
sativa [41], Arabidopsis thaliana [42] and Picea glauca
[43] (Spermatophytes) (Figure 1). We have also included
analysis from brown algae, Ectocarpus siliculosus
(Phaeophyceae) [44] and the red algae, Cyanidioschyzon
merolae (Rhodophyta) [45] (Figure 1). Red algae represent a cell lineage with a primary plastid endosymbiosis
that is proposed to have been derived from the same
event that gave rise to the plastids in green plants, but
diverged from the green plant lineage early after this
endosymbiotic event [46]. Brown algae have obtained
their plastids via a secondary endosymbiosis, and contain four plastid envelope membranes. Thus, plastid proteins are first targeted to the outer membrane via a
hydrophobic signal sequence and secondary targeting
signals mediate uptake into plastids [47].

Results and Discussion
Translocase of the Outer Membrane (TOM)

The TOM complex represents the gateway into mitochondria, through which almost all mitochondrial proteins pass (exceptions include Fis1 [48]). It has been
characterized from yeast, Neurospora, mammals and
plants, in particular Arabidopsis. In addition to being
purified from Arabidopsis, functional studies on the
Tom20 receptor components show that all three isoforms can be deleted, resulting in a reduced rate of
import for several precursor proteins, but no deleterious
phenotypic lesions. The complex typically contains 7

Carrie et al. BMC Plant Biology 2010, 10:249
http://www.biomedcentral.com/1471-2229/10/249

Page 3 of 15

Homo sapiens
Saccharomyces cerevisiae
Dictyostelium discoideum
Oryza sativa

Opisthokonts
Amoebozoa

Arabidopsis thaliana
Picea glauca
Selaginella moellendorffii

Green Plants

Physcomitrella patens
Chlamydomonas reinhardtii
Chlorella variabilis
Cyanidioschyzon merolae
Ectocarpus siliculosus
1600

1400

1200

1000
800
600
Million years ago

400

200

Red algae
Brown algae

0

Figure 1 Overview of the evolutionary relationship of organisms used in this study. The taxonomy database at NCBI was used to draw a
phylogenetic tree, which was visualized using PHY-PI [91]. The timeline is based upon [57].

subunits, Tom70, 40, 22, 20 7, 6, and 5, but in Arabidopsis Tom22 is replaced by Tom9 and no orthologue
to Tom70 can be identified. Arabidopsis contain a protein termed OM64 that is not present in yeast or mammals, which appears to play a role as an import
receptor.
The TOM complex fulfills the vital function of specifically recognizing mitochondrial proteins from the pool
of all proteins synthesized in the cytosol. Of the seven
components characterized biochemically to be present
in the TOM complex from yeast, only Tom40 is conserved between yeast, mammals and plants (Figure 2,
Additional file 1). While Tom40 is a b-barrel protein,
there is no significant sequence similarity with bacterial
b-barrel proteins [30], nonetheless, hidden Markov
model searches define this as a universal component of
all mitochondria, including mitosomes in Entamoeba
histolytica and Giardia intestinalis [49,50].
Tom22 has been shown to fulfill a central receptor
role in yeast, and insertional inactivation in yeast results
in a strong impairment of mitochondrial biogenesis,
compared to the other two preprotein receptors characterized, Tom20 and Tom70 [51]. Searching the genome
of the red algae C. merolae for Tom22-like proteins
identified a protein with a predicted molecular mass of
20 kDa. This protein displays sequence identity and a
similar domain structure to the yeast Tom22 (Figure 2,
Figure 3b). Thus, the TOM complex of C. merolae
appears to be similar to that of D. discoideum, in that it
contains a single receptor Tom22-like protein [49]. In
contrast, green plants and E. siliculosus do not contain a

Tom22 protein. Rather, they contain a Tom9 protein
domain component (Figure 2, Additional file 1). Plant
Tom9 is predicted to be structurally similar to yeast and
mammalian Tom22, except that it lacks the cytosolic
receptor domain [30]. Thus, Tom22 has either lost the
receptor domain to form Tom9 or been replaced by a
different protein. Irrespective of the mechanisms by
which Tom9 arose, it appears that green plants have
lost the Tom22 receptor. The presence of the Tom22
receptor component in C. merolae and D. discoideum
suggests that it represented a universal mitochondrial
receptor component prior to the divergence event that
gave rise to plants verse animals and fungi.
None of the receptor proteins characterized in yeast
or mammalian systems, Tom20, Tom70 and Tom22, are
present in green plants [52,53] (Figure 2). The evolutionary situation for Tom22 is outlined above, and
although a Tom20 receptor protein is present in plants,
it represents a case of convergent evolution that has
been previously well described [27,54,55], thus, plant
and yeast Tom20 proteins are not orthologous (Figure 2,
Additional file 1). The third receptor component,
Tom70, is only present in animals and fungi [45] (Figure 1). Tom70 is not present in any green plant genome
[29], a variety of searches in this study failed to detect
any Tom70 like sequences in the green plant genomes
interrogated. However, in the genome of the brown
algae E. siliculosus, a protein with a similar domain
structure to Tom70 was identified (Figure 3C). This
Tom70 like protein contains an N-terminal transmembrane domain and 11 Tetratricopeptide repeat (TPR)

Carrie et al. BMC Plant Biology 2010, 10:249
http://www.biomedcentral.com/1471-2229/10/249

A)

Page 4 of 15

Ectocarpus

Yeast

Cyanidioschyzon

Yeast

70

70
20

OM

5

40

7 6

40

40

7

22

22
9
Poplar
Zea
Glycine
Rice
Arabidopsis

Picea
Selaginella
Chlamydomonas
Physcomitrella

Chlorella

64

20

OM

40

20

7
9

5

40

20

7 6
9

5

40

7 6
9

B)

Figure 2 Diversity of the TOM complex in plants. A) Schematic diagrams of the TOM complex from a selection of plant species using the
TOM complex from yeast as a reference. B) A table depicting components of the TOM complex in a variety of organisms. The pink color refers
to proteins that are conserved across all organisms and likely have a common ancestor. The lime green colored proteins are specific to the plant
lineage. The pale green proteins are proteins that have an unknown origin. Yeast - Saccharomyces cerevisiae, Ectocarpus - Ectocarpus siliculosus
(Es), Cyanidioschyzon - Cyanidioschyzon merolae (Cm), Chlorella - Chlorella variabilis (Cv), Picea - Picea glauca (Pg), Selaginella - Selaginella
moellendorffii (Sm), Chlamydomonas - Chlamydomonas reinhardtii (Cr), Physcomitrella - Physcomitrella patens (Pp), Rice - Oryza sativa (Os),
Arabidopsis - Arabidopsis thaliana (At), Human - Homo sapiens, Poplar -Populus tricocarpa (Pt), Glycine - Glycine max (Gm) Zea - Zea mays (Zm).

Carrie et al. BMC Plant Biology 2010, 10:249
http://www.biomedcentral.com/1471-2229/10/249

Page 5 of 15

64
t OM
Atm

PtToc64-2
PtT
oc
Vv
64
To
-1
c6
4

A)

64
M
tO
m
Pt

Vv

4-2
c6
To
Gm Toc64-1
Gm

mt

OM

64

Gmm

AtToc64

tOM6

GmmtOM64-2
-3
64
tOM
m
Gm

Chloroplastic

Os

To
c

64

PgToc64

tO
m

mtO
Zm

Os

64

oc6
PpT

0.2

c
To

Sm

M
64
M6
4

Mitochondrial

4-1

4

B)

C)

ScTom70

TM

AtOM64

TM

EsTom70

1

2

3

4

5

6

7 8

9 10 11

Amidase

TM

1

2

3

1

4

5

6

2

7

617 AA

3

8

603 AA

9 10 11

739 AA

Figure 3 Analysis of components of the plant TOM complex. A) Phylogenetic tree of the chloroplastic Toc64 and mitochondrial OM64
sequences from plants. Only sequences which showed the characteristic domain structure of Toc64, an N-terminal transmembrane domain
followed by an amidase like domain with 3 TPR repeats at the C-terminus, were used for phylogenetic analyses. No such proteins were
identified in Ectocarpus siliculosus, Cyanidioschyzon merolae, Chlamydomonas reinhardtii and Chlorella variabilis. For most plant species a clear
Toc64 and mtOM64 homologue can be identified however only 1 sequence can be identified in Physcomitrella patens, Selaginella moellendorffii
and Picea glauca which all branch closest to the Toc64 chloroplastic proteins. B) Sequence alignment of the Saccharomyces cerevisiae Tom22
with the Arabidopsis thaliana Tom9 and Cyanidioschyzon merloae Tom22. While most plants contain only the Arabidopsis Tom9 like protein
Cyanidioscyzon contains a full Tom22 receptor, which shows a high similarity with the yeast Tom22. C) The domain organization of the yeast
Tom70, Arabidopsis mtOM64 and an EsTom70 like protein. TM - transmembrane domain and the numbers correspond to the TPR repeats. At Arabidopsis thaliana, Vv - Vitis vinifera, Gm - Glycine max, Pt - Populus tricocarpa, Os - Oryza sativa, Pp - Physcomitrella patens, Zm - Zea mays, Sc Saccharomyces cerevisiae, Cm - Cyanidioschyzon merolae, Es - Ectocarpius siliculosus, Sm - Selaginella moellendorffii and Pg - Picea glauca.

Carrie et al. BMC Plant Biology 2010, 10:249
http://www.biomedcentral.com/1471-2229/10/249

motifs similar to yeast Tom70 [29]. However, the level
of sequence identity is low (20%), and it is unclear if
this protein represents a Tom70 orthologue.
A protein with a predicted molecular mass of 64 kDa
(OM64) is found on the outer membrane of mitochondria in Arabidopsis, displaying ~70% sequence identity
with the Toc64 protein (translocase of the outer envelope of chloroplasts) from plastids [56] (Figure 3A). In
plant mitochondria, this protein has been shown to be
involved in the import of some precursor proteins [53].
Analysis of various plant genomes reveals that OM64
appears to be present only in a sub-set of vascular
plants and is absent in P. glauca and S. moellendorffii,
as well as lower plant groups represented by C. reinhardtii, C. variabilis, P. patens, E. siliculosus and
C. merolae (Figure 2A). Tom7 represents an interesting
case in that it is absent in C. merolae, but present in all
other plants and eukaryotes (Figure 2). TBlastx and hidden Markov model based searches of all red algae genomes available failed to find this component [50]. Thus
even if it was not annotated in the genome sequence of
C. merolae these searches should detect its presence.
However, it cannot be ruled out that it may have been
missed in the sequencing and/or assembly of the
C. merolae genome. Tom5 and Tom6, proteins of
approximately 50 amino acids long, were not detected
in C. variabilis, using either plant or yeast interrogation
sequences in searches. However, the small size of Tom5
and 6 proteins means that it is difficult to define their
evolutionary relationship across wide phylogenetic gaps.
Tom7, on the other hand appears to be orthologous
across all groups, with the exception that it cannot be
found in C. merolae. Thus suggesting that the small
TOM proteins may be lineage specific, as is the case of
the Tom20 receptor.
It is evident that the TOM complex of plants displays
diversity with respect to the receptor components present. While Tom40 is universally present, the presence
of Tom20 is only evident in green plants, Tom70 is only
present in E. siliculosus, and OM64 appears to have
arisen by a relatively recent evolutionary event as it is
only present in a variety of monocot and dicot plants
examined and could not be detected in P. glauca and
S. moellendorffii (Figure 2A and 3A). The brown algae
E. siliculosus, contains a Tom70 type receptor. As there
is no Tom70 like sequences in green plants [29], the
Tom70 type receptor was either derived from the specific host in the symbiosis that led to the formation of
brown algae, or alternatively, it may represent a case of
convergent evolution, as has been observed between
green plants and Opisthokonts for the Tom20 receptor
[27,55].
An analysis of the mitochondrial protein import apparatus in a variety of plants reveals that C. merolae

Page 6 of 15

clearly contains a Tom22 type receptor in contrast to all
other plant lineages. Thus, this component may either
have been lost from brown algae and green plant
lineages or the presence of a Tom22 type receptor in
C. merolae represents another case of convergent evolution. As brown algae are proposed to have been derived
from red algae, after the latter branched from green
plants [57], the Tom22 receptor would have to be lost
independently in green plants and brown algae. However, caution needs to be exercised, as the sequence of
E. siliculosus may not be fully representative of all
brown algae.
The question of how red algae solve the sorting problem between plastids and mitochondria may relate to
the binding substrates of the receptors, that is, the targeting signals. Analysis of plastid targeting signals from
all plant lineages reveals that red algae (and the other
primary plant lineage, glaucophytes) contain a phenylalanine residue within a few amino acids of the
N-terminus, which is in a hydrophobic context [58].
This ‘ancestral’ plastid targeting motif is not present in
plastid targeting signals in green plants [58], and thus
the differentiation of plastid and mitochondrial targeting
signals in green plants differs to red algae. In red algae,
the “phenylalanine containing” transit peptide may serve
as a means for mitochondria and plastid targeting signals to be recognized or rejected by plastidic or mitochondrial receptors respectively.
Sorting and Assembly Machinery of the Outer
Mitochondrial Membrane (SAM)

The SAM complex is required for the insertion of
b-barrel and a-helical proteins into the outer membrane
[52]. The insertion of b-barrel proteins into the mitochondrial outer membrane is conserved from bacteria to
mitochondria and plastids, where Omp85, Sam50 and
Toc75 are orthologous b-barrel proteins that are essential for this process [59,60]. However, apart from this
central component, there are no other conserved components identified for the insertion of b-barrel proteins
into membranes from bacteria to mitochondria and
plastids (Figure 4A and 4B). In yeast, four additional
components are involved; Sam35, Sam37, Mdm10 and
Mim1, with Sam35 representing an essential component.
As the SAM complex has not been biochemically characterised from mammalian or plant systems, any additional components are unknown in these systems. The
genome of D. discoideum has a gene encoding Sam50,
but lacks the other components identified in yeast. As
D. discoideum is an amoeba that diverged from Opisthokonts after this lineage had split from plants, this suggests that the additional components observed in yeast
arose after the lineage divergence of plants from other
groups. Although additional components are likely to be

Carrie et al. BMC Plant Biology 2010, 10:249
http://www.biomedcentral.com/1471-2229/10/249

Page 7 of 15

A)

Cyandioschyzon

Yeast

35

Selaginella
Chlorella
Picea
Poplar
Zea
Glycine
Ectocarpus
Chlamydomonas
Physcomitrella
Rice
Arabidopsis

37
Metaxin
50

OM

50

50
mdm
10
1

im

m

B)

Yeast Sam37

GST2

H
KAR

V
N
L
K
H

Q

S

50

49

48

47

46

45

Q

44

I

43

I

42

39

38

M KH

A
V ES N
L FSV
TL LN L EA
A
M

M

41

MN

F

40

Y

S

L
DY

H
E

37

34

R

RYW

F

I

36

V

N
R

35

S

32

V

31

30

29

28

27

S

LVQA

L
G

E

S
V
P
H
H
DAG

33

Y

F

26

22

21

20

E
V
P
N
A

19

I

18

16

15

D
TSV
N

H
E
K

23

S

V

T
N
T

M
A
E
A
D

14

9

I Y SDLPWP I GK I L WKQ VKQ LG I

YT
S
C
T I KS
LRDN RQCRK
H
E
V
S
C
H

12

V
I

17

T

A
E
F

25

G A

G D SS

A

SQ
L
L
V
8

N

11

I

S

VEE
N

VC

7

S

P

0

24

WL A YELWV

T AD L

M

1

10

41

40

MEME (no SSC)18.10.2010 01:02

13

2

6

L

5

P
M

A
H

39

37

36

35

I

38

A
F

E
E
A

34

3

T

S

4

P

F
N

S

bits

T

L

N
P
AV
S

V

S
Y
Q I Q
E
D
E
T
H
VW
CTS
N I
S
AS I E
P
T
W

33

28

Motif 1
Amino acids 10-51

N
LDFH
Y

D

R

1

F

4

N
VH

S

GAR

G
T
S
Q
C
A
L

32

M
27

26

25

24

22

21

19

18

17

N
K

16

15

14

V

AF
H

Arabidopsis Metaxin

P

QV

L

F
TQ
L
S I AS
E

T

V

S

23

I

I
FSNF

SVL

A

L
S S
13

9

12

8

11

7

10

G
M
E
Y
D

Q

V
T

20

A

S

6

5

I
A

G

C

A
V PASW
4

0

A

TW
YKS
R

Human Metaxin2

TM

LV RKP FGLPT CP CLPAY I YLRKLA

T

V
1

1

2

2

3

bits

3

GST2

31

GST1
M1 M2

4

TM

GST2

3

GST1

2

GST1

30

TM

29

C)

MEME (no SSC)18.10.2010 01:02

Motif 2
Amino acids 104-154

Figure 4 The SAM complex of plants. A) Schematic representation of the SAM complexes found in yeast and plants. B) Table indicating the
presence or absence of a component of the SAM complexes found in plants. C) Representation of the different domains of yeast Sam37,
Human Metaxin and Arabidopsis metaxin proteins. All three share similar Glutathione S-transferase (GST) domains. However the location of the
transmembrane domains (TM) differs. A motif analysis search of plant metaxin sequences identified Motif 1 to regions between amino acids 37
and 79 and motif 2 between amino acids 104 and 155 in Arabidopsis. These motifs appear only in plant like Metaxins and near a region
required for protein binding. Colors and abbreviations are the same as Figure 2.

Carrie et al. BMC Plant Biology 2010, 10:249
http://www.biomedcentral.com/1471-2229/10/249

present in the SAM complexes from plants, they are
unlikely to be orthologous to the components in yeast.
In Arabidopsis, a protein called Metaxin has been
shown to be involved in the import of b-barrel proteins
into the outer membrane. The deletion of Metaxin is
not lethal in Arabidopsis, although plants are sterile and
grow poorly [53]. Deletion of Metaxin results in a large
up-regulation of transcript abundance for genes encoding the mitochondrial b-barrel proteins porin and
Tom40, with an accumulation of porin observed in the
cytosol, indicating that Metaxin plays a role in the insertion of b-barrel proteins in plants. Mammalian genomes
contain two genes encoding Metaxin, in fact the plant
Metaxin protein was identified using blast searches of
the mammalian Metaxin protein [53] (Figure 4C). Mammalian Metaxin has also been shown to be involved in
the import of b-barrel proteins into the outer membrane
of mitochondria [61]. Mammalian Metaxin does identify
with Sam37 in a blast search, although the sequence
identity is very low (Additional file 1). A number of features distinguish plant and animal Metaxins from
Sam37 in yeast. Firstly, human and Arabidopsis Metaxins are anchored to the outer membrane in the opposite
orientation compared to Sam37 (Figure 4C). Yeast
Sam37 is anchored to the mitochondrial outer membrane by an N-terminal transmembrane domain,
whereas human and Arabidopsis Metaxins contain
C-terminal transmembrane domains. Secondly, human
and Arabidopsis Metaxins contain conserved glutathione
S-transferase (GST) domains (Figure 4C). Plant and animal Metaxins are distinguished by the fact that Metaxin
is not found in a complex with human Sam50 [61],
whereas plant Metaxin is in a complex with plant
Sam50 (Duncan and Whelan - unpublished data). In
Arabidopsis Metaxin there are two conserved motifs in
a region critical for binding that are only found in plant
type Metaxins (Figure 4C motif 1 and 2). It is also of
interest to note that while Trypanosomes do contain a
Metaxin like protein [62], there are no Metaxin or
Sam37 like proteins identified in D. discoideum [49] or
C. merolae in this study. The presence of a Metaxin
protein in E. siliculosus may be derived from the host
cell. Thus, plant and animal Metaxins may be orthologous, but functions are likely to have diverged over
time. Biochemical characterization of the plant SAM
complex would provide information on the accessory
proteins of this complex and provide a clearer picture of
the evolutionary nature of the accessory subunits in this
complex.
Intermembrane space - Mitochondrial intermembrane
space import and Assembly (MIA) and Tiny TIMs

The intermembrane space contains two sets of proteins
that are essential for cell viability in yeast. The tiny TIM

Page 8 of 15

proteins 8, 9, 10 and 13 appear to be present in a wide
variety of eukaryotes (Figure 5A). They play an essential
role in the import of carrier proteins into the inner
membrane and also the assembly of b-barrel proteins
into the outer membrane [52]. It has been proposed
that they arose from an ancestral protein present in the
original host that housed the mitochondrial endosymbiont [63]. There are eukaryotes that lack the small
Tims (Trichomonas vaginalis and Encephalitozoon cuniculi) or only contain one small Tim protein (Cryptosporidium hominis) [64,65], indicating that they are not
absolutely essential, even though these organisms contain carrier type proteins on the inner membrane that
should require these components for import. Thus, the
lack of small Tims is likely to be a derived situation
associated with the presence of highly modified mitochondria (i.e. mitosomes) in these organisms.
The MIA pathway is the most recently described
import pathway for mitochondrial proteins. It consists
of two essential proteins in yeast, Mia40 and Erv1,
which catalyse the oxidative folding of proteins when
they enter the intermembrane space. Substrates of this
pathway are proteins that contain conserved cysteine
residues that undergo oxidative protein folding in the
intermembrane space. Both Mia40 and Erv1 are essential proteins in yeast, with Mia40 proposed to act as the
intermembrane space receptor for proteins [66]. Whilst
detailed structural and mechanistic analysis has been
carried out on this system in yeast [67], little is known
about the components in other organisms. Interestingly
the apparent lack of a gene encoding Mia40 in trypanosomes suggests that this pathway may display variations
between species [68].
For the MIA machinery, orthologues of Mia40 and
Erv1 are present in yeast, humans and plants (Figure
5A). Although Hot13 has been reported to be widespread in eukaryotes, analysis of the proteins identified
indicates it is ~600 amino acids long and most likely a
transcription factor in green plants. Brown algae contain
a protein similar to yeast Hot13 (Figure 5A, Additional
file 1). However, given the small size of this protein
with conserved metal domains it is unclear if it is orthologous to the yeast protein. Although plant Erv1 and
Mia40 are orthologous to their yeast counterparts, the
primary structure of the protein differs (Figure 5B), suggesting possible mechanistic differences. Deletion of
Mia40 in Arabidopsis is not lethal, and in fact normal
growth and development are observed [69]. Erv1 is
essential in Arabidopsis and analysis of the primary
sequence indicates that the arrangement of cysteines differs to that in yeast (and humans). Arabidopsis Erv1 is
similar to that found in Trypanosoma brucei and a protein called Erv2 that is located in the endoplasmic reticulum of yeast (which operates without a Mia40 like

Carrie et al. BMC Plant Biology 2010, 10:249
http://www.biomedcentral.com/1471-2229/10/249

Page 9 of 15

A)

B)

Yeast Erv2p (196 AA)
CXXC

FAD

C-16-C

CGC

Yeast Erv1p (189 AA)
C30/C33

CXXC

FAD

C-16-C
Mia40 essential

Human Erv1 (205 AA)
C71/C74

CXXC

FAD

C-16-C

Arabidopsis Erv1 (191 AA)
CXXC

FAD

C-16-C

CXXXXC
Mia40 non essential

Rice Erv1 (194 AA)
CXXC

FAD

C-16-C

CXXXXC

Trypanosome Erv1 (273 AA)
CXXC

FAD

C-16-C

CXXXC
No known Mia40

E. cuniculi (162 AA)
CXXC

FAD

C-16-C

CXC

Figure 5 Components of the MIA and IMS protein import apparatus of plants. A) A table displaying the components of the small Tim
proteins and MIA pathway components of plants. B) Schematic diagram of the different Erv1 sequences found in different organisms. The grey
region represents the most conserved region between different Erv1 sequences containing the redox centre, the FAD binding site and two
conserved cysteine domains (CXXC and C-16-C). The location of the third cysteine pair differs between organisms which seems to be dependent
on either a Mia40 protein being present or whether that Mia40 is essential or not. Abbreviations are the same as Figures 2 and 4.

Carrie et al. BMC Plant Biology 2010, 10:249
http://www.biomedcentral.com/1471-2229/10/249

protein) (Figure 5B). Given that the import of the small
Tim proteins and carrier proteins is normal in Arabidopsis plants that lack Mia40 [69], mechanistically Erv1
can function without Mia40 in oxidative protein folding
in the intermembrane space. Analysis of the genome of
D. discoideum indicates that Mia40 is present [68]. As
Mia40 is absent in Trypanosoma brucei, Encephalitozoon
cuniculi [65,68], and the brown algae E. siliculosus
(Figure 5A, Additional file 1), this suggests that the presence of Mia40 in plants is a primitive situation, but its
function(s) differ in various lineages.
Translocases of the inner membrane (TIMS)

The inner mitochondrial membrane contains two translocases, the TIM17:23 complex that is responsible for
the import of proteins via the general import pathway,
and the TIM22 complex, that is responsible for the
import of carrier proteins into the inner membrane. The
TIM17:23 complex is responsible for the import of proteins that contain N-terminal targeting signals into or
across the inner mitochondrial membrane [23]. This
complex contains 9 components in yeast and several of
the components are essential in yeast [52]. Tim23 forms
a presequence and voltage sensitive channel [70], while
Tim17 plays a crucial role in voltage sensing [71,72].
The TIM17:23 complex can be divided into the PAM
complex, the presequence assisted motor consisting of
five subunits (Tim44, HSP70, Pam 16, 17 and 18) and
the membrane components of Tim17, 23, 21 and 50.
The TIM22 translocase is responsible for the import of
proteins that contain internal targeting signals and contain multiple (4 or 6) transmembrane spanning regions
into the inner membrane [23]. In contrast to the
TIM17:23 complex, the mechanistic details of how it
operates are not yet fully understood. However Tim22
has been shown to have channel activity that is only
active in the presence of a substrate protein [73]. In
yeast it contains three accessory proteins, Tim54, 18
and 12 [52]. However no details on the composition of
this complex in other organisms have been reported.
In contrast to the TOM complex on the outer membrane, eight of the nine components of the Tim17:23
complex are conserved between yeast, humans and
plants (the only difference being that yeast contain a
Pam17 protein not present in humans and plants)
(Figure 6A, Additional file 1). It has been previously
proposed that the channel forming subunits of this complex, Tim23 and Tim17, are derived from amino acid
transporters in bacteria, specifically LivH, and defined a
family of proteins termed PReprotein and Amino acid
Transporters (PRAT) [74]. It seems that originally there
was one PRAT type protein that subsequently diverged
to give rise to the three different PRAT proteins typically found in mitochondria [75]. In some organisms a

Page 10 of 15

single PRAT protein exists, which is likely a derived
condition where the other PRAT proteins have been
lost [65]. Additionally, not all subunits of the TIM17:23
complex are observed in all organisms, i.e. the absence
of Tim50 in D. discoideum [49], suggests that accessory
subunits can be lost.
In the case of the TIM22 complex, the translocase
responsible for the import of metabolite carriers, or
multiple spanning proteins of the inner membrane,
including the PRAT proteins themselves, only the
Tim22 component is conserved. In fact Tim18 and
Tim54, along with Tim12, are only found in yeast and
not in other organisms including plants. Thus, the additional components of this translocase are yet to be characterized in other organisms.
Although the TIMs seem to be better conserved in
terms of orthology compared to the TOM complex,
there are notable differences in plants. Firstly, the family
of PRAT proteins has greatly expanded in plants compared to yeast and mammals. In Arabidopsis there are
17 members, rice has greater than 24 members and
examination of C. reinhardtii and P. patens reveal 5 and
21 members respectively. Some of these PRAT proteins
are located in plastids, while others are found in mitochondria [76]. In addition to the greater number of
PRAT proteins in plant genomes, Tim17 in plants varies
in size from 133 amino acids to 252 amino acids, the
difference to yeast Tim17 of 158 amino acids is at the
C-terminal end of the protein (Additional file 1).
A C-terminal extension is found on Tim17 proteins
from C. merolae through to Arabidopsis. It has been
shown in Arabidopsis that this C-terminal extension is
exposed on the outer surface of the outer membrane
[36] and Arabidopsis Tim17 can only complement a
yeast Tim17 mutant if this extension is removed [36].
In order to investigate possible function(s) of the
Tim17 C-terminal extension in plants we conducted a
motif search on all the identified Tim17 extensions.
A distinct motif was detected (Figure 6C). Using this
motif in blast searches identifies a number of different
nucleic acid binding proteins (Additional file 2). This
suggests that Tim17 in plants may be able to bind RNA
and/or DNA. As plant mitochondria import tRNAs and
have recently been shown to bind mRNA [22,77], suggests a possible role for Tim17 in the binding and/or
import of nucleic acids into plant mitochondria.
Mitochondrial processing peptidase(s)

Mitochondria require a number of peptidases to remove
the targeting signals from proteins before or after they
are assembled into functional protein complexes. These
peptidases range from activities that remove the targeting signals, such as mitochondrial processing peptidase
(MPP) and intermediate processing peptidase (IMP)

Carrie et al. BMC Plant Biology 2010, 10:249
http://www.biomedcentral.com/1471-2229/10/249

Page 11 of 15

A)

OM

Cyanidioschyzon

Yeast

50

21

17

IM

44

50

23

70

17
16
17
18
om

50

21

23
16
18

70

44

17

23

Ectocarpus
Chlamydomonas
Physcomitrella
Rice
Arabidopsis
Selaginella
Chlorella
Poplar
Zea
Glycine

16
18

44

70

M1

243 Amino acids

B)

C)

TM

LESFDAPPVP

N

I
P P
S
T
S
M

15

13

12

11

T
K

14

T S

E NT

SSG

9

D

F

10

H
N
Y
N
L
D
M

4

0

T
KSE I

E KV
R
K
3

1

2

bits

2

1

3

8

4

TM

7

TM

6

TM

5

AtTim17-2

MEME (no SSC)18.10.2010 01:51

Motif 1
Figure 6 The TIM17:23 and TIM22 complexes of plants. A) A schematic representation of the TIM17:23 complex from yeast and plants.
B) A table displaying the components of the TIM17:23 and TIM22 complexes in all plant species analysed. C) A diagram showing Arabidopsis
Tim17-2 that contains four transmembrane domains (TM) and an extra C-terminal extension. This C-terminal extension is found in all higher
plant species and motif analysis of the C-terminal extension identified to highly conserved regions of which both are related to nucleic acid
binding proteins. Colors and abbreviations are the same as Figures 2 and 4.

Carrie et al. BMC Plant Biology 2010, 10:249
http://www.biomedcentral.com/1471-2229/10/249

[23], to the removal of a single amino acid from proteins that have already had the targeting signal removed
[78], to the presequence degrading peptidase (PreP) that
degrades the targeting signals once they have been
removed [34].
Plant mitochondria contain a number of orthologous
proteins in comparison with the various processing peptidases of yeast mitochondria (Additional file 1). At a
sequence level the processing peptidases found in plant
mitochondria look similar to those of yeast, however,
functional investigations have revealed a number of differences. One major difference between yeast and plants
is the location of the mitochondrial processing peptidase
(MPP). In yeast both a and b MPP subunits are located
in the matrix, however, in plants it has been demonstrated that they are integrated into the cytochrome bc1
complex located in the inner membrane [31,32]. Additionally, the presequence degrading peptidase of yeast is
located in the intermembrane space whereas in plants it
is located in the matrix [79,80]. Interestingly, the presequence degrading peptidase of plant mitochondria is
also dual targeted to plastids where it degrades plastid
targeting signals [79].
In terms of the processing site recognition by MPP in
plants, it has been reported that the majority of plant
mitochondrial presequences fall into 2 classes. In class 1
the processing signal is a -2 Arg residue while in class 2
presequences the signal is a -3 Arg residue [20,81], similar to what has been reported for yeast and mammals
[23]. However, it has been demonstrated that in fact
there is a second processing step in yeast [82], a novel
peptidase called intermediate cleavage peptidase (Icp55)
was found to process mitochondrial presequences after
MPP, cleaving only 1 amino acid from the N-terminus,
turning the proposed -2 cleavage signal into a -3 cleavage signal [78]. It is tempting to speculate that as
plants contain an orthologue of Icp55 that the same
cleavage is occurring, however this awaits experimental
confirmation. Despite the orthology between many plant
peptidases and those in other organisms it is still necessary to define their specific functions in plants. It has
been demonstrated that the plant orthologue of the
rhomboid protease from yeast does not carry out the
same processing roles/activities in plants such as Arabidopsis [83].

Conclusions
The plant mitochondrial import apparatus displays
many differences compared to other non-plant organisms and between plant groups. The TOM complex in
plants displays the most variability in that as many as
five different TOM complexes exist in plants when red
algae, brown algae and green plants are considered.
Even in the green plant lineage variation is observed

Page 12 of 15

with OM64 only being present in monocot and dicot
plants. While the composition of the other protein complexes may appear more conserved, the lack of biochemical characterization of these complexes in any plant
group means that the presence of plant specific accessory subunits in various lineages cannot be judged.
Additionally for some proteins, such as Mia40 and
Tim17, functions have expanded in plants compared to
those characterized in yeast.

Methods
The protein sequences for all of the known mitochondrial protein import components from Saccharomyces
cerevisiae (Tom20, Tom70, Tom71, Tom40, Tom22,
Tom5, Tom6, Tom7, Sam50, Sam37, Sam35, Mdm10,
Mim1, Mia40, Erv1, Hot13, Tim9, Tim10, Tim8, Tim13,
Tim12, Tim22, Tim54, Tim18, Tim23, Tim17, Tim50,
Tim21, mtHsp70, Mge1, Tim44, Pam18, Mdj2, Pam16,
Pam17, MPPa, MPPb, Oct1, Imp1, Imp2, Som1, Yta12,
Yta10, Yme1, Mgr1, Mgr3, Pcp1, Icp55, Oxa1, Mba1,
Cox18, Pnt1, Mss2, Mdj1, Hsp60, Hsp10, Hsp78 and
Zim17) were downloaded from the NCBI protein database (http://www.ncbi.nlm.nih.gov/protein/). The
Metaxin protein sequences were also obtained from
Homo sapiens. Using the above protein sequences Blastp
[84] searches of the protein sequences from Physcomitrella patens, Selaginella moellendorffii, Chlamydomonas
reinhardtii, Arabidopsis thaliana, Oryza sativa, Zea
mays, Vitis vinifera, Glycine max and Populus tricocarpa
were performed using the Phytozome (http://www.phytozome.net) database. Blastp [84] searches of Cyanidioschyzon merolae were performed using the Cyanidioschyzon
merolae genome project website (http://merolae.biol.s.utokyo.ac.jp/). Blastp [84] searches of Ectocarpus siliculosus were performed at the Bioinformatics online genome
annotation system website (http://bioinformatics.psb.
ugent.be/webtools/bogas/overview/Ectsi). Blastp [84]
searches of Chlorella variabilis NC64A genome [38] was
performed at the Chlorella genome website (http://genome.jgi-psf.org/ChlNC64A_1/ChlNC64A_1.home.html).
To identify mitochondrial import components of Picea
glauca tblastn [84] searches were carried on EST
sequences [43] at the NCBI website (http://blast.ncbi.
nlm.nih.gov/Blast.cgi).
All multiple sequence alignments were carried out
using MAFFT [85] and visualized using Multiple align
show (http://www.bioinformatics.org/sms/multi_align.
html). The program IQPNNI [86] was used to reconstruct a maximum likelihood phylogeny assuming the
Whelan and Goldman model [87]. Phylogenetic trees
were finally visualized using the program Geneious
(http://www.geneious.com).
TMpred (http://www.ch.embnet.org/software/TMPRED_
form.html), TMHMM (http://www.cbs.dtu.dk/services/

Carrie et al. BMC Plant Biology 2010, 10:249
http://www.biomedcentral.com/1471-2229/10/249

Page 13 of 15

TMHMM/), and DAS (http://www.sbc.su.se/~miklos/
DAS/) [88] were used in the prediction of transmembrane regions. TPR repeats were predicted using
TPRpred (http://toolkit.tuebingen.mpg.de/tprpred) [89].
Motif analysis was performed using MEME (http://
meme.nbcr.net/meme4_4_0/cgi-bin/meme.cgi) using
default parameters for all plant like Metaxin sequences
and sequences of the plant Tim17 extensions [90].

11.

Additional material

14.

9.
10.

12.
13.

Additional file 1: Supplementary table 1. The mitochondrial import
machinery of plants.

15.

Additional file 2: Supplementary table 2. The top 50 proteins
identified using the conserved motifs on the C-terminal of plant
Tim17 in a Blastp search.

16.

Abbreviations
Erv1: Essential for respiration and vegetative growth 1; FAD: Flavin adenine
dinucleotide; GDP: Guanosine diphosphate; GTP: Guanosine diphosphate;
GST: Glutathione S-transferase; Hot13: Helper of Tim protein 13; Icp55:
Intermediate cleavage peptidase of 55 kDa; Mdm10: Mitochondria
distribution and morphology protein 10; MIA: Mitochondrial import and
assembly; Mim1: Mitochondrial import 1; MPP: Mitochondrial processing
peptidase; OM64: Mitochondrial outer membrane protein of 64 kDa; Omp85:
Outer membrane protein of 85 kDa; PRAT: Preprotein and amino acid
transporter; TIM: Translocase of the inner membrane; TOC: Translocase of the
outer envelope of chloroplasts; TOM: Translocase of the outer membrane;
TPR: Tetratricopeptide repeat; SAM: Sorting and assembly machinery.
Acknowledgements
This work was supported by an Australian Research Council Centre of
Excellence Grant CEO561495.
Authors’ contributions
CC carried out the data analysis with the help of MM. JW oversaw the
analysis, design and implementation. CC, MM and JW drafted the
manuscript. All authors read and approved final manuscript.
Received: 26 August 2010 Accepted: 16 November 2010
Published: 16 November 2010
References
1. Cavalier-Smith T: Predation and eukaryote cell origins: a coevolutionary
perspective. Int J Biochem Cell Biol 2009, 41(2):307-322.
2. Dyall SD, Brown MT, Johnson PJ: Ancient invasions: from endosymbionts
to organelles. Science 2004, 304(5668):253-257.
3. Mentel M, Martin W: Energy metabolism among eukaryotic anaerobes in
light of Proterozoic ocean chemistry. Philos Trans R Soc Lond B Biol Sci
2008, 363(1504):2717-2729.
4. Deusch O, Landan G, Roettger M, Gruenheit N, Kowallik KV, Allen JF,
Martin W, Dagan T: Genes of cyanobacterial origin in plant nuclear
genomes point to a heterocyst-forming plastid ancestor. Mol Biol Evol
2008, 25(4):748-761.
5. Gould SB, Waller RF, McFadden GI: Plastid evolution. Annu Rev Plant Biol
2008, 59:491-517.
6. Theissen U, Martin W: The difference between organelles and
endosymbionts. Curr Biol 2006, 16(24):R1016-1017, author reply R10171018.
7. Huang CY, Ayliffe MA, Timmis JN: Direct measurement of the transfer rate
of chloroplast DNA into the nucleus. Nature 2003, 422(6927):72-76.
8. Adams KL, Palmer JD: Evolution of mitochondrial gene content: gene
loss and transfer to the nucleus. Mol Phylogenet Evol 2003,
29(3):380-395.

17.

18.

19.

20.

21.

22.

23.
24.
25.

26.

27.

28.
29.

30.

31.

32.

33.

Martin W, Herrmann RG: Gene transfer from organelles to the nucleus:
how much, what happens, and Why? Plant Physiol 1998, 118(1):9-17.
Lithgow T, Schneider A: Evolution of macromolecular import pathways in
mitochondria, hydrogenosomes and mitosomes. Philos Trans R Soc Lond
B Biol Sci 2010, 365(1541):799-817.
Alverson AJ, Wei X, Rice DW, Stern DB, Barry K, Palmer JD: Insights into the
evolution of mitochondrial genome size from complete sequences of
Citrullus lanatus and Cucurbita pepo (Cucurbitaceae). Mol Biol Evol 2010.
Bonen L: Cis- and trans-splicing of group II introns in plant
mitochondria. Mitochondrion 2008, 8(1):26-34.
Palmer JD, Herbon LA: Plant mitochondrial DNA evolves rapidly in
structure, but slowly in sequence. J Mol Evol 1989, 28:87-97.
Wolfe KH, Li WH, Sharp PM: Rates of nucleotide substitution vary greatly
among plant mitochondrial, chloroplast, and nuclear DNAs. Proc Natl
Acad Sci USA 1987, 84(24):9054-9058.
Schmitz-Linneweber C, Small I: Pentatricopeptide repeat proteins: a
socket set for organelle gene expression. Trends Plant Sci 2008,
13(12):663-670.
Richardson AO, Palmer JD: Horizontal gene transfer in plants. J Exp Bot
2007, 58(1):1-9.
Day DA, Whelna J, Millar AH, Siedow J, Wiskich JT: REgulation of the
alternative oxidase in Plants and Fungi. Aus J Plant Physiol 1995,
22:497-509.
Considine MJ, Holtzapffel RC, Day DA, Whelan J, Millar AH: Molecular
distinction between alternative oxidase from monocots and dicots. Plant
Physiol 2002, 129(3):949-953.
Somerville C, Browse J, Ohlrogge J: Lipids. In Biochemistry and Molecular
Biology of Plants. Edited by: Buchanan BB, Gruissem W, Jones RL. Wiley;
2002:456-527.
Zhang XP, Glaser E: Interaction of plant mitochondrial and chloroplast
signal peptides with the Hsp70 molecular chaperone. Trends Plant Sci
2002, 7(1):14-21.
Chew O, Whelan J: Just read the message: a model for sorting of
proteins between mitochondria and chloroplasts. Trends Plant Sci 2004,
9(7):318-319.
Michaud M, Marechal-Drouard L, Duchene AM: RNA trafficking in plant
cells: targeting of cytosolic mRNAs to the mitochondrial surface. Plant
Mol Biol .
Neupert W, Herrmann JM: Translocation of proteins into mitochondria.
Annu Rev Biochem 2007, 76:723-749.
Soll J, Schleiff E: Protein import into chloroplasts. Nat Rev Mol Cell Biol
2004, 5(3):198-208.
Jansch L, Kruft V, Schmitz UK, Braun HP: Unique composition of the
preprotein translocase of the outer mitochondrial membrane from
plants. J Biol Chem 1998, 273(27):17251-17257.
Werhahn W, Niemeyer A, Jänsch L, Kruft V, Schmitz UK, Braun HP:
Purification and characterization of the preprotein translocase of the
outer mitochondrial membrane from Arabidopsis. Identification of
multiple forms of TOM20. Plant Physiology 2001, 125:943-954.
Perry AJ, Hulett JM, Likic VA, Lithgow T, Gooley PR: Convergent evolution
of receptors for protein import into mitochondria. Curr Biol 2006,
16(3):221-229.
Lister R, MW M, Whelan J: The Mitochondrial Protein Import Machinery of
Plants (MPIMP) database. Nucleic Acids Reserach 2003, 31:325-327.
Chan NC, Likic VA, Waller RF, Mulhern TD, Lithgow T: The C-terminal TPR
domain of Tom70 defines a family of mitochondrial protein import
receptors found only in animals and fungi. J Mol Biol 2006,
358(4):1010-1022.
Macasev D, Whelan J, Newbigin E, Silva-Filho MC, Mulhern TD, Lithgow T:
Tom22’, an 8-kDa trans-site receptor in plants and protozoans, is a
conserved feature of the TOM complex that appeared early in the
evolution of eukaryotes. Mol Biol Evol 2004, 21(8):1557-1564.
Braun HP, Emmermann M, Kruft V, Schmitz UK: The general mitochondrial
processing peptidase from potato is an integral part of cytochrome c
reductase of the respiratory chain. EMBO J 1992, 11(9):3219-3227.
Glaser E, Dessi P: Integration of the mitochondrial-processing peptidase
into the cytochrome bc1 complex in plants. J Bioenerg Biomembr 1999,
31(3):259-274.
Brumme S, Kruft V, Schmitz UK, Braun HP: New insights into the coevolution of cytochrome c reductase and the mitochondrial processing
peptidase. J Biol Chem 1998, 273:13143-13149.

Carrie et al. BMC Plant Biology 2010, 10:249
http://www.biomedcentral.com/1471-2229/10/249

34. Bhushan S, Ståhl A, Nilsson S, Lefebvre B, Seki M, Roth C, McWilliam D,
Wright SJ, Liberles DA, Shinozaki K, et al: Catalysis, subcellular localization,
expression and evolution of the targeting peptides degrading protease,
AtPreP2. Plant Cell Physiology 2005, 46:985-996.
35. Lister R, Mowday B, Whelan J, AH M: Zinc-dependent intermembrane
space proteins stimulate import of carrier proteins into plant
mitochondria. Plant J 2002, 30:555-566.
36. Murcha MW, Elhafez D, Millar AH, Whelan J: The C-terminal region of
TIM17 links the outer and inner mitochondrial membranes in
Arabidopsis and is essential for protein import. J Biol Chem 2005,
280(16):16476-16483.
37. Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB,
Terry A, Salamov A, Fritz-Laylin LK, Marechal-Drouard L, et al: The
Chlamydomonas genome reveals the evolution of key animal and plant
functions. Science 2007, 318(5848):245-250.
38. Blanc G, Duncan G, Agarkova I, Borodovsky M, Gurnon J, Kuo A, Lindquist E,
Lucas S, Pangilinan J, Polle J, et al: The Chlorella variabilis NC64A Genome
Reveals Adaptation to Photosymbiosis, Coevolution with Viruses, and
Cryptic Sex. Plant Cell .
39. Rensing SA, Lang D, Zimmer AD, Terry A, Salamov A, Shapiro H,
Nishiyama T, Perroud PF, Lindquist EA, Kamisugi Y, et al: The
Physcomitrella genome reveals evolutionary insights into the conquest
of land by plants. Science 2008, 319(5859):64-69.
40. Banks JA: Selaginella and 400 million years of separation. Annu Rev Plant
Biol 2009, 60:223-238.
41. The map-based sequence of the rice genome. Nature 2005,
436(7052):793-800.
42. Arabidopsis Genome Initiative. Nature 2000, 408(6814):796-815.
43. Pavy N, Paule C, Parsons L, Crow JA, Morency MJ, Cooke J, Johnson JE,
Noumen E, Guillet-Claude C, Butterfield Y, et al: Generation, annotation,
analysis and database integration of 16,500 white spruce EST clusters.
BMC Genomics 2005, 6:144.
44. Cock JM, Sterck L, Rouze P, Scornet D, Allen AE, Amoutzias G, Anthouard V,
Artiguenave F, Aury JM, Badger JH, et al: The Ectocarpus genome and the
independent evolution of multicellularity in brown algae. Nature
465(7298):617-621.
45. Matsuzaki M, Misumi O, Shin IT, Maruyama S, Takahara M, Miyagishima SY,
Mori T, Nishida K, Yagisawa F, Yoshida Y, et al: Genome sequence of the
ultrasmall unicellular red alga Cyanidioschyzon merolae 10D. Nature
2004, 428(6983):653-657.
46. Keeling PJ: The endosymbiotic origin, diversification and fate of plastids.
Philos Trans R Soc Lond B Biol Sci 2010, 365(1541):729-748.
47. Bolte K, Bullmann L, Hempel F, Bozarth A, Zauner S, Maier UG: Protein
targeting into secondary plastids. J Eukaryot Microbiol 2009, 56(1):9-15.
48. Dimmer KS, Rapaport D: Proteomic view of mitochondrial function.
Genome Biol 2008, 9(2):209.
49. Dolezal P, Dagley MJ, Kono M, Wolynec P, Likic VA, Foo JH, Sedinova M,
Tachezy J, Bachmann A, Bruchhaus I, et al: The essentials of protein
import in the degenerate mitochondrion of Entamoeba histolytica. PLoS
Pathog 2010, 6(3):e1000812.
50. Likic VA, Dolezal P, Celik N, Dagley M, Lithgow T: Using hidden markov
models to discover new protein transport machines. Methods Mol Biol
2010, 619:271-284.
51. van Wilpe S, Ryan MT, Hill K, Maarse AC, Meisinger C, Brix J, Dekker PJ,
Moczko M, Wagner R, Meijer M, et al: Tom22 is a multifunctional
organizer of the mitochondrial preprotein translocase. Nature 1999,
401(6752):485-489.
52. Chacinska A, Koehler CM, Milenkovic D, Lithgow T, Pfanner N: Importing
mitochondrial proteins: machineries and mechanisms. Cell 2009,
138(4):628-644.
53. Lister R, Carrie C, Duncan O, Ho LH, Howell KA, Murcha MW, Whelan J:
Functional definition of outer membrane proteins involved in preprotein
import into mitochondria. Plant Cell 2007, 19(11):3739-3759.
54. Likic VA, Perry A, Hulett J, Derby M, Traven A, Waller RF, Keeling PJ,
Koehler CM, Curran SP, Gooley PR, et al: Patterns that define the four
domains conserved in known and novel isoforms of the protein import
receptor Tom20. J Mol Biol 2005, 347(1):81-93.
55. Lister R, Whelan J: Mitochondrial protein import: convergent solutions for
receptor structure. Curr Biol 2006, 16(6):R197-199.
56. Chew O, Lister R, Qbadou S, Heazlewood JL, Soll J, Schleiff E, Millar AH,
Whelan J: A plant outer mitochondrial membrane protein with high

Page 14 of 15

57.

58.
59.
60.

61.

62.

63.

64.

65.

66.
67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

amino acid sequence identity to a chloroplast protein import receptor.
FEBS Lett 2004, 557(1-3):109-114.
Yoon HS, Hackett JD, Ciniglia C, Pinto G, Bhattacharya D: A molecular
timeline for the origin of photosynthetic eukaryotes. Mol Biol Evol 2004,
21(5):809-818.
Patron NJ, Waller RF: Transit peptide diversity and divergence: A global
analysis of plastid targeting signals. Bioessays 2007, 29(10):1048-1058.
Gentle IE, Burri L, Lithgow T: Molecular architecture and function of the
Omp85 family of proteins. Mol Microbiol 2005, 58(5):1216-1225.
Paschen SA, Waizenegger T, Stan T, Preuss M, Cyrklaff M, Hell K, Rapaport D,
Neupert W: Evolutionary conservation of biogenesis of beta-barrel
membrane proteins. Nature 2003, 426(6968):862-866.
Kozjak-Pavlovic V, Ross K, Benlasfer N, Kimmig S, Karlas A, Rudel T:
Conserved roles of Sam50 and metaxins in VDAC biogenesis. EMBO Rep
2007, 8(6):576-582.
Schneider A, Bursac D, Lithgow T: The direct route: a simplified pathway
for protein import into the mitochondrion of trypanosomes. Trends Cell
Biol 2008, 18(1):12-18.
Dolezal P, Likic V, Tachezy J, Lithgow T: Evolution of the molecular
machines for protein import into mitochondria. Science 2006,
313(5785):314-318.
Gentle IE, Perry AJ, Alcock FH, Likic VA, Dolezal P, Ng ET, Purcell AW,
McConnville M, Naderer T, Chanez AL, et al: Conserved motifs reveal
details of ancestry and structure in the small TIM chaperones of the
mitochondrial intermembrane space. Mol Biol Evol 2007, 24(5):1149-1160.
Waller RF, Jabbour C, Chan NC, Celik N, Likic VA, Mulhern TD, Lithgow T:
Evidence of a reduced and modified mitochondrial protein import
apparatus in microsporidian mitosomes. Eukaryot Cell 2009, 8(1):19-26.
Riemer J, Fischer M, Herrmann JM: Oxidation-driven protein import into
mitochondria: Insights and blind spots. Biochim Biophys Acta 2010.
Endo T, Yamano K, Kawano S: Structural basis for the disulfide relay
system in the mitochondrial intermembrane space. Antioxid Redox Signal
2010, 13(9):1359-1373.
Allen JW, Ferguson SJ, Ginger ML: Distinctive biochemistry in the
trypanosome mitochondrial intermembrane space suggests a model for
stepwise evolution of the MIA pathway for import of cysteine-rich
proteins. FEBS Lett 2008, 582(19):2817-2825.
Carrie C, Giraud E, Duncan O, Xu H, Wang Y, Clifton R, Murcha MW,
Filipovska A, Rackham O, Vrielink A, et al: Characterisation of the disulfide
relay system of Arabidopsis thaliana mitochondria reveals that Erv1 is
the only known essential component. 2010.
Truscott KN, Kovermann P, Geissler A, Merlin A, Meijer M, Driessen AJ,
Rassow J, Pfanner N, Wagner R: A presequence- and voltage-sensitive
channel of the mitochondrial preprotein translocase formed by Tim23.
Nat Struct Biol 2001, 8(12):1074-1082.
Martinez-Caballero S, Grigoriev SM, Herrmann JM, Campo ML, Kinnally KW:
Tim17p regulates the twin pore structure and voltage gating of the
mitochondrial protein import complex TIM23. J Biol Chem 2007,
282(6):3584-3593.
Meier S, Neupert W, Herrmann JM: Conserved N-terminal negative
charges in the Tim17 subunit of the TIM23 translocase play a critical
role in the import of preproteins into mitochondria. J Biol Chem 2005,
280(9):7777-7785.
Peixoto PM, Grana F, Roy TJ, Dunn CD, Flores M, Jensen RE, Campo ML:
Awaking TIM22, a dynamic ligand-gated channel for protein insertion in
the mitochondrial inner membrane. J Biol Chem 2007,
282(26):18694-18701.
Rassow J, Dekker PJ, van Wilpe S, Meijer M, Soll J: The preprotein
translocase of the mitochondrial inner membrane: function and
evolution. J Mol Biol 1999, 286(1):105-120.
Clements A, Bursac D, Gatsos X, Perry AJ, Civciristov S, Celik N, Likic VA,
Poggio S, Jacobs-Wagner C, Strugnell RA, et al: The reducible complexity
of a mitochondrial molecular machine. Proc Natl Acad Sci USA 2009,
106(37):15791-15795.
Murcha MW, Elhafez D, Lister R, Tonti-Filippini J, Baumgartner M, Philippar K,
Carrie C, Mokranjac D, Soll J, Whelan J: Characterization of the preprotein
and amino acid transporter gene family in Arabidopsis. Plant Physiol
2007, 143(1):199-212.
Eliyahu E, Pnueli L, Melamed D, Scherrer T, Gerber AP, Pines O, Rapaport D,
Arava Y: Tom20 mediates localization of mRNAs to mitochondria in a
translation-dependent manner. Mol Cell Biol 30(1):284-294.

Carrie et al. BMC Plant Biology 2010, 10:249
http://www.biomedcentral.com/1471-2229/10/249

Page 15 of 15

78. Vogtle FN, Wortelkamp S, Zahedi RP, Becker D, Leidhold C, Gevaert K,
Kellermann J, Voos W, Sickmann A, Pfanner N, et al: Global analysis of the
mitochondrial N-proteome identifies a processing peptidase critical for
protein stability. Cell 2009, 139(2):428-439.
79. Bhushan S, Lefebvre B, Stahl A, Wright SJ, Bruce BD, Boutry M, Glaser E:
Dual targeting and function of a protease in mitochondria and
chloroplasts. EMBO Rep 2003, 4(11):1073-1078.
80. Kambacheld M, Augustin S, Tatsuta T, Muller S, Langer T: Role of the novel
metallopeptidase Mop112 and saccharolysin for the complete
degradation of proteins residing in different subcompartments of
mitochondria. J Biol Chem 2005, 280(20):20132-20139.
81. Huang S, Taylor NL, Whelan J, Millar AH: Refining the definition of plant
mitochondrial presequences through analysis of sorting signals, Nterminal modifications, and cleavage motifs. Plant Physiol 2009,
150(3):1272-1285.
82. Schneider G, Sjoling S, Wallin E, Wrede P, Glaser E, von Heijne G: Featureextraction from endopeptidase cleavage sites in mitochondrial targeting
peptides. Proteins 1998, 30(1):49-60.
83. Kmiec-Wisniewska B, Krumpe K, Urantowka A, Sakamoto W, Pratje E,
Janska H: Plant mitochondrial rhomboid, AtRBL12, has different substrate
specificity from its yeast counterpart. Plant Mol Biol 2008, 68(1-2):159-171.
84. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment
search tool. J Mol Biol 1990, 215(3):403-410.
85. Katoh K, Kuma K, Toh H, Miyata T: MAFFT version 5: improvement in
accuracy of multiple sequence alignment. Nucleic Acids Res 2005,
33(2):511-518.
86. Vinh le S, Von Haeseler A: IQPNNI: moving fast through tree space and
stopping in time. Mol Biol Evol 2004, 21(8):1565-1571.
87. Whelan S, Goldman N: A general empirical model of protein evolution
derived from multiple protein families using a maximum-likelihood
approach. Mol Biol Evol 2001, 18(5):691-699.
88. Cserzo M, Wallin E, Simon I, von Heijne G, Elofsson A: Prediction of
transmembrane alpha-helices in prokaryotic membrane proteins: the
dense alignment surface method. Protein Eng 1997, 10(6):673-676.
89. Karpenahalli MR, Lupas AN, Soding J: TPRpred: a tool for prediction of
TPR-, PPR- and SEL1-like repeats from protein sequences. BMC
Bioinformatics 2007, 8:2.
90. Bailey TL, Elkan C: Fitting a mixture model by expectation maximization
to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol 1994,
2:28-36.
91. Fredslund J: PHY.FI: fast and easy online creation and manipulation of
phylogeny color figures. BMC Bioinformatics 2006, 7:315.
doi:10.1186/1471-2229-10-249
Cite this article as: Carrie et al.: An in silico analysis of the mitochondrial
protein import apparatus of plants. BMC Plant Biology 2010 10:249.

Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color ﬁgure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit

</pre>
</body>
</html>