Báo cáo khoa học: Conserved pore-forming regions in polypeptidetransporting proteins pot

Conserved pore-forming regions in polypeptide-
transporting proteins
Suncana Moslavac
1
, Oliver Mirus
1
, Rolf Bredemeier
1
,Ju
¨
rgen Soll
1
, Arndt von Haeseler
2,3
and Enrico Schleiff
1
1 Botanik, LMU Mu
¨
nchen, Germany
2 Institut fu
¨
r Informatik, Heinrich-Heine Universita
¨
tDu
¨
sseldorf, Germany
3 Neumann Institute for Computing, Forschungszentrum Ju
¨
lich, Germany
Transport of solutes or macromolecules such as pro-
teins across membranes requires a proteinaceous chan-

nel or transporter. Besides their way of action, these
proteins can be divided according to their substrates or
to their secondary structure of the membrane domain.
In terms of secondary structure a-helical or b-sheet
channels can be differentiated [1]. Both types of chan-
nels show a high neighbourhood correlation according
to the fold [2] suggesting similar folds of the mem-
brane-inserted domains. In the past, much attention
was given to the a-helical channels [3–5]. However,
recently ion channels formed by the b-sheets moved
into the focus of interest [6,7]. While analyzing these
channels it became obvious that they emerged from
outer membrane proteins of prokaryotic endo-
symbionts, as these proteins were the only b-barrel
type membrane proteins found in bacteria [6]. This
class of proteins is present in organellar membranes of
eukaryotic organisms, like in the outer mitochondrial
membrane [8] emerged from a-proteobacteria [9], in
the outer envelope of chloroplasts [10,11] emerged
from cyanobacteria [9] and maybe even in the peroxi-
somal membrane [12]. The peroxisomal b-barrel pro-
tein might be an indication either of the discussed
endosymbiotic origin (for example [13]) or of a redis-
tribution of proteins within the cell as a result of the
gene transfer of the other two endosymbiotic events
[14]. Most of the b-barrel type channels of eukaryotes
belong to the porin type family. Recent research
revealed that b-barrel type channels are also involved
in the translocation of polypeptides [15], in the assem-
bly of proteins in the outer membrane of endosymbio-

tic organelles [16–19] or in the assembly of proteins in
the outer membrane of bacteria [7,20,21].
One polypeptide-transporter that was found in
Bordetella pertussis is FhaC, which secretes the main
Keywords
endosymbiosis; Omp85; protein
translocation; pore-forming domains; Toc75
Correspondence
E. Schleiff, Botanik, LMU Mu
¨
nchen,
Menzinger Str. 67, Room 223, 80638
Mu
¨
nchen, Germany
Fax: +49 89 17861185
Tel: +49 89 17861182
E-mail:
(Received 04 November 2004, revised 27
November 2004, accepted 14 January 2005)
doi:10.1111/j.1742-4658.2005.04569.x
Transport of solutes and polypeptides across membranes is an essential
process for every cell. In the past, much focus has been placed on helical
transporters. Recently, the b-barrel-shaped transporters have also attracted
some attention. The members of this family are found in the outer bacterial
membrane and the outer membrane of endosymbiotically derived organ-
elles. Here we analyze the features and the evolutionary development of a
specified translocator family, namely the b-barrel-shaped polypeptide-trans-
porters. We identified sequence motifs, which characterize all transporters
of this family, as well as motifs specific for a certain subgroup of proteins of

this class. The general motifs are related to the structural composition of
the pores. Further analysis revealed a defined distance of two motifs to the
C-terminal portion of the proteins. Furthermore, the evolutionary relation-
ship of the proteins and the motifs are discussed.
Abbreviation
EBS, exact b-sheet.
FEBS Journal 272 (2005) 1367–1378 ª 2005 FEBS 1367
adhesin, namely haemagglutinin [22,23]. This is an
outer membrane protein of various Gram-negative
pathogens and facilitates translocation of polypeptides
[22]. A further member of the family of accessory outer
membrane proteins involved in secretion of haemoly-
sins or adhesins in various Gram-negative pathogens is
ShlB first found in Serratia marcescens [24]. Despite the
homology between FhaC and ShlB, the proteins are
not exchangeable indicating the molecular specificity
of the transporters [25]. Structural modelling of FhaC
[26] and ShlB [27] suggests long loop regions at the
N-terminus, whereas the C-terminal portion seems to
be involved in pore formation. It was further estab-
lished that ShlB has two functions. On one hand, the
channel formed by ShlB facilitates the translocation of
ShlA [28]. On the other hand it activates ShlA by chan-
ging the conformation of this substrate and thereby
inducing the transfer of phosphatidylethanolamine
[29–31] required for the activation of the enzyme ShlA.
A third class of translocators is formed by the
Omp85 ⁄ D15 homologues. Omp85 is an essential com-
ponent for outer membrane biogenesis in the Gram-
negative bacterium Neisseria meningitidis . Similarly to

the ShlB family, it seems that Omp85 has two func-
tions: the assembly of outer membrane proteins [32]
and the translocation of lipids [33]. Voulhoux and
coworker [32] further suggested a b-barrel-shaped
membrane structure. However, little more is known
about these proteins.
Toc75, the 75-kDa subunit of the translocation com-
plex of the outer envelope of chloroplasts of Pisum sat-
ivum, is one member of this polypeptide-transporting
family found in the endosymbiotic organelle chloro-
plast. It is one of the major proteins of this membrane
and acts as the protein translocation channel [34]. In
contrast to the other identified polypeptide transport-
ers, the translocation of proteins requires the action of
assisting proteins like Toc159 [35]. Similar to FhaC,
ShlB and Omp85, structural modelling of Toc75 from
Pisum sativum [36] and Toc75-V from Arabidopsis
thaliana [11] suggests a b-barrel type structure of the
protein. Furthermore, it was proposed that the Toc75
family might have evolved from the ShlB [37,38] or the
Omp85 class [32]. Recently, a protein of the outer
membrane of the second endosymbiotically derived
organelle, the mitochondrion, was identified to belong
to this distinct family of polypeptide-transporting pro-
teins. The protein was termed Sam50 [17], Tob55 [18]
or mitochondrial Omp85 homologue [19]. This protein
facilitates the assembly of proteins into the outer mito-
chondrial membrane [17–19].
Here we present an analysis of this transport protein
family. We observed a putative motif in the N-terminal

region, but with a lower reliability than a conserved
motif in the C-terminal region. We provide evidence
that this conserved motif is specific for polypeptide-
transporting proteins and that it is involved in pore
formation. The possible function is discussed.
Results
Motifs in b-barrel-shaped polypeptide-transporting
proteins b-barrel type proteins are divided in several
subclasses regarding their structural or functional fea-
tures as discussed elsewhere [1]. Herein we have
analyzed 71 b-barrel-shaped proteins with putative
polypeptide-transporting function (Table S1). For our
analysis and to test the specificity of the identified
domains we included 10 members of the b-barrel-
shaped FepA family. These proteins are known to
facilitate iron transport across the outer membrane of
bacteria and are not involved in polypeptide transport
[39].
Analyzing the b-barrel-shaped polypeptide-trans-
porting proteins using the ‘motif alignment and search
tool’ [40] we identified four motifs (Fig. 1, Table 1)
in the selected proteins (Table S1). The respective
sequence with highest probability is shown in Fig. 1A.
The motifs are not found in the sequences of the mem-
bers of the FepA class with the exception of the pro-
tein in Pseudomonas putida KT2440 where motif 3 and
4 were detected, however, with the highest P-value of
the whole set of analyzed proteins. The P-value of an
identified motif within the target sequence is computed
as following: the match score of the identified motif

within the target sequence with the position-specific
scoring matrix generated by MEME facilitating a hid-
den Markov model for the motif is calculated. This
match score is then compared to the match score of a
randomly generated string of amino acids generated
from the background letter frequencies (Table S2) of
the used sequence pool (Table S1). The P-value is esti-
mated as the fraction of random strings that have
match scores bigger or equal than the score of the
putative motif in the target sequence. The threshold to
select sequences containing a specific motif was set cor-
responding to a P-value of 10
)10
for highest certainty
(Table S1, bold) and 0.001 for low certainty for the
presence of the motif within a sequence (Table S1).
The latter value would correspond to an appearance of
a motif with a score better than the threshold once
every 1000 sequences randomly generated using the
same amino acid frequency as in the sequence pool.
According to these limits we conclude that all identi-
fied motifs are restricted to the polypeptide transport-
ers, as they cannot be found in the solute transporting
Domains of the Omp85 like proteins S. Moslavac et al.
1368 FEBS Journal 272 (2005) 1367–1378 ª 2005 FEBS
FepA family. Furthermore, pair-wise-motif correlation
[40] revealed no significant overlap between the identi-
fied motifs as the correlation value was in the range of
0.16–0.28 and therefore smaller than the threshold of
0.6 suggested by MEME. The first and the second

motif is a polypeptide stretch of 150 amino acids.
Motif 1 comprises a sequence where the amino acids
of 58% of the sequence are similar and 37% of all
amino acids are identical in all candidates analyzed
(Fig. 1B). In motif 2, slightly more amino acids are
defined in their features (60%) but fewer amino acids
are identical (33%, Fig. 1B). Motif 1 is present in the
C-terminal region, whereas motif 2 was identified in
the N-terminal portion of the proteins. In contrast
to motif 3 or motif 4, motifs 1 and 2 could only be
identified in few polypeptides (Fig. 1C, Table S1).
Interestingly, both motifs 1 and 2 were found almost
exclusively in proteins of the Oma87 homologues
(Table S1, Fig. 6), a protein class with so far unknown
function [41]. In our subsequent investigation we have
focused on motif 3 and motif 4, as these were charac-
teristic for the whole family.
Motifs 3 and 4 are present in almost all investigated
polypeptide transporters (Fig. 1C, Table S1). In total,
53 of the analyzed polypeptide transporters contain
motif 3 and 51 of the analyzed transporters motif 4
(Fig. 1C). The phylogenetic distribution of the motifs
is displayed in Fig. 6. In particular the polypeptide
transporters of the Oma87 family of higher organisms
do not contain the identified motifs 3 or 4. Motif 3
comprises 43 amino acids and motif 4, 30 amino acids
(Fig. 1A). In the identified motifs of the class 3, 79%
of all amino acids are similar and 44% of the amino
acids are identical (Fig. 1B). In contrast, the sequences
representing motif 4 are more diverse. We observed

that in certain positions (positions 1, 3, 4, 18, 20, 24,
25, 26 and 30) two defined but different amino acids
were placed (Fig. 1B). However, besides this splitting
between these two amino acids, the position is clearly
defined. Taking these amino acid positions into
account, we have 70% of all positions defined by a
class of amino acids and 23% defined by a specific
amino acid.
A
B
C
Fig. 1. Identification of four independent
motifs in the class of b-barrel polypeptide
transporters. (A) The motif sequence of the
candidate with lowest P-value is shown. For
motif 1 the region of Rno-Oma87 (Rattus
norvegicus), for motif 2 the region of Mmu-
Oma87 (Mus musculus), for motif 3 the reg-
ion of Npu2024 (Nostoc punctiforme) and
for motif 4 Gem-Oma87-II (Geobacter
metallireducens) are shown. (B) The result-
ing consensus motif is given. X stands for
any amino acids, bold for the specific amino
acid and normal letter for any amino acid of
this class. (C) The percentage of proteins
with identified motif (grey, left scale) and
the total number of identified motifs (black,
right scale) in a pool of 81 protein
sequences, including 10 proteins not
transporting polypeptides, is shown.

Table 1. Obtained log likelihood values (llr) and E-values for the
obtained motifs.
Motif number Length (aa) llr E-values
1 150 2557 8.9e-540
2 150 2517 7.9e-519
3 43 1764 2.1e-388
4 30 1310 3.3e-260
S. Moslavac et al. Domains of the Omp85 like proteins
FEBS Journal 272 (2005) 1367–1378 ª 2005 FEBS 1369
Analysis of the two C-terminal motifs 3 and 4
To gain insight into the function of the detected motifs
we analyzed the physicochemical parameter of the two
motifs. Strikingly, both motifs consist of two b-barrel
regions according to the exact b-sheet (EBS) score
(Fig. 2A,C). The EBS score is based on the amino acid
distribution in membrane segments of b-barrel proteins
[42]. These two transmembrane b-sheet regions can
also be seen by analysis of the alternating hydropho-
bicity profile (Fig. 2B,D). In here, the hydrophobicity
values of the amino acids according to the octanole
scale of White and Wimley [43] were used to calculate
the alternating hydrophobicity as a typical signature of
membrane-inserted b-sheets [44,45]. Additionally, all
motifs were analyzed by mcmbb, a program probing
for a b-barrel conformation [46]. Of all identified
sequences of the class 3, 49% were selected by the
program (Table S3). When only the sequences with
a P-value below e-10 were analyzed, 95% of all
sequences were selected by mcmbb to form a trans-
membrane b-sheet structure. Using the same procedure

for all sequences of motif 4 revealed a prediction rate
of 60% for all selected motifs and a rate of 79% for
all motifs with a P-value below e-10. This strongly
supports the notion that the detected motifs indeed
represent transmembrane regions. To further support
this statement, the topology of all sequences represent-
ing either motif 3 or motif 4 was analyzed using Pred-
TMBB [47,48]. Subsequently, the percentage of all
amino acids in sheet conformation for a specific posi-
tion within the motif was calculated either for all
sequences found (Fig. 2E, solid line) or for motifs
with a P-value below e-10 (Fig. 2E, dashed line).
Analyzing motif 3, two regions were identified, where
for most of the sequences a transmembrane b-sheet
was predicted (Fig. 2E, left). For motif 4, two regions
in such a conformation were also observed (Fig. 2E,
right); the second transmembrane segment was not
present as frequently as the first segment. This is in
line with the observation that the EBS score of this
transmembrane sheet is not as high as for the first
predicted sheet (Fig. 2C). Nevertheless, when the
sequences with a P-value below e-10 were analyzed,
more then 60% of all sequences contained a sheet in
this region (Fig. 2E, right). Therefore, comparing the
prediction based on the statistical analysis (Fig. 2A,C)
with that achieved by the hidden-Markov-model based
method (Fig. 2E) shows that the same regions of the
motifs were predicted to form a transmembrane
b-sheet structure (compare Fig. 2A,E, left; Fig. 2C,E,
right). However, for motif 4, the scores of the models

generated by Pred-TMBB are slightly shifted toward
the C-terminus. We can conclude that both motifs
represent structural units composed of the two
b-sheets, respectively.
Fig. 2. Physicochemical parameter of the two motifs. The EBS
score (A, C) and the alternating hydrophobicity (B, D) of motif 3 (A,
B) and motif 4 (C, D) were calculated according to [11]. The values
of the region including the 10 amino acids in front and behind the
motifs are shown. Black lines show the average of the analyzed
motifs and the grey line the weighted average (according to the
methods used). The motif length is shown on top. The proposed
loop regions are shown in black and membrane segments are
shown in grey. (E) Sequences representing motif 3 (left) or motif 4
(right) were used for topology prediction by
PRED-TMBB [46,47]. The
percentage of the prediction of a specific position to be in trans-
membrane b-sheet conformation for all sequences (grey solid line)
or for all sequences with a P-value below e-10 (black dashed line)
is shown.
Domains of the Omp85 like proteins S. Moslavac et al.
1370 FEBS Journal 272 (2005) 1367–1378 ª 2005 FEBS
We further analyzed the positioning of the two motifs
with regard to the amino acid sequence of the target
proteins. We first looked at the relative positioning (nor-
malized to the amino acid length of the protein) either
to the N-terminus or to the C-terminus. Here, no signifi-
cant cluster could be observed. Next, the absolute dis-
tance (in amino acids) of the start of the motif to both
termini was analyzed. Again no direct relation of the
positioning to the N-terminus could be observed. In

contrast, the spacing to the C-terminus of the proteins is
highly conserved (Fig. 3). We found a distance of the
starting amino acid of motif 3 from the C-terminus of
118 amino acids (Fig. 3A) and a distance of the starting
amino acid of motif 4 from the C-terminus of 40 amino
acids (Fig. 3B). Taking the length of motif 3 (43 amino
acids) this further implies an almost constant spacing
between the C-terminus of motif 3 and the N-terminus
of motif 4 of about 35 amino acids.
Taking this into account, we analyzed the existing
topological models of Nme-I-Omp85 [32], Bpe-FhaC
[26] and Ath-Toc75-V [11]. Aligning the region inclu-
ding the motifs 3 and 4 (Fig. 4; black box above
sequence, motif 3; black box below sequence, motif 4)
revealed that Nme-I-Omp85 has enlarged loop regions
(Fig. 4) in motif 3, which explains the high P-value for
this motif of 4.3e-6. However, earlier individually pro-
posed transmembrane segments (Fig. 4, grey frames
under the sequence) align very well with the exception
of one missing segment in Bpe-FhaC (first segment,
Fig. 4) and an additional segment in Ath-Toc75V
(fourth segment, Fig. 4). Furthermore, the proposed
transmembrane segments are in agreement with the
physicochemical parameter analysis (Fig. 2) for the
whole set of sequences representing the motifs 3 and 4.
The analysis of the exact b-barrel score [11,42] or of
the alternating hydrophobicity [45,49] revealed that in
motif 3 and 4 two transmembrane b-strand segments
exist (Fig. 2, grey boxes above). This is in agreement
with the analysis of the motif sequences facilitating

hidden-Markov-model based methods (Fig. 2E and
not shown). In addition, the previously identified
motifs by Eckart et al. [50] (Fig. 4, dashed boxes) sub-
sequently confirmed by Voulhoux [32] (Fig. 4, open
box), and Gentle [19] (Fig. 4, grey box), are in this
region and cover most of the transmembrane segments
(Fig. 4). We therefore conclude that in contrast to the
previously identified POTRA motif [51], which was
postulated to represent a polypeptide-binding motif,
and to the motifs 1 and 2, which are specific for the
Oma87 family, the motifs 3 and 4 are related to the
general pore-forming region. It might therefore be that
the regulation of the translocation of polypeptides
through the channel is rather conserved and defined by
these two identified domains and specificity gained by
the accompanied N-terminal region of the protein.
The identification of new Toc75-related proteins
Using the proposed motifs 3 and 4 we have searched for
proteins belonging to this family in Arabidopsis thaliana.
On the base of this search we identified two putative
members of this family, namely Ath-P1 and Ath-P2
(At3g44160, At3g48620). The mRNA encoding the pro-
teins was detectable in roots, flowers and flower stalks
(Fig. 5A, lanes 4,2 and 3). The mRNA level of Ath-P1
in flowers was comparable to that in flower stalks,
whereas the mRNA level of Ath-P2 was slightly lower
(Fig. 5A, lanes 2 and 3). For both proteins, almost no
mRNA was detectable in leaves. This result was con-
firmed by Affymetrix gene ship analysis [52]. Here, how-
ever, only the gene expression of both genes together

A
B
Fig. 3. Absolute positioning of the identified motif in regard to the
C-terminus of the investigated sequences. (A) The positioning of
the starting amino acid of motif 3 in relation to the C-terminus and
(B) the positioning of the starting amino acid of motif 4 in relation
to the C-terminus was analyzed. The number of identified distances
is shown as bars and analyzed by Gaussian distribution shown as
line. The inset shows an enlargement of the peak region.
S. Moslavac et al. Domains of the Omp85 like proteins
FEBS Journal 272 (2005) 1367–1378 ª 2005 FEBS 1371
could be analyzed, as both genes are annotated to the
same spot of the ATH1 genome ship. However, a 10
times lower expression of both genes in combination
was observed in leaves when compared to Ath-Toc75-III
(Fig. 5B). In addition, the diurnal expression of Ath-P1
and Ath-P2 did not differ drastically from that of Ath-
Toc75-III further suggesting a tissue-dependent differen-
tial expression rather than a differential expression
during the daily cycle. This might suggest a differenti-
ated function of the two proteins in comparison to
Ath-Toc75-III and Ath-Toc75-V (Fig. 5). Both proteins
Ath-P1 and Ath-P2 are smaller in size (47 and 36 kDa,
respectively). Sequence comparison of Ath-P1 and Ath-
P2 with Ath-Toc75-III and Ath-Toc75-V revealed that
both proteins lack the N-terminal domain, which was
proposed to form long soluble loops [11] and to contain
the POTRA motif [51]. The phylogenetic tree (Fig. 6)
indicates a close relationship between Ath-P1 and
Ath-Toc75-V, which constitute a cluster with 96%

support value, whereas the phylogenetic affiliation of
Ath-P2 remains unresolved. It has to be investigated
whether these proteins Ath-P1 and Ath-P2 assemble
polypeptide transporters and, if so, how recognition of
the polypeptides is achieved.
Analysis of the evolutionary relation of the b-barrel
proteins b-barrel proteins present in eukaryotes have
most likely evolved from the proteins of the outer
membrane of Gram-negative bacteria. However,
recently the relation between certain proteins found in
mitochondria, namely Sam50 ⁄ Tob55, or chloroplasts,
namely Toc75, with the proteins of the Omp85
class was discussed [17–19,32]. Interestingly, in
Sam50 ⁄ Tob55 only the motif 1 was identified with
high probability (Table S1) suggesting a relation to the
protein class Oma87 but not to Omp85. This relation
is further substantiated by the phylogenetic tree
(Fig. 6). The five Oma87 sequences from Metazoa con-
stitute a sister group to Ncr-Tob55 and Sce-Sam50,
which receives high support. Therefore it might be spe-
culated that these Oma87 proteins actually assemble
the homologues of the mitochondrial polypeptide
transporter.
Fig. 4. Motif positioning in the models of Toc75-V, Omp85 and FhaC. Shown is the amino acid sequence of Ath-Toc75-V starting at amino
acid 561, Nme-I-Omp85 starting at amino acid 643 and Bpe-FhaC starting at amino acid 463. Grey highlighted sequence regions indicate
the proposed transmembrane segments according to the individual proposed model. The black boxes above and below the alignment show
the position of the identified motif 3 (top) and motif 4 (bottom). The motifs identified by Eckart et al. [53] are shown with dashed boxes, the
motifs subsequently identified by Voulhoux et al. [30] or Gentle et al. [19] are shown as open boxes or grey boxes, respectively.
A
B

C
Fig. 5. mRNA expression of different members of the Toc75 family. (A) The mRNA level for Ath-P2, Ath-P1, Ath-Toc75-V and Ath-Toc75-III
was analyzed using mRNA isolated from leaves (L, lane 1), flowers (F, lane 2), flower stalks (FS, lane 3) and roots (R, lane 4). To confirm the
loading of mRNA the amount of 18S RNA and the RT-PCR efficiency for actin (not shown) was probed. (B) The gene expression of Toc75-III
and P1 & P2 in combination in leaves was determined by Affymetrix expression analysis. Signals were normalized as described previously
[49]. (C) The diurnal expression of Toc75-III (solid line) and P1 & P2 in combination of complete plants is shown. The black bar on top indi-
cates the dark cycle and the white bar indicates the light cycle of plant growth.
Domains of the Omp85 like proteins S. Moslavac et al.
1372 FEBS Journal 272 (2005) 1367–1378 ª 2005 FEBS
However, one should note that the Oma87 sequences
from bacteria (Eco, Gme, Hso, Nar, Pmu, Rru, Vvu)
are not clustered in the tree. The proteins of the Toc75
class revealed a high homology to the consensus of
motif 3 and 4 as found for the members of the Omp85
class. However, the Toc75-class is not a homogeneous
group from a phylogenetic perspective, and the phylo-
genetic relationship to other protein classes remains
Fig. 6. Phylogenetic analysis of the 71 sequences. Consensus tree as estimated from 25 000 intermediate trees. The numbers on the bran-
ches indicate the quartet puzzling support. Species name and sequence name abbreviations are shown in Table S1.
S. Moslavac et al. Domains of the Omp85 like proteins
FEBS Journal 272 (2005) 1367–1378 ª 2005 FEBS 1373
unclear. Almost all proteins assigned to the Toc75-
class containing the sequence motifs 3 and 4 are in a
weakly supported group (58%). Within this group four
supported subgroups (support values larger than 80%)
are discernable, the largest one is concentrated around
Ath-Toc-III, the main protein translocation channel in
A. thaliana, and consists of representatives from higher
plants (Ath, Osa, Psa). The second grouping comprises
of a collection of proteins from Nostoc sp., the third

again consists of proteins from cyanobacteria (Pmar,
Ssp). As already mentioned, the final subgroup, with
highest support value clusters the newly identified
protein Ath-P1, which only contains motif 3, with
Ath-Toc75-V, whereas P2 does not belong to this
phylogenetic family even though it contains both iden-
tified motifs.
The polypeptide transporters with adhesin selectivity
(ShlB and FhaC) are evolutionary closely related with
the exception of ShlB from Bordetella pertussis (Bpe-
ShlB), Haemophilus influencae (Hin-HuxB) and Xantho-
monas axonopodis (Xax-ShlB). These three proteins
together with Omp85 from Geobacter sulfurreducens
(Gsu-Omp85) form a separated branch with almost no
support. The proteins assigned to the Omp85 family,
however, do not form a single phylogenetic group.
This might reflect that many of those proteins were
annotated just based on low sequence homologies
without any functional information.
Discussion
Recently, a new class of membrane proteins was
defined according to their function as polypeptide
transporters. Besides the members of the family of
accessory outer membrane proteins involved in secre-
tion of haemolysins or adhesins in various Gram-
negative pathogens, proteins of the endosymbiotic
organelles belong to the increasing list of such proteins
[53]. On top of the functional relation of these pro-
teins, the initiated sequence analysis of the b-barrel-
type proteins involved in polypeptide transport

revealed a possible structural relation of the proteins.
First, a cluster of short motifs in the C-proximal
region of such proteins was identified [50]. This cluster
was partly confirmed to be present in the Omp85 fam-
ily [19,32]. Comparison with these earlier motif predic-
tions revealed that motif 3 was partly identified by
Voulhoux [32] (Fig. S1B). However, in the previous
work the motif was limited to 20 amino acids. Motif 4
shows a significant overlap with the motif identified by
Gentle et al. [19]. Interestingly, in line with the motif
prediction by Eckart et al. [50] analyzing Toc75 homo-
logues, the work of Gentle [19] (Fig. S1B) suggests a
prolongation of the motif towards the N-terminus.
Motifs 3 and 4 are found with highest reliability in the
proteins homologous to Toc75 proteins (Table S1),
however, incorporation of the other polypeptide trans-
porters revealed that the conserved motif for the entire
family is not as long as when only Toc75 proteins were
analyzed (not shown). Both motifs are present in a
defined distance from the C-terminus of the proteins
(Fig. 3). This underlines that during evolution these
specific pore-forming regions remained conserved
(Fig. 6). In addition, the motifs do not only reflect
similar sequence features, but also structural features
as determined by their physicochemical parameters
(Fig. 2). Both motifs define two transmembrane
b-sheets. The conservation of transmembrane sheets
suggests a similar gating and translocation behaviour
of the member of this family. Therefore, the N-
terminal region would be essential for the fine tuning

of the specific function of the individual protein.
This hypothesis has to be confirmed in future. Further-
more, guided by this notion it should be suggested that
the proposed topological models of FhaC [26] and
Omp85 [32] should contain an additional transmem-
brane domain (Fig. S2).
Furthermore, we identified one motif with N-ter-
minal proximity. Such a region localized in the N-ter-
minus was previously identified [51] and termed
POTRA for polypeptide transport-associated domain.
Voulhoux and coworker [32] also identified a similar
but shorter consensus sequence. It was proposed that
this region might have a function in polypeptide trans-
port. However, the herein identified motif 2 does not
represent the identified POTRA motif, but shows a
certain overlap with the C-terminal portion of this
earlier proposed consensus sequence (Fig. S1). The two
motifs 1 and 2 are limited to a certain subset of pro-
teins of the Oma87 family. Remarkably, the same
motif 1 is found in the Sam50 or Tob55 protein. This
observation and the evolutionary relation of Sam50
and Tob55 to certain members of the Oma87 family
(Fig. 6) together suggest a functional relation of the
identified group. However, the phylogenetic tree also
underlines that the given nomenclature for Oma87
or Omp85 proteins requires further investigation to
understand their functional relations. Based on the
large distance of this family to the iron transporter
FepA (Fig. 6), we came to the conclusion that the class
of polypeptide-transporting proteins must have evolved

from a common branch during evolution.
Taking our proposal and those of others, the poly-
peptide transporters can be identified by at least three
signature sequences, namely POTRA, motif 3 and
motif 4, which are not present in proteins transporting
Domains of the Omp85 like proteins S. Moslavac et al.
1374 FEBS Journal 272 (2005) 1367–1378 ª 2005 FEBS
solutes, like the proteins of the FepA class. The
POTRA motif, however, is less well defined as we
could not identify it using a larger pool of sequences.
Based on motifs 3 and 4 two new members of the
family were identified in Arabidopsis thaliana (Fig. 5),
whose function has to be explored in future.
Experimental procedures
Sequence selection and motif detection
The amino acid sequences of the proteins listed in Table S1
were identified by homology searches at http://www.
ncbi.nlm.nih.gov/BLAST/ [54] taking one member of the
protein class described. Sequences were controlled for
redundancy and further analyzed by the ‘multiple EM for
motif elicitation’ (MEME) program at http://meme.
sdsc.edu/meme/website/meme.html ([40] and references
therein). The sequences were analyzed for the presence of
motifs using the following parameter: any number of repeti-
tion; maximum of motifs find to 5; minimum length was set
to 20 amino acids and maximum length to 200 amino acids.
The selection procedure to identify motifs within MEME is
based on the statistical significance of the log likelihood
ratio (Table 1) of the occurrence of the motif within the
user defined range. The E-value of a single motif is an esti-

mate of the number of motifs (Table 1) that would have an
equal or higher log likelihood ratio, if the sequences had
been generated randomly according to the 0-order back-
ground hidden Markov model consisting of the frequencies
of the letters in the training set (Table S2).
Analysis of the physicochemical parameter
The distance of the starting amino acid of the motifs to the
different termini was calculated using the individual amino
acid length of each sequence. The distribution was analyzed
by a Gaussian function by least square fit analysis using
the incorporated tool of sigma plot. The EBS score and
the alternating hydrophobicity were calculated for each
identified motif individually as described [11]. The average
was calculated using sigma plot. For the weighted average,
the results were normalized according to their significance
by calculating the significance (S) as follows: S ¼ ) log(p).
The b-sheet topology of the motifs was predicted using the
following programs: proftmb ([55] accessible at the web
address />mcmbb [46] at />pred-tmbb [47,48] at />PRED-TMBB/. The output of the last program was ana-
lyzed by calculating the percentage of predicted transmem-
brane b-sheet formation for each amino acid position in the
motif of either all selected motifs (Table S1) or all motifs
with a P-value below e-10 (Table S1, bold numbers).
RT-PCR analysis
Arabidopsis thaliana (ecotype Columbia) were grown on
plates suggested by Murashige and Skoog [56]. Plant
growth was achieved in a 16 h light at 21 °C and 8 h dark
at 16 ° C cycle. Seedlings were transferred to soil after
18 days and growth was continued under the same condi-
tions. Different body parts (as indicated) were collected

from 55-day-old flowering plants [51]. Total RNA from the
individual plant material was isolated using RNeasy Plant
Mini Kit (Qiagen, Hilden, Germany) according to the pro-
tocol recommended. RT-PCR reactions were performed
using 20 ng RNA and the SuperScript One Step RT-PCR
Kit with Platinum Taq (Invitrogen, Karlsruhe, Germany)
as described by the manufacturer. The reverse transcription
reaction was performed for 30 min at 45 °C followed by 40
PCR cycles.
Gene chip analysis
RNA extraction and gene chip analysis using Affymetrix
ATH1 Arabidopsis genome chip (Affymetrix, High Wyco-
mbe, UK) was performed as described [51]. The data
for the diurnal gene expression were downloaded from
(o/narrays/experimentpage.
pl?experimentid ¼ 60).
Alignments and phylogenetic analysis
The alignments were performed using clustalw at the
BCM Search launcher ( />multialign/multialign.html [57]). The alignment of 71
sequences of the proteins according to Table S1 comprises
1549 aligned positions. After visual inspection and deletion
of sites with too many gaps 432 sites were used for a phylo-
genetic analysis (see supplementary material). The align-
ment was further modified for presentation using the
boxshade program ( />BOX_form.html).
The tree-puzzle program [58] was applied in its parallel
version 5.2 [59] to infer a maximum likelihood based phylo-
genetic gene tree (). We used the
JTT model with constant rates across sites. Twenty-five
thousand puzzling steps were applied and the resulting con-

sensus tree was used for the analysis.
Acknowledgements
We are grateful to Dr S. Smith (University of Edin-
burgh, UK) for his permission to use his Affymetrix
data for the diurnal cycle and to A. Vojta for permis-
sion to his Affymetrix data. We are grateful to Dr H.
A. Schmidt (FZ-Ju
¨
lich) for help with the computation
on the JUMP supercomputer at the ZAM ⁄ NIC of the
S. Moslavac et al. Domains of the Omp85 like proteins
FEBS Journal 272 (2005) 1367–1378 ª 2005 FEBS 1375
Research Center Ju
¨
lich. This work was supported by
grants to E.S. from the Deutsche Forschungsgemeinsc-
haft (SFB-TR01), Fonds der Chemischen Industrie
and Volkswagenstiftung and to A.v.H. from the Deut-
sche Forschungsgemeinschaft (SFB-TR01).
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Supplementary material
The following material is available from http://www.
blackwellpublishing.com/products/journals/suppmat/E JB/
EJB4569/EJB4569sm.htm
Appendix 1 contains the following material:
Fig. S1. Alignment of the motifs with previously pub-
lished regions.
Fig. S2. Comparison of the structural models of
Sam50, FhaC, Omp85 and Toc75-V.
Table S1. List of protein sequences analysed.
Table S2. Letter frequency in the dataset.
Table S3. Prediction by MCMBB.
Domains of the Omp85 like proteins S. Moslavac et al.
1378 FEBS Journal 272 (2005) 1367–1378 ª 2005 FEBS