A Draft of the Human Septin Interactome
Marcel Nakahira1,2., Joci Neuby Alves Macedo3., Thiago Vargas Seraphim2, Nayara Cavalcante3,
Tatiana A. C. B. Souza1, Julio Cesar Pissuti Damalio3, Luis Fernando Reyes3, Eliana M. Assmann1,
Marcos R. Alborghetti1,2, Richard C. Garratt3, Ana Paula U. Araujo3, Nilson I. T. Zanchin4, João A. R. G.
Barbosa5, Jörg Kobarg1,2*
1 Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, Brasil, 2 Departamento de Bioquı́mica-Programa de Pós-graduação
em Biologia Funcional e Molecular, Universidade Estadual de Campinas, Campinas, Brasil, 3 Centro de Biotecnologia Molecular Estrutural, Universidade de São Paulo, São
Carlos, Brasil, 4 Centro de Biologia Molecular e Engenharia Genética e Faculdade de Ciências Aplicadas, Universidade Estadual de Campinas, Campinas, Brasil, 5 PósGraduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brası́lia, Brası́lia, Brasil
Abstract
Background: Septins belong to the GTPase superclass of proteins and have been functionally implicated in cytokinesis and
the maintenance of cellular morphology. They are found in all eukaryotes, except in plants. In mammals, 14 septins have
been described that can be divided into four groups. It has been shown that mammalian septins can engage in homo- and
heterooligomeric assemblies, in the form of filaments, which have as a basic unit a hetero-trimeric core. In addition, it has
been speculated that the septin filaments may serve as scaffolds for the recruitment of additional proteins.
Methodology/Principal Findings: Here, we performed yeast two-hybrid screens with human septins 1–10, which include
representatives of all four septin groups. Among the interactors detected, we found predominantly other septins,
confirming the tendency of septins to engage in the formation of homo- and heteropolymeric filaments.
Conclusions/Significance: If we take as reference the reported arrangement of the septins 2, 6 and 7 within the
heterofilament, (7-6-2-2-6-7), we note that the majority of the observed interactions respect the ‘‘group rule’’, i.e. members
of the same group (e.g. 6, 8, 10 and 11) can replace each other in the specific position along the heterofilament. Septins of
the SEPT6 group preferentially interacted with septins of the SEPT2 group (p,0.001), SEPT3 group (p,0.001) and SEPT7
group (p,0.001). SEPT2 type septins preferentially interacted with septins of the SEPT6 group (p,0.001) aside from being
the only septin group which interacted with members of its own group. Finally, septins of the SEPT3 group interacted
preferentially with septins of the SEPT7 group (p,0.001). Furthermore, we found non-septin interactors which can be
functionally attributed to a variety of different cellular activities, including: ubiquitin/sumoylation cycles, microtubular
transport and motor activities, cell division and the cell cycle, cell motility, protein phosphorylation/signaling, endocytosis,
and apoptosis.
Citation: Nakahira M, Macedo JNA, Seraphim TV, Cavalcante N, Souza TACB, et al. (2010) A Draft of the Human Septin Interactome. PLoS ONE 5(11): e13799.
doi:10.1371/journal.pone.0013799
Editor: Gustavo Goldman, Universidade de São Paulo, Brazil
Received July 6, 2010; Accepted October 13, 2010; Published November 2, 2010
Copyright: ß 2010 Nakahira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Fundação de Amparo a Pesquisa do Estado São Paulo (FAPESP), CAPES: Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior, Conselho
Nacional de Pesquisa e Desenvolvimento (CNPq) and the Laboratório Nacional de Biociências-Centro Nacional de Pesquisa em Energia e Materais (LNBio-CNPEM).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail:
[email protected]
. These authors contributed equally to this work.
nomenclature in which a variant of SEPT6, for example, would
be indicated as SEPT6_v1. SEPT9 is particularly variable in this
respect [5].
Mammalian septins have been classified into 4 different groups,
based on their amino acid sequences. At present several different
nomenclatures exist [2,6–8]. The simplest of these makes use of a
reference septin for each group. Thus, the SEPT2 group (also
called group 2B [2] or group III [7]) contains SEPT1, SEPT4 and
SEPT5 as well as SEPT2. Similarly, the SETP3 group (alternatively called group 1A or group I) consists of SEPT3, SEPT9 and
SEPT12 and, the SEPT6 group (also called group 1B or group II)
contains: SEPT6, SEPT8, SEPT10, SEPT11 and SEPT14. The
SEPT7 group, which includes in addition to SEPT7 also SEPT13
(group IV), is normally considered to form the fourth independent
group. However, the latter two septins have sometimes been
Introduction
Septins belong to the GTPase superclass of P-loop NTPases.
Specifically, they belong to the TRAFAC class which includes the
Ras-like superfamily, Myosin-kinesin superfamily, and translation
factor superfamily [1]. They are found in all eukaryotes, from
yeast to mammals, except in higher plants [2] but the number of
septin genes found in different species varies considerably. For
example Saccharomyces cerevisiae, has seven septin genes (Cdc3,
Cdc10. Cdc11. Cdc12, Shs1, Spr28, Spr3), whilst Caenorhabditis
elegans has only two (Unc59 and Unc61), Drosophila melanogaster five
(Pnut, Sep1, Sep2, Sep4. Sep5), and Mus musculus thirteen (Sept1Sept9, Sept10a, Sept10b, Sept11, Sept12) [2–4]. In humans, so
far 14 septin genes have been reported. However, many of them
present several splice variants, which are designated by a
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to identify septin interactors using the yeast two-hybrid system have
been made to date for the individual septins 5, 8, 9 and 14, so far no
large scale analysis has been attempted [7,25,33–35].
Therefore we set out to perform yeast-two hybrid screens of the
human septins 1–10, representing all 4 septin groups. In summary,
we found that all septins, except SEPT10, interacted predominantly
with other septins, principally those from other groups. The only
exception came from the members of the SEPT2 group, which also
interacted with partners from the same group. Most, although not
all, of the results were confirmative in a reciprocal sense. For
example, SEPT3 when used as a bait molecule identified SEPT6 as
a partner and when SEPT6 was used as the bait, SEPT3 was
identified. Most interestingly, the results on the whole seemed to
conform to the proposed trimeric arrangement in a group format.
This means that if we organize the two trimers in the following
arrangement: Group 3/7-6-2 - 2-6-3/7, we can assign the great
majority of the individually found interactions and all pair wise
group interactions in this model are covered by statistical data which
demonstrate that the experimental observed distribution of the
fished septin clones is not random. If this interpretation of the trimer
arrangement and the ‘‘group rule’’ holds true through additional
biochemical experiments we can propose a range of new possible
septin trimers, whose existence in cells should be tested.
All septins also interacted with other non-septin proteins. Those
common to several septins or septin groups can be largely attributed
to the ubiquitin and sumoylation cycles, transport and motor
activity, cell division/cell cycle, and protein phosphorylation. Novel
individual septin or septin family specific functions include: apoptosis
(SEPT3 and SEPT6), transcription (SEPT8 and SEPT7) and DNA
repair and splicing (SEPT3), among others. Our data shed new light
on septin function and provide a wealth of new information to form
new hypotheses, especially with respect to septin trimer and filament
formation and the association of septins within new cellular contexts.
Future biochemical, structural and cellular functional studies are
required to test the newly proposed hypotheses.
considered to form a sub-group of the SEPT2 group (part of group
2B) with which they are closely related phylogenetically [2–4].
Septins have molecular masses that vary between 30 and
65 kDa and present a high sequence identity in their conserved
central GTPase domains, featuring typical Ras like GTPase motifs
and P-loop signatures found in most GTPases [2,6,9]. This central
GTP-binding domain generally also contains a short polybasic
region prior to the P-loop (or Walker A box). This has been shown
to bind to phospholipids and may be responsible for mediating
interactions with membranes [9]. The central domain is flanked by
an amino-terminal domain which is variable in both sequence
length and identity and a carboxy-terminal domain which also
varies considerably from one septin to another, but frequently
contains a coiled-coil region, possibly involved in mediating
protein-protein interactions. In the case of mammalian septins,
only members of the SEPT3 group lack the coiled-coil signature.
One of the most remarkable features of septins is their ability to
engage in the formation of homo- and hetero-meric filaments,
which seem to be important for mediating their cellular functions.
Aside the expected and demonstrated roles of these filaments in
intracellular transport processes and cellular movements, especially in the context of cell division, the septin filaments have been
predicted to serve also as scaffolds for the docking of other
regulatory or signaling proteins [10].
Several studies have now shown that the typical filamentous form
of mammalian septins appears to systematically involve a
heterotrimer as its core module [10–12]. The complexes described
so far vary, however, in size and composition. Biochemical studies
reported to date suggest the existence of several different trimeric
complexes including SEPT4-SEPT5-SEPT8 [7], SEPT7-SEPT11SEPT9b [13], SEPT5-SEPT7-SEPT11 [14] and SEPT3-SEPT5SEPT7 [15,16]. The most well studied of all is that of human septins
2, 6 and 7 [17] which remains the only septin complex to have been
solved crystallographically to date. Its structure, at 4.0 Å was
reported together with that of a fragment of human SEPT2 which
lacks 46 residues of the predicted coiled-coil region at the Cterminus [18]. These structures showed that the GTPase domain is
responsible for polymerization and septin-septin interactions at the
so called G and NC interfaces result in the assembly of linear nonpolar polymers. More recently, the crystal structure of the GTP
bound form of SEPT2 was reported at 2.9 Å resolution, revealing
that GTP binding induces a conformational change in the switch
regions directly affecting the G interface and indirectly, the NC
interface [19]. Moreover, GTP binding/hydrolysis and the nature
of the bound nucleotide influence the stability of the interface in the
heterooligomeric and polymeric state, as well as filament assembly
and disassembly.
Septins have been shown to be functionally involved in a diversity
of processes ranging from cytokinesis [12,20], cell membrane
dynamics [21,22], signal transduction cascades, cellular signaling
events [6,23], cell cycle control and others [1,24]. Moreover, septins
have been shown to regulate bacteria-host interactions [25] and to
be important determinants for yeast virulence [26–28]. Finally,
septin dysfunction has been associated with several human
pathologies, including cancer [29], Parkinson’s disease [30],
Alzheimer’s disease [31] and hereditary neuralgic amyotrophy [32].
Despite all the documented progress, the exact molecular
mechanisms of the septins, and their cellular and physiological
functions are still poorly understood. As a first step in approaching
molecular and cellular functions a comprehensive description of the
interacting protein partners for the septins would be very valuable.
This is especially important if we consider the possible role of the
different septin filaments as docking or scaffolding platforms to
mediate additional new functions. Although some isolated attempts
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Results and Discussion
The interactions of septins with other septins
Analysing the overall result of our initiative to characterize
septin interacting proteins on a broad scale, one first is impressed
by the large number of septin prey proteins that were discovered
for all septin baits, with the sole exception of SEPT10. Although
the latter did not interact with any other septin when used as bait,
it was however selected in reasonable quantity by both SEPT4
(23% of the interacting septins) and to a lesser extent by SEPT7
(2% of the interacting septins). The complete data is given in
resumed form in Table 1, Figure 1 and Figure S1. Please refer to
Table S1 for complete primary data.
Members of the SEPT6 group, with the exception of SEPT10,
had a clear tendency to interact with other septins instead of with
other non-septin proteins (Fig. 1). For example, SEPT6 had only
12% non-septin interactors and SEPT8 only 24%. A similar result
is observed for SEPT7, for which only 9% of the identified
interactors were non-septins. Several other septins, including
SEPT2, SEPT4 and SEPT3, showed the opposite tendency and
tended to interact with a relatively large number of non-septin
partners. SEPT2 interacted with 54%, SEPT4 with 48% and
SEPT3 with an impressive 80% of non-septin preys.
Interestingly, several septins showed a marked preference for a
particular septin as partner from among those identified. SEPT2
predominantely interacted with SEPT6 (38%), SEPT9/SEPT9(1269) mostly with SEPT6 (39%) and SEPT6, SEPT8 and SEPT7
all interacted predominantly with SEPT9 (62%, 40% and 61%,
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Human Septin Interactome
further taking into consideration Kinoshitas prediction [6] that in
the trimer format septins from within a given group may
substitutes for one another (Figs. 2 and 3).
Analysis of the results for the SEPT6 group members as baits,
given the known arrangement of the trimer, reveals interesting
new insights. SEPT6 occupies the central position in the trimer
structure and Kinoshita’s prediction suggests that other members
of the group, namely SEPT8, SEPT10, SEPT11 and SEPT14
should be competent substitutes at this position of the filament.
Hence, the SEPT6 group members could be predicted to be
sandwiched between SEPT2 and SEPT7 group members. Based
on this assumption it would be expected to find them largely
interacting with septins from the latter two groups but never with
members of their own. The number of clones corresponding to
an interaction between SEPT6 group members and SEPT2
group members is statistically significant (p,0.001) as is that
between SEPT6 and SEPT3 group members and between
SEPT6 and SEPT7 group members. [Fig. 3, (a)] (Please refer to
Materials and Methods section for details of the statistical
analysis).
The only exception is SEPT10, which seems to behave
differently altogether, since, when used as bait, we did not identify
any septin partners at all. However, consistent with the above
scenario, SEPT10 was identified as a binding partner of both
SEPT4 (a SEPT2 group member) and SEPT7 in the reverse
experiments. In summary these findings are entirely in accordance
with what would be expected from the trimer model and allow us
to propose new possible trimer configurations, which can be
summarized as 7-8-1/2/4/5 or 7-6-1/4/5. Although we are
unaware of any direct experimental evidence reporting these
particular trimeric arrangements it is worth mentioning that a
complex of SEPT5, SEPT7 and SEPT11 (a very close relative of
SEPT6) has been reported recently [14]. The physiological
relevance of such potential complexes clearly depends on other
factors such as the tissue specific expression of different septins and
their splice variants [36].
Notably, of all the 10 septins analyzed, SEPT6 followed by
SEPT8 showed both the largest number of interacting clones as
well as the greatest number of different identified candidate
interactors. Since the majority of clones found to interact
represented other septins (88% and 76% for SEPT 6 and 8,
respectively), this may reflect the fact that the members of this
group always occupy the central position of the filament’s trimeric
unit. In co-purification studies we were able to demonstrate that
GST-SEPT6 co-purified with septins 1, 2, 3, 5, 7 and 9, when both
are expressed together in E. coli, thereby confirming the majority
of the interactions given in Table 1 (Nakahira et al., unpublished
observations). It has been recently shown that septins of this group
(SEPT 6, 8 and 11) show a lower rate of GTP hydrolysis when
compared to SEPT2 (Souza et al., unpublished observation). Most
interestingly, septins of this group have the key Ser residue in the
G1 motif substituted by a Thr residue, possibly indicating why the
septins of this group have a less efficient rate of hydrolysis. This
speculation has some support in the finding that in the crystal
structure of the 7-6-2 hetero-trimer GDP is observed bound to
SEPT2 and SEPT7 but GTP to SEPT6 [18].
A very interesting result was obtained when the SEPT2 group
members were used as bait [Fig. 2, Fig. 3(b)]. In this case the
majority of preys belong to the SEPT6 group (46, 38, 52 and 65%
respectively, for baits SEPT1, SEPT2, SEPT4 and SEPT5). This is
consistent with what is expected from the canonical 7-6-2 trimer,
where SEPT2 makes direct contact with SEPT6. Again the
preference of SEPT2 group members for partners from SEPT6
group is statistically significant (p,0.001, Fig. 3b).
Table 1. Correlation of septin prey clones (columns) fished by
a given bait septin (lines).
Prey:
Bait:
1
2
4
5
6
1
3
2
1
1
14
2
1
1
10
11
3
3
9
7
15
5
4
5
8
1
6
8
5
5
12
8
3
6
9
5
2
1
8
6
3
7
3
1
96
9
49
22
10
11
3
6
9
5
7
1
3
11
5
3
1
1
34
The numbers are the number of identified clones. Bold and underlined numbers
emphasizes those preys that also fished the corresponding prey when they
were used as bait (reciprocal fishing). The baits and prey are listed regarding to
membership in the four septin groups: 1,2,4,5 (group 2); 6,8,10,11 (group 6), 3,9
(group 3) and 7 (‘‘group 7’’).
doi:10.1371/journal.pone.0013799.t001
respectively). For the whole table of groups of baits vs. groups of
prey septins we obtained with Fischers exact test a p value of
0.0005, diagnosed as significant (Table S1). In other words, the
data are far from being randomly distributed. This can also be
seen directly from Fig. 2, where islands and clusters can be easily
observed in between many blank regions.
Although there was not always a strict reciprocity in the sense that
each bait which picked up a given septin prey was also found as a
prey when the interacting septin was used as bait, several interesting
tendencies can nevertheless, be detected. In Table 1 the number of
fished septin clones is listed in accordance with their group
membership which aids in emphasizing a certain clustering of
blocks of interactions which can be readily observed (clone numbers
in bold and underlined). Block 1 corresponds to the use of SEPT6
and SEPT8 as bait. Both these septins picked up predominantly
septins of the SEPT2 group (Block1a) and members of group 3/9
and 7 (Block1b), but never septins of the same group (SEPT6). Block
2 refers to the SEPT2 group when used as bait, which preferentially
picked up septins of the SEPT6 group (Block2a). Most interestingly,
for both of these blocks there is also a considerable degree of
reciprocity (bold underlined numbers in Table 1), which lends an
additional degree of confidence to the results. For example bait
SEPT6 fishes prey SEPT3/9/7 and in reverse bait septin 3/9/7
fished prey SEPT 6. A third block also corresponds to the use of the
SEPT2 group septins as bait and which had a weak but consistent
tendency to pick up preys of the same group: SEPT1 interacted with
SEPT1/2/4/5; SEPT2 with SEPT4, and SEPT5 with SEPT2/5.
The latter is the only example of self interactions among members of
the same group, detected during this study. These intra-group
interactions observed for the SEPT2 members are in stark contrast
to what is observed for the SEPT6 group members and are probably
pertinent to filament formation as described below.
Comparison of the septin-septin interactions within the
format of the septin trimer
The interaction results described in the previous paragraph gain
an interesting new perspective when analyzed in the light of the
structural data obtained for the septin trimer 7-6-2 [18] and
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Figure 1. Summary of protein-protein interactions found for human septins 1-10. The values are given as a percentage of clones from the
total number of confirmed interacting clones sequenced and identified. See color code for septin specification. Septins are grouped according to the
four groups SEPT2, SEPT3, SEPT6 and SEPT7 from left to right.
doi:10.1371/journal.pone.0013799.g001
A small but consistent fraction of the preys belonged to the
SEPT2 group were found as partners of septins of the same group
(8%, for each of SEPT1, SEPT2 and SEPT5, when used as bait).
This makes SEPT2 group members an exception as the only
septins which interacted with members of their own group and
may reflect the fact that the first stage of polymerization appears to
be the formation of hexamers (or dimers of trimers) in which one
copy of SEPT2 makes contact with a second via what has been
called an NC interface [18] (Fig. 4A). Furthermore, SEPT2 when
expressed and purified as a dimer, although curiously this appears
to use the G interface rather than NC [19]. Together, this further
suggests that SEPT1, 4, and 5 can substitute SEPT2 in the trimer
format and reinforces the proposed 7-6-1/4/5 combinations
described above. It is interesting to note that none of the SEPT2
group septins picked up SEPT7, as would be also expected, given
that there is no direct contact made between them in the trimer.
However, during the reverse experiment SEPT7 identified a small
number (7%) of SEPT2 group clones. These may correspond to
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real hits corresponding to hetero-polymers of different arrangement or may be artifacts. It is known for example that SEPT2
alone is able to form continuous polymers similar to those seen in
the 7-6-2 complex, in which it makes use of both G and NC
interfaces. The G interface may be promiscuous in that it is not
observed in the hetero-polymer and similar promiscuity may be
observed with other septins leading to possible artifacts. What is
noteworthy, is the fact that the vast majority of observed
interactions are consistent with the 7-6-2 filament and with
Kinoshita’s conjecture [6].
Besides identifying the expected binding partners predicted
from the trimeric arrangement, SEPT6 and SEPT8 also interacted
with SEPT3 group members, notably SEPT9 (Fig. 3a, p,0.001).
Furthermore, in the reverse sense, when SEPT3 and SEPT9 were
used as bait, they preferentially fished out preys belonging to the
SEPT6 group (39% in the case of SEPT9 for example, Fig. 3c,
p,0.001), consistent with previous studies [13,37]. Since the
SEPT3 group is not present in the canonical 7-6-2 trimer, this
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Human Septin Interactome
Figure 2. Three dimensional column diagram of the number of clones of septin prey proteins fished by septin bait proteins in
group wise organization. Number of fished clones for each prey septin (Y-axis), was plotted against bait (X-axis) and prey septins (Z-axis), where
the septins have been ordered in a group wise fashion (See Table S1 for raw data of clone numbers). The whole distribution is significantly
(p = 0.0004998) different from a random distribution, as can also be verified visually, since the clone numbers group into ‘‘islands’’ in between various
blank areas. When we compared groups in a one-to-one and reciprocal fashion we obtained significant p-values (p,0.001), suggesting a non-random
distribution, for the following pairs of groups: group 2 vs. group 6, group 6 vs. group 3/9, group 6 vs. group 7, and group 3/9 vs. group 7.
Furthermore, we can still analyze the occurrence of interactions among septins of the same group. The result in this case is obvious: members of
groups 6, 3/9 and 7 never interacted with themselves or with other members of the same group. The only exception is group 2, which members
tended to interact with other group 2 members. See also Fig. 3.
doi:10.1371/journal.pone.0013799.g002
already described in the literature (e.g. 767, 969). An intriguing
possibility is that we never observed these homotypical septin
interactions in our yeast two hybrid assay, because the latter is
limited to one-to-one interactions among individual septins. It may
be possible that 767 and 969 predominantly interact in the
context of trimer-trimer or hexamer-hexamer interactions at the
final stage of filament formation. This may be due to conformational changes which could occur at the septins 3, 7 and 9 when
the hexamer units are formed. Septin 7 in a hexamer context
would therefore gain then the capacity to interact with itself.
Either way, these potential new filaments need to be verified in vivo
and evaluated by further biochemical studies in vitro.
SEPT9 and SEPT7 identified one another as mutual partners at
very high percentages. 23% of the prey clones identified by SEPT9
corresponded to SEPT7, whilst in the reverse direction the rate was
even higher (61% of septin clones, Fig. 1). Again this distribution is
significantly different from a random distribution (Fig. 3c,d), and the
preferential interaction was reciprocal: septin bait 7 fished septin 9
(p,0.001) and septin bait 9 fished septin 7 prey (p,0.001). These
results strongly suggest a physiological significance and possibly
raises the intriguing question of how they may participate in
filament formation. Since SEPT6 occupies the central position,
one possibility is that the SEPT3 group members may replace
either SEPT2 or SEPT7. If they were to occupy the position
normally occupied by SEPT7, this would lead to 3-6-2 and 9-6-2
as possible new trimeric arrangements (Fig. 3c, Fig. 4B). However,
this would be incompatible with the coiled coil C-termini which
are expected to project laterally, at a 90 degree angle, from the
filament (Fig. 4A,B), since it would leave the SEPT6 coiled coil
unpaired. On the other hand the coiled-coil pairing may have only
a stabilizing function, since we know that the filament formation is
primarily based on GTPase domain interactions [18,19]. An
alternative would be for the SEPT3 group to substitute SEPT2 at
the center of hexameric unit (Fig. 4C). This would lead to
combinations of the type 7-6-3 or 7-6-9. Included in the latter
group is the combination 7-11-9b which has been experimentally
reported [13].
Although we did not observe homotypical interactions of the
type: septins 767, 363 or 969 in our yeast two hybrid screens the
above mentioned models (Fig. 4) synthesize our data with those
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Figure 3. Representation of the two-hybrid septin-septin results in the light of the format of the trimer/hexamer SEPT7/SEPT6/
SEPT2 – SEPT2/SEPT6/SEPT7 [18]. Assuming that members of the same group may serve as substitutes [6] and taking into account the structural
arrangement found for the crystal of the trimer/hexamer SEPT 7/6/2, interesting observations can be made. The bait septins employed in the twohybrid system are given on the left. In green in the schematic figure the preferentially found prey septins are indicated and assigned to likely
positions in the hexamer scheme. The three dimensional column diagrams on the right refer to Fig. 2. The data that differ significantly from a random
distribution have been circled to indicate the experimental basis on which each structural arrangement (monomer interfaces) is based. From top to
bottom: (a) is based on the following statistical comparison: group 6 vs. group 2 (no random distribution: p, 0.001), group 6 vs. group 3/9 (p,0.001),
group 6 vs. 7 (p,0.001). (b): group 2 vs. group 6 (p,0.001), (c): group 3/9 vs. group 6 (p,0.001), group 7 vs. group 9/3 (p,0.001), (d): group 7 vs.
group 6 (p,0.001), group 7 vs. group 9/3 (p,0.001). None of the septins fished members of its own family, except group two members (b). The
group pairings with statistically significant clone distributions were indicated at the right side of the figure (e.g. 667, 3/966 etc.). By comparison of
the letters color codes it can be seen that all of these group pairs were reciprocal. For example bait septin 6 group fished group 2 septins (a) and vice
versa (b) (p ,0.001). As initially proposed by Kinoshita there may be substitutions among different members of the same septin group. The sequence
of listed septins from left to right reflects a descending order of frequency of clones with septins that were found to interact (e.g. for SEPT6 bait: 9,3,7
and 5,1,4,2).
doi:10.1371/journal.pone.0013799.g003
variants in different tissues [8,36]. Full length SEPT9 interacted
only with SEPT6 and SEPT7, but its N-terminal region used alone
as bait, only picked up non-septin proteins as interaction partners
(see discussion below). Since SEPT9 lacks the C-terminal coiled
coil domain, its interaction with other septins is likely to occur via
the GTPase domain.
Besides the foregoing discussion SEPT7 also detected some
clones of SEPT4 and SEPT1 (together 7%). These are not
anticipated by the canonical 7-6-2 filament and may suggest a
tendency to participate in other trimeric or even dimeric or
tetrameric assemblies upon multimerization or filament formation.
SEPT7 is expressed in most tissues and is therefore expected to be
more involved in basic processes such as cell division. It belongs to
the group of septins with the fewest members (SEPT7 and 13,
only) and is present in almost all of the heterofilaments described
to date. It may therefore turn out to be a fundamental element for
filament formation.
imply filament assembly, which is different from the canonical 7-6-2
arrangement. Surka and coworkers [38] described the immunoprecipitation of a complex containing septins 2, 6, 7 and 9 from
HeLa cells, therefore suggesting that septin complexes may involve
more than three components. This was confirmed more recently by
Mostowy and coworkers [25] in which SEPT11 was replaced by
SEPT6. A possible arrangement of SEPT7 and 9 in the filament is
given in Figure 4D, where our own data are combined with data
from the literature.
The SEPT3 members tested, especially SEPT3 itself, showed
the lowest number of interactions with other septins but a
relatively large number of interactions with other proteins (80%,
Fig. 1). SEPT3 is highly expressed in neurons [8], which may
imply a specialized function in these cells that may not be limited
to septin filament formation, although it has been found in
complex with SEPT6 members [13,37]. SEPT9, on the other
hand, is expressed ubiquitously, but occurs in many different
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Human Septin Interactome
show that the sumoylation process may be relevant for the
regulation of septin functions, since SEPT3, SEPT6 and SEPT8
all picked up SUMO1 and/or the Sumo ligase PIAS3. Sumoylation
of proteins has been shown to regulate the assembly and disassembly
of protein complexes, their localization, stability and various other
functions [39–41]. Previous data from the literature have
demonstrated that yeast septins interact with SUMO and it has
been suggested that sumoylation may be key for assembly of human
septin filaments [42]. In yeast the septin ring formation depends
critically on septin sumoylation [42–44].
Further functional contexts which are common to more than
one septin or even to more than one septin group include: (a)
Microtubular transport/intracellular trafficking/endocytosis (septins 2, 3, 5, 7 and 8); (b) Cell motility (septins 3, 6 and 8); (c) cell
division/cell cycle (septins 1, 6, 8 and 10); (d) apoptosis (septins 3, 9
and 6) and (e) regulation through kinases/phosphorylation
(signaling) (septins 2, 3, 4 and 6) (Table 2, Figure 5).
Selected non-septin interactors
Some of the septins used as baits interacted with non-septin
proteins or members of the same family of proteins that have been
previously reported to interact with another septin. For example,
the protein CENP-F was identified as an interactor of SEPT8
(Table 2). Interestingly, SEPT7 has been previously found to
interact with CENP-E [45]. In that case it was reported that
interference with SEPT7 iRNA prevents the correct localization of
CENP-E to the kinetochore and abnormal chromosomal segregation [45]. Based on these and other experiments it was
established that SEPT7 and CENP-E form an interacting pair in
cells and that this interaction is not only important for the septin
filament assembly but also for the correct formation of the
kinetochore complex and is therefore essential for the mitotic
spindle checkpoint [12,45–48]. We may speculate that another
such pair of interactors may be the proteins SEPT8 and CENP-F,
whose interaction we describe here for the first time. This may
suggest that SEPT8 in addition to other previously reported
septins (SEPT2, SEPT7), is important for chromosome segregation
and mitotic progression. Since this particular combination of
septins (SEPT 7-8-2, Fig. 4) is expected to form viable filaments
(see above discussion), this may imply that complex formation is
relevant for fulfilling this physiological role.
Another individual interaction found, which merits discussion is
that of SEPT5 and SNX6, since this may involve a coiled-coil
interaction between the C-terminal domain of SEPT5 and one or
both of the coiled-coil regions present in the C-terminal region of
SNX6 [49,50]. Coiled-coil domains (CC) are predicted to occur in
up to 10% of eukaryotic proteins and are related to a wide array of
different functions but serve predominantly to promote proteinprotein interaction [51,52]. SNX6 contains a Phox (PX) domain
involved in phosphoinositol binding and members of the SNX
family of proteins, like septins, are involved in intracellular
trafficking. SNX members have been found in oligomeric
complexes with other proteins where their interactions were
mediated by both the PX and CC domains [49,50]. The interaction
of SEPT5 and SNX6 seems to be biologically relevant since several
other proteins that have been described to interact with septins are
also related to intracellular trafficking and/or exocytosis.
It is of interest to note that both SNX6 and CENP-E appear to
interact with septins via a coiled coil domain. This may imply that
under certain circumstances, acting individually or even within the
context of certain heterofilaments, not all of the septin coiled coils
are satisfied. Some combinations of septins may leave coiled coils
available for interaction with non-septins partners (e.g. Fig. 4B).
Figure 4. Schematic representation of possible septin-septin
interactions within putative filaments, based on a combination
of our yeast two hybrid assay’s data and previously published
data. (A) the canonical or standard filament taken from the crystal
structure of the 7-6-2 complex [18] showing the trimeric and hexameric
cores and the NC and G interface. The 7-6 dimer is believed to be
stabilized by a long coiled coil at an NC interface, whilst the 2-2 dimer is
similarly stabilized by a short coiled coil. (B) possible arrangement for a
filament composed of members from the SEPT3, SEPT6 and SEPT2
groups. SEPT3 group contains both septin 3 and 9. This arrangement
would leave the SEPT6 coiled coil unpaired and ‘‘free’’ for interaction
with other coiled-coil containing proteins (see text for details). (C)
possible arrangement for a filament composed of members from the
SEPT3, SEPT6 and SEPT7 groups. (D) possible position for SEPT9
compatible with the observed yeast two-hybrid data and data from the
literature [38] (see also Fig. 3c,d).
doi:10.1371/journal.pone.0013799.g004
SEPT11 (which is very similar to SEPT6), although not used as
a bait by us here, was found as a prey in significant proportions
when SEPT1, 4, 5 and 3 were used as bait and in relatively smaller
proportion in the case of SEPT5 (Fig. 1). These interactions are
consistent with the canonical filament where SEPT11 would
occupy the central position of the trimeric unit. Curiously, neither
septins 12, 13 nor 14 were ever picked up by any of our bait
septins. This may either suggest that they are poorly expressed in
the libraries tested or that these septins, like SEPT10 have no
strong tendency to interact with other septins.
Common non septin-interactors
Aside the many septins found to interact with most of our baits,
we observed varying quantities of non-septin proteins as interaction
partners in all cases. These belonged to both structural and
functional classes of proteins (Table 2, Figure S1). Most interestingly, some of them were repeatedly found with several different
septins (e.g. UBE2I, SUMO or PIAS). In fact the latter proteins,
functionally associated with the ubiquitin and sumo-cycles were
found as interactors for all septins except SEPT7, SEPT9 and
SEPT10. This indicated that at all members of the SEPT2 group
and at least some members of the SEPT3 and SEPT6 groups have a
propensity to interact with proteins from the sumo- and ubiquitincycles and seems to suggest possible protein degradation pathways
relevant for human septins (Fig. 5). Furthermore, these findings
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Human Septin Interactome
Table 2. Summary table of all septin interacting proteins identified and their functional assignments.
Septin preys
(In order of
frequency)
Predominant
septin prey
group
Non-septin preys in order
of frequency (shared
preys)
Functions of non-septins
2
Septin 9,6,11,
1,2,4,5
Group 6
UBE2I, CEP110, SKA1
Protein degradation, cell division
2
2
Septin 6, 4
Group 6
ANKZF1, DCTN2, CCDC45,
PC-S/K-I, MAP3K12, ribosomal
S6 Kinase like, UBE2I
Transport, Motor activity,
microtubule-based process,
kinase
4
2
Septin 11, 10,
6, 8
Group6
UBE2I, VEGFR-1, CASC3
Phosphorylation/Tyrosine kinase
receptor, Protein degradation
5
2
Septin 6, 8,
11, 5, 2
Group6
UBE2I, SNX6
Protein degradation, Intracellular
Trafficking
T
9 (9N)
3 (3/9)
Septin 6, 7
Group 6
FLNA, SH3KBP1
Cytoskeletal organization, apoptosis
T
3
3 (3/9)
Septin 6, 11
Group 6
UBE2I, CASP8AP2, PIAS3, TDG,
ABCB10, ACTB, EIF 4A, EXOSC9,
GABA-RAPL2, HNRNPH3, MYO1B,
PLK2, PRDX2, RPL14, RPS24,
STMN2, SUMO1, TMEM93, IFT27,
TDG
Protein degradation, Cell motility,
Apoptosis, DNA repair, SUMO ligase,
Neuron differentiation, Splicing
process, Phosphorylation, Translation,
Signal transduction, kinase,
Endocytosis
T
6
6
Septin 9, 5, 7,
1, 2, 4, 3
Group 3
UBE2I, SUMO1, CASP8AP2,
PIAS3, TOPORS, ACTR2, HIPK3,
MDH1
Apoptosis, Cell cycle, SUMO cycle
activity, Protein modification process,
Ubiquitin cycle, Cell motility, Cell
division, Protein degradation, kinase
8
6
Septin 9, 7, 4,
2, 5, 1
Group 3
C1QBP, CERCAM, CENP-F, SH2B3,
UFD1L, CAPRIN1, ERP29, FAM89B,
HDAC11, KIF14, LCP1, LMNB1,
PBXIP1, PIAS3, SMARCC2,
ZNF451
Immune response, Cell division, Cell
adhesion, cell motility, SUMO ligase
activity, Cell cycle, intracellular
protein transport, Transcription,
microtubule motor activity, Cell
differenciation, ubiquitin cycle
10
6
–
PLZF-ZBTB16
cell cycle, transcription
7
7
Septin 9, 6, 4,
1, 10, 11
RALBP1, ANKRD12, ZNF451
Endocytosis, Transcriptional
Septin
bait
Group
1
Group 3
Overlapping functions
T
M
C
U
P
C
U
P
C
U
C
U
C
M
P
G
P
T
M
C
U
C
U
C
U
C
G
C
Overlapping functions: T = Transport, endocytosis and cytoskeleton, M = Motoractivity, P = Phosphorylation, G = GAP Ras, C = Cell division, Cell cycle, U = Ubiquitin /
Sumo cycles (Pias, Ube2I).
doi:10.1371/journal.pone.0013799.t002
In yeast it has been reported that septins recruit SDKs (septin
dependent kinases) [53–55] indicating that phosphorylation of
septins may be an important factor in the regulation of their
activities. SDKs function as regulators of septin filament assembly
and for the correct positioning and alignment of the microtubules
during the budding process. Other kinases such as Hsl1 and Gin4
seem to be critical for the transition from G2 to M during the cell
cycle [53,56,57] and their direct interaction with septins seems to
be essential for their correct localization and activity. In our
screens we identified two additional kinases that may represent
candidates for additional points of regulation of septin activity.
HIPK3 (Homeodomain interacting protein kinase 3) was found as
an interactor for SEPT6 and PLK2 (Polo-like kinase 2) as an
interactor of SEPT3 (Table S1). Although in both cases only a
single clone was identified, the result may be significant. Based on
the usually weak and transient interaction between kinases and
their substrates, it is not expected that this type of interaction
appear with considerable frequency in yeast two-hybrid screens. It
is worth pointing out that PLK2 is like the functionally related
SDKs mentioned above, associated with the regulation of the
transition from G2 to M [58]. Should this interaction prove to be
of physiological relevance our data suggest that aside the SDKs
other kinases are involved in the functional regulation of septins
during this part of the cell cycle.
In the case of the HIPK3 kinase, a possible biological link is less
evident and there are only very few publications on the functions of
Figure 5. Non-septin interactors of the septins 1-10 grouped by
functional categories. The group wise clustering of functional
contexts is evident. The most predominant functions are emphasized
only in order not to pollute the figure with excessive information.
Septins are shown clustered into the four groups: SEPT2, SEPT3, SEPT6
and SEPT7 from top to bottom in different colors.
doi:10.1371/journal.pone.0013799.g005
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Human Septin Interactome
interactions [18]. Furthermore, this finding indicated that the
identified new septin-septin interactions may indeed represent
physiological relevant combinations. For the other, non-septin
preys, we were able to identify the following overall trend: smallsize prey proteins tend to be present rather completely, while for
the larger proteins, often only a restricted interacting region could
be identified. This may imply that for larger proteins, only specific
domains or modules are responsible for septin recognition.
The first crystal structure of a septin complex [18] provided
considerable food for thought with respect to the way in which this
apparently redundant family of proteins can potentially form a
myriad of different filaments. Here we provide the first large scale
yeast two-hybrid study which addresses this question. We have
provided a large body of experimental evidence which for the most
part corroborates the speculation made by Kinoshita [6] that
septins are substitutable within their given groups. New potential
non-septin partners have also been described. What controls
assembly and the physiological requirement for such potential
diversity are questions which badly need addressing.
this kinase currently available. It has been identified to be associated
with the cytoplasmic domain of the FAS receptor, although its
activity was not found to influence cell death directly [59].
Another interesting finding is the identification of the two
proteins IFT27 (former name RabL4) and RalABP1 which
interacted with SEPT3 and SEPT7 respectively. IFT27 is a Raslike GTPase protein that contains the five consensus sequences
needed for GTP-binding and GTPase activity. IFT27 can bind to
GTP and may act at the end of the cytokinesis where Rab family
members are involved in vesicle trafficking required to complete
this process [60]. Since septins are also involved in vesicle
trafficking and cytokinesis, the interaction with IFT27 may be
biologically relevant and should be further tested experimentally.
RalABP1 is a member of the Ras GTPase superfamily and the
latter is GTPase-activator protein, involved in the regulation of
endocytosis during interphase. Since the discovery that septins
bind and hydrolyze GTP, although at a rather slow rate, it has
been speculated that other interacting proteins may act as GAPs
(GTPase activating proteins), that through promoting GTP to
GDP hydrolysis may regulate septin function. This could alter the
septins conformation and may influence its interaction with other
proteins, including their assembly into septin filaments. RalABP1
has been already reported to act as a GAP on other GTPases and
RabL4 could be another candidate for a septin GTPase regulatory
protein. The GAP Rho has been previously shown to act on the
mammalian SEPT9b [35].
Finally, we also found two interesting non-septin interactors of
the SEPT9 N-terminal region: filamin A (FLNA) and SH3-domain
kinase binding protein 1 (SH3KBP1). The former one is directly
involved in actin cytoskeleton organization [61] and the interaction
between FLNA and SEPT9 could represent a novel physical and
functional connection between septin and actin filaments.
SH3KBP1, the second interactor, is an adaptor protein involved
in many processes, from cytoskeleton remodeling and vesiclemediated transport to signal transduction and cell death [12,62–65].
Recently, many proteins involved in cytoskeleton and membrane
processes, including septins, were found to interact with SH3KBP1
by mass spectrometry analysis [66]. Our results confirm these
previous studies since the N-terminal domain of SEPT9 picked up
SH3KBP1 in the two-hybrid assay. In addition, an interactor of
septins, cytoskeleton components and plasma membranes called
anillin [67] was found to interact with SH3KBP1 [66]. Since anillin
and septins interact directly [17] and both interact with SH3KBP1,
our findings suggest that the septins-SH3KBP1 interaction could be
involved in cytokinesis and plasma membrane processes, such as
vesicle trafficking. Although our findings provide possible clues for
better understanding septin filament assembly and regulation,
further experimental studies are clearly essential.
Materials and Methods
Plasmid construction
Oligonucleotides were designed to amplify and sub-clone the
cDNAs encoding the amino acid sequences of the human Septins 2–
10 studied here. Full length SEPT1 was picked up as a prey in a two
hybrid screen with SEPT6. Its cDNA was subsequently sub-cloned
in the pBTM116 vector to perform a screen with SEPT1 as bait. In
all cases full length cDNAs were amplified, except for SEPT9, where
in addition to the full length protein we also used a construct that
spans the N-terminal region alone (amino acids 1- 269). A second
exception was that of the full length version of SEPT4, which
showed auto-activation of the reporter genes. In this case we
employed for the screen a construct which lacked the N-terminal
domain (aa 124-478) and no longer resulted in auto-activation. All
septin cDNAs were isolated from a human fetal brain cDNA library
(Clontech). These were cloned in frame with lexA into the poly
linker of the bait vector pBTM116 which had the ampicilin
resistance marker changed to a kanamycin resistance marker to
facilitate recovery of the prey plasmid from the co-transformed baitplasmid pACT2, contains an Amp resistance marker. Furthermore,
the modified plasmid contained some additional restriction enzyme
sites in the cloning site to facilitate cloning. A total of 11 baits
cDNAs were successfully cloned. These include: hSEPT1
(NM_052838), hSEPT2 (NM_004404), hSEPT3 (NM_019106),
hSEPT4 (lacking the N-terminal domain and hence corresponding
to amino acid residues 124–478) (NM_080416), hSEPT5
(NM_002688), hSEPT6 (NM_015129), hSEPT7 (NM_001788),
hSEPT8 (NM_001098811.1), hSEPT9 (NM_006640) transcript
variant 3, protein: isoform c, hSEPT9 N-terminal region (amino
acid residues 1-269 transcript variant 3, protein: isoform c), and
hSEPT10 (NM144710). These are no new cell lines but only cDNA
clones obtained by in vitro experiments as described above.
Prey regions involved in Protein-Protein Interactions
After DNA sequencing of the interacting prey plasmids and
analysis of their sequences we found that a large fraction of the
clones encode full-length or almost full length proteins. As
expected however many clones also encode only protein fragments
that may contain specific protein domains.
In case of the septin preys found to interact with septin baits it
was especially obvious that the majority of clones encoded full
length proteins (Table S1, e.g, SEPT3: interacted with SEPT6(4427), SEPT11(7-429). Most interestingly, even if the prey septins
were not full length proteins, they almost always encoded for the
central GTPase domain, for example SEPT6, which interacted
with SEPT9(129-568), SEPT5(39-369) and SEPT7(65-437), and
SEPT4(101-478). This clearly confirms the known fact that the
central GTPase domain is important for mediating septin-septin
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Basic yeast procedures and two hybrid screen
The yeast two-hybrid screens [68] of two different cDNA
libraries were screened for all 11 bait proteins; a human fetal brain
library and a human leukocyte library (both from Clontech). We
used the yeast strain L40 (trp1-901, his3D200, leu2-3, ade2
LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lac GAL4) and the baits
described above fused to the bacterial LexA protein in the slightly
modified vector pBTM116 [69]. Since SEPT4 auto-activated the
yeast reporter genes, a construct spanning amino acids 124-478
was used, which showed no auto-activation. This construct
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Human Septin Interactome
contains the GTPase and C-terminal domains of SEPT4, but lacks
the relatively large N-terminal domain.
Yeast cells were transformed according to procedures provided
by Clontech. The autonomous activation test for HIS3 was
performed in minimal medium plates in the absence of tryptophan
and histidine but containing varying concentrations of 3-AT (3amino-1,2,4-triazole). None of the septin bait constructs, except
SEPT4, showed auto-activation.
For the library scale screens the competent L40 yeast cells were
first transfected with the bait construct as described previously
[70]. The recombinant cells were then, in a second round of
growth, transfected with the library plasmid. For the interaction
screen these double transfected cells were then plated on minimal
medium plates in the absence of tryptophan, leucine, and histidine
and containing 5 mM 3-AT, to suppress non specific background
growth. At least 1 million co-transfectants were plated and
analyzed in total for each bait septin. Typically this meant that
at least 20 plates of 15 cm diameter were screened for each of the
two libraries. The number of clones growing varied greatly from
septin to septin bait, ranging from few clones for SEPT10 to
several thousand clones in case of SEPT6. Recombinant pACT2
plasmids of growing colonies were isolated and subsequently
transformed in E. coli for plasmid amplification and isolation. Prey
plasmid DNA was extracted and sequenced with an automatic
DNA sequencer (Model 16-capillary 3130xl Genetic Analyzer,
Applied Biosystems). The corresponding Accession numbers of the
DNA sequences identified are given in the Supplementary Table
S1. As no new sequences have been obtained no new sequence
data have been deposited in the GenBank.
When only a relatively small number of colonies grew (,100-),
plasmid DNA was extracted and sequenced from all such colonies.
In cases where a relatively large number of colonies were obtained
(.1000), plasmid was extracted and sequenced for at least 200
colonies (SEPT6 and SEPT8). All plasmids were submitted to
confirmation assays in the yeast (see next paragraph) and only those
which proved to be positive were considered for subsequent analyses
and are presented here (see next paragraph). In some cases up to
50% of the initially sequenced clones did not give positive results in
the confirmation assay and all of these were discarded from further
analysis. All clones shown in the supplementary tables 1–10 have
been confirmed.
10 mM MgCl2, 50 mM 2-mercaptoethanol, pH 7.0) containing
1 mg.mL21 5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-Gal).
After incubation at 37uC for 30 min to 1 h the formation of a blue
color was evaluated. Only clones that had an unambiguous blue
color were considered true positives. These interactions were
considered to be confirmed and the corresponding clones were
included in the data analysis presented in the tables and figures of
this report. Colonies that remained white or faintly blue were
excluded from further analyses.
Yeast interaction confirmation assay
Table S1 Characteristics of interacting proteins for septins 1 to
Cloning, protein expression and purification for
confirmatory interaction assays
For expression in E. coli of either the bait proteins or the
corresponding preys identified above, the encoding cDNAs were subcloned into the expression vectors pET28 and/or pGEX as described
[70]. Orientation and correctness of DNA sequences were confirmed
by DNA sequencing. The recombinant proteins were either
expressed in fusion with GST or 6xHis tags according to standard
protocols [71]. Subsequently, the fusion proteins were purified on
glutathione-Uniflow resin (Clontech) or HiTrap chelating resin (GE
Healthcare) as described [72]. In vitro binding assays/pull down assays
were performed as described previously [73].
Statistical analysis
In the case of the septin bait’s interactions with other prey
septins (Fig. 2, 3) we performed group wise statistical analysis using
a Fischers exact test for counts employing the free software R,
version 2.11.1 [74]. First, septins were grouped into four groups,
according to their sequence similarity: group 2 (septins 1,2,4,5),
group 6 (septins 6,8,10,11), group 3/9 (septins 3 and 9), ‘‘group’’ 7
(only septin 7). Then the number of interacting clones was
summed for each member inside the same group (see raw data
plotted in Fig. 2 and also Table S1) and tested for a random
distribution. Subsequently, we compared groups in a one-to-one
and reciprocal fashion in a similar manner. Finally, an analysis was
performed for the occurrence of interactions among septins of the
same group. Please refer to the supplementary material for
detailed results of the statistical analysis.
Supporting Information
septin 10 as predicted from the clones retrieved in the yeast twohybrid system screenings. The Tables appear in sequence of the
septin protein used as bait in the screen. HFB: Human fetal brain
cDNA library screened, LEU: human leukocyte cDNA library
screened. The number of clones obtained is indicated as a total as
well as the numbers for the leukocyte and human fetal brain
library separately. The gene accession number, main assigned
protein function, specific present protein domains and references
are also given. Septins are listed first followed by non-septins, in
both cases in order of decreasing frequency (i.e., the number of
identified clones, independent of being either identical or not. See
table for discrimination if available). Results of statistical analyses
are given after the tables.
Found at: doi:10.1371/journal.pone.0013799.s001 (0.39 MB DOC)
Extracted plasmids from positive clones during the initial
screening were used to transform L40 yeast cells previously
transformed with the appropriate bait. The interactions were
confirmed in yeast cells using the b-galactosidase assay. The
control of assay was performed with an empty bait vector
(pBTM116-lexA alone). Clones that did not confirm the growth
and blue color production when co-transformed with their bait
septins were discarded from further analysis (false positives). Any
prey plasmids that had alone (in absence of the specific bait vector
and presence of ‘‘empty’’ bait vector only) the capacity to promote
growth or the production of blue color was also discarded (false
positive). All clones reported in the Table S1 have been confirmed.
b-Galactosidase assay for the confirmation of interactions
Figure S1 A protein interaction network of the human septins 1-
For confirmation of the potential interaction between the septin
bait and the fished prey clones in a one-to-one fashion, bGalactosidase activity in yeast cells was determined using the filter
assay method. Yeast transformants (Leu+, Trp+, His+) grown on
minimal medium were transferred onto filter papers. The paper
disks were incubated for 3 min in liquid nitrogen, thawed and
soaked with Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4,
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10. The network consists of a total of proteins (colored nodes,
including the septin baits and its interacting partners identified in
the yeast two-hybrid screens) and the interactions connecting them
(grey links). The nodes are colored based on the GO biological
process as indicated in the legend. The network was generated
using the Osprey 1.2.0. software (http://biodata.mshri.on.ca/
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Human Septin Interactome
and Heloize de Souza for helping with the two-hybrid screens of SEPT1 and
7. We also thank Elaine Cristina Teixeira, who helped with the two-hybrid
screens of SEPT3 and 9. Thanks to Napoleão Fonseca Valadares for critical
reading of the manuscript. We also like to thank Roberto Zulli (Hemocentro,
UNICAMP) for expert help with statistical analysis.
osprey/). The proteins that interacted with septin are involved in
Carbohydrate Metabolism, Cell Cycle, Cell Organization and
Biogenesis, DNA Damage Response, DNA metabolism, DNA
Repair, Metabolism, Protein amino acid phosphorylation, Protein
biosynthesis, Protein transport, RNA Localization, RNA processing, Signal transduction, Transcription, Transport.
Found at: doi:10.1371/journal.pone.0013799.s002 (0.11 MB DOC)
Author Contributions
Conceived and designed the experiments: JK. Performed the experiments:
MN JNAM TVS NC TACBS JCPD LFR EMA MA. Analyzed the data:
MN JNAM TVS NC TACBS JCPD LFR MA RCG APUA NITZ JARGB
JK. Wrote the paper: MN TVS RCG JK.
Acknowledgments
We thank Maria Eugenia R. Camargo, Elaine Cristina Teixeira, Tereza
Cristina Lima Silva and Adriana Cristina Alves Pinto for technical assistance
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