 |
INTRODUCTION |
Septins form a class of eukaryotic guanine nucleotide-binding
proteins that were first identified in the budding yeast,
Saccharomyces cerevisiae, and congregate in a ring at the
bud neck during cell division (1-4). Temperature-sensitive mutations
in four of the septin genes produce cell cycle arrest and cytokinesis
defects when the yeast is grown at the non-permissive temperature. One essential function of septins at the bud neck may be as a diffusion barrier that helps segregate mother and daughter cell membrane components (5-7). The septin ring also provides a structural scaffold
for the localization of many proteins to the bud neck (3). Septins
purified from yeast form filaments of variable length that are 7-9 nm
in diameter, although the physiological significance of the filament
structure remains unclear (8). The filaments show a periodicity of
about 32 nm, which may represent an octamer comprising two copies of
the four different septin subunits. Similar filaments have also been
isolated from Drosophila and mammalian brain tissue (9, 10).
However, the mechanism by which the filaments assemble from septin
monomers is completely unknown. The Drosophila filaments
appear to be composed of three different septins. Two of these septins,
Pnut and Sep1, have been localized to the cleavage furrow in dividing
cells, and Pnut has been implicated in cytokinesis, although only in
certain cell types, and RNA interference of Pnut expression has an
unusually low penetrance (11-13).
At least 10 mammalian septins have been identified to date, but most
have not been studied in any detail. (Note that many have been given
multiple different names, which can cause confusion in the literature
and, for this reason, we will use a standard nomenclature in this
report, in accordance with Human Genome Organization (HUGO) and Mouse
Genomic Nomenclature (MGN) committee guidelines; see Ref. 14). The
murine Sept2 (previously called Nedd5) localizes to the contractile
ring of dividing cells, and the injection of anti-Sept2 antibodies
partially inhibits cytokinesis (15). Mammalian septins have also been
implicated in exocytosis and are associated with the exocyst proteins
Sec6 and Sec8 (10). Sept5 (previously called Cdcrel-1 or Pnutl) binds
syntaxin and copurifies with synaptic vesicles (16). Sept5 also
copurifies with syntaxin-4 in a complex from platelets, and platelets
from a Sept5 knockout mouse show misregulation of secretion and
aggregation (17), although the mice appear otherwise normal (18).
In interphase cells, mammalian septins are organized into filamentous
structures that often partially colocalize with actomyosin stress
fibers (15). Sept2, Sept6, and Sept7 each colocalize with one another,
suggesting that they are part of a complex, and these proteins also can
be copurified in a 1:1:1 stoichiometry using the Borg3 protein as an
affinity matrix (19). Borg3 is a small adapter protein that is part of
a family of downstream effectors for the small GTPase Cdc42 (20, 21). A
short, conserved motif within the Borgs (residues 83-110 in Borg3),
called the BD3 domain, is both necessary and sufficient for binding
septins, and, when expressed in cells, this domain induces a pronounced aggregation of septin filaments (19). Full-length Borg3 induces a less
pronounced reorganization of septins, and this effect is inhibited by a
gain-of-function mutant of Cdc42. Therefore, Borgs can function as
adapters that link the Ras-like GTPase Cdc42 to the septin GTP-binding
proteins. It is of interest that in budding yeast, which does not
contain any Borg-like genes, Cdc42 has also been implicated in the
regulation of septin organization at the bud neck (22, 23). The
mechanism by which Borgs bind to and regulate septins is not understood
and has been frustrated by the lack of information on septin
oligomerization and filament assembly. Moreover, it has not been clear
whether the Borgs bind to one specific septin, to all septins, or
rather to a particular supramolecular assembly of septins.
To address this issue, we have developed methods for the expression of
septin heterodimers and trimers in bacteria. We find that, whereas
monomers are very unstable, the dimers and trimers are soluble and
stable and can bind guanine nucleotides. The coiled-coil domains of the
septins are required for these interactions. Interestingly, only the
Sept6/Sept7 heterodimer can bind Borg3. Septin trimers can assemble
into filaments of a diameter similar to that seen in native structures
isolated from yeast and flies. We find that septin dimerization is
associated with GTP hydrolysis but that Borg3 does not regulate this
assembly step.
 |
MATERIALS AND METHODS |
Septin Expression in Escherichia coli--
Individual septins
were cloned into the BamHI site of the Novagen pET30 vector
so as to attach in-frame His6 + S-peptide tags (HS)1 to their N termini. The
septin open reading frames were then excised by digestion with
NcoI/NotI and subcloned into a second bacterial
vector called ptacNT, which contains an ampicillin resistance marker, a
ptac promoter, and no epitope tag (NT). To create a bicistronic vector,
the pET30-Sept constructs were digested with XbaI/NotI, which excises the HS-septin open
reading frame plus the upstream ribosome binding site. This fragment
was cloned into ptacNT-Sept downstream of the first septin. Thus, the
second septin in the vector is expressed from an internal ribosomal
entry site (IRES) and possesses an HS tag, whereas the first septin
lacks a tag (for example, ptacNT-Sept7-HS-Sept6).
To express three or four proteins simultaneously, we used a second
compatible vector that contained a p15A origin of replication and
chloramphenicol and tetracycline resistance genes (pACYC184, from New
England Biolabs). Expression cassettes for the septins were ligated
into the BamHI site within the TetR gene of this vector. For example, Sept2 was first subcloned into pET30a and then
amplified by PCR using primers that flank the T7 promoter and
terminator sequences of pET30a and that attach BclI sites to
each end of the product. BclI is cohesive with
BamHI. Therefore, this PCR product was digested with
BclI and subcloned into the BamHI site in the
p15A vector. The resulting plasmid generates an HS-tagged Sept2
protein. A similar strategy was used to express untagged septins by
subcloning from ptacNT into the p15A vector. In all cases, bacterial
strain BL21(DE3) Gold was co-transformed with the p15A vector plus a
second vector (ptacNT), and selected on plates containing both
ampicillin and chloramphenicol. Protein expression was induced by
addition of isopropyl-1-thio-
-D-galactopyranoside to
liquid cultures to a concentration of 0.5 mM plus 2%
ethanol at 18 °C. Cells were ruptured using a French press, and
the septins were purified over Ni2+-nitrilotriacetic
acid-agarose beads (Qiagen).
DNA encoding various fragments of the septins was amplified by PCR and
subcloned into the vectors described above. GST-Borg3 and GST-BD3 were
expressed as described previously (19).
To assemble septin heterodimers in vitro, monomers were
prepared in small quantities from 100 ml of liquid culture and mixed immediately after elution from the Ni2+-agarose beads after
>10-fold dilution into a binding buffer (25 mM HEPES, pH
7.4, 150 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, 10% glycerol, 10 mM
-mercaptoethanol, and 0.1% bovine serum albumin).
Dynamic Light Scattering--
Protein solutions (~1.0 mg/ml)
were cleared by centrifugation at 14,000 × g for 10 min immediately prior to measurements. Light scattering was performed
using a DynaPro-MS/X instrument, with DYNAMICS control and analysis
software (Proteins Solutions, Inc.).
Electron Microscopy--
The purified Septin complex
(Sept2-Sept6-Sept7) was diluted to 100 µg/ml in a buffer containing
50 mM HEPES, pH 7.4, 250 mM NaCl, and 5 mM MgCl2. The protein solution was applied onto
glow-discharged, carbon-coated grids and incubated for 5 min. The
sample was then washed with the dilution buffer and stabilized in the
same buffer with 0.5% glutaraldehyde for 2 min. After rinsing with
water, the grid was stained with 1% uranyl acetate in 30% ethanol and examined in an electron microscope (Philips CM12). For rotary shadowing
of the protein complex, the specimen was prepared as described above
but on a native mica surface, and the sample was directly shadowed
without staining.
Guanine Nucleotide Binding--
Septins were passed over a PD-10
column (Pharmacia) to exchange them into binding buffer (50 mM MOPS, pH 7.4, 150 mM NaCl, 2 mM
-mercaptoethanol, 5 mM MgCl2, 100 µg/ml
bovine serum albumin). The proteins (20 µg) were incubated with
[
-32P]GTP or [
-32P]GDP (3000 Ci/mmol)
for the specified times, at 30 °C. Binding was assessed by filtering
through nitrocellulose membranes as described previously (20). To
assess guanine nucleotide binding and hydrolysis to mixed monomers, 30 µg of each freshly prepared HS-tagged monomer was mixed in the
presence of 2 µCi of either [
-32P]GTP or
[
-32P]GTP (3000 Ci/mmol) in 1 ml of binding buffer and
incubated at 30 °C. Aliquots (50 µl) were removed and washed
through nitrocellulose filters at intervals.
GTP hydrolysis was measured after loading 20 µg of heterotrimeric
septins with [
-32P]GTP for 3 h and then passing
them through a Centrisep spin column equilibrated with binding buffer
to remove free nucleotides. The septins were then incubated for various
times, and samples were analyzed for guanine nucleotide state by thin
layer chromatography on polyethyleneimine-cellulose plates. EDTA (10 mM) and SDS (2%) were added to the samples to release the
nucleotides. Plates were developed in 0.75 M potassium
phosphate, pH 3.4, as described previously (24), and radioactive spots
were detected and quantified using a Amersham Biosciences PhosphorImager.
 |
RESULTS |
Expression of Recombinant Septin Monomers, Dimers, and Trimers in
E. coli--
A fundamental problem in studying septins has been the
absence of systems for their expression as stable, soluble proteins in
bacteria. We were able to express small quantities of each of the
Sept2, 6, and 7 monomers (Fig.
1A), but the majority of the
protein in each case remained in the bacterial pellet after lysis under
all conditions tested, and the proteins in the soluble fraction tended
to precipitate out of solution after purification. However, using a
bicistronic vector, we were able to co-express pairs of septins that
were soluble and could be purified over Ni2+-agarose in a
~1:1 stoichiometry when a HS epitope tag was attached to one member
of each pair (Fig. 1B). All three pairs of Sept2, Sept6, and
Sept7 could be expressed in this manner. Binding of the untagged
partner did not represent nonspecific association with the
Ni2+-agarose, because enterokinase treatment to cleave the
HS tag resulted in the loss of both proteins from the beads
(e.g. Fig. 1C). Finally, by co-transformation of
the bacteria with a bicistronic vector plus a second, compatible
plasmid that expresses the third septin, we could copurify all three
proteins on Ni2+-agarose beads at roughly a 1:1:1
stoichiometry. An example is shown in Fig. 1D, where Sept2
was tagged and the Sept6 and Sept7 were untagged.

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 1.
Bacterial expression of mammalian septin
monomers, dimers, and trimers. Proteins were expressed in E. coli strain BL21(DE3) from pET30a (monomers,
panel A), a bicistronic vector (dimers,
panel B), and from two compatible vectors
(trimer, panel D). One component in each case was
fused to an N-terminal HS tag, and the proteins were purified on
Ni2+-agarose beads. Proteins were separated by SDS-PAGE and
stained with Coomassie Brilliant Blue dye. In panel C the
purified dimer was incubated with enterokinase for 2 h to cleave
the HS tag prior to binding to the Ni2+-agarose beads.
D, septin heterotrimer, purified over
Ni2+-agarose beads using the His6 tag on
Sept2.
|
|
When the purified septin trimer was analyzed by dynamic light
scattering, two distinct peaks were observed, i.e. a major
one corresponding to a hydrodynamic radius of ~14 nm and a minor peak corresponding to a radius of ~74 nm (Fig.
2A). The major peak is
consistent with a molecular size of about 256 kDa, assuming a spherical
shape. This peak may correspond to a septin hexamer, which would have a
calculated molecular size of about 280 kDa. The other peak represents
very large particles (>5 MDa) and may represent aggregates or
filaments. In contrast, a Sept6-Sept7 complex displayed a single peak
radius of ~4 nm corresponding to a molecular mass of ~87 kDa, which
is consistent with the two proteins forming a heterodimer.

View larger version (95K):
[in this window]
[in a new window]
|
Fig. 2.
Formation of septin heterotrimeric filaments
in bacteria. A, dynamic light scattering of recombinant
Sept6/7 dimers and Sept2/6/7 trimers (~1.0 mg/ml) purified from
bacterial extracts. Scattering was measured using a DynaPro-MS/X
instrument. B-D, electron micrographs of recombinant
Sept2/6/7 filaments. In panels B and
D, septins were applied onto carbon-coated grids, washed,
and stabilized with 0.5% glutaraldehyde for 2 min. After rinsing with
water, the grid was stained with 1% uranyl acetate in 30% ethanol and
examined in a Philips CM12 electron microscope. In panel C,
the specimen was rotary shadowed on a native mica surface without
staining.
|
|
To determine whether the septin trimer preparation contains filaments,
the purified proteins were visualized by electron microscopy. The
proteins were negatively stained on carbon film (Fig. 2, B and D) or were metal-shadowed (Fig. 2C). In each
field, long filaments were visible. These filaments are ~10 nm in
diameter. The filament shown in Fig. 2D is ~850 nm in
length. A larger field is shown at lower magnification in Fig.
2B, which contains filaments with a variety of lengths. No
filaments were observed when a Sept6-Sept7 dimer was examined after
similar treatment. For comparison, the septin filaments from yeast are
7-9 nm in diameter (8). Those from Drosophila are also ~7
nm in diameter, and those from mammalian brain tissue are ~8.25 nm in
diameter (9, 10). A significant difference between the recombinant and
purified septin filaments is that the latter show a periodicity along
the length of the filament with a unit length of about 25-30 nm,
whereas our recombinant Sept2/6/7 filaments do not. This difference and
the low percentage of trimers that assemble into filaments in bacteria
together suggest that we are lacking a stabilizing component. One
possibility is that this component is a fourth septin, such as MSF or
E-septin (now called Sept9), which was detected, although at low
stoichiometry, in our initial pull-down experiments with GST-Borg3
(19). Nonetheless, our data show that septin filaments can form
spontaneously when expressed in bacteria. Moreover, septin monomers are
highly unstable but can form stable heterodimers.
Guanine Nucleotide Binding by Recombinant Septins--
To test the
functionality of the recombinant septins, we assayed guanine nucleotide
binding. Initially, septins were incubated with
[
-32P]GTP or [
-32P]GDP and then bound
to nitrocellulose filters. Under a variety of conditions (± MgCl2 or EDTA at 4 °C or 30 °C) we could not detect
32P binding to Sept6 but found a low level of binding to
Sept7 and Sept2 (not shown). Others have also reported GTP-binding to
Sept2 and indicated that the nucleotide has a high off-rate (15). These
data are consistent with our conclusion that the septin monomers are
unstable and may be largely unfolded. The three septin heterodimers and
the trimer all bound [
-32P]GTP in a
time-dependent fashion (Fig.
3A). When diluted into a
solution containing 1 mM unlabeled GTP, the 32P
was slowly released with a similar half-time for all the complexes of
~150 min at 30 °C (Fig. 3B). Nucleotide binding to the
trimer continued for at least 24 h at 30 °C, by which time the
complex was labeled to a stoichiometry of about 10% (assuming that
each subunit is capable of binding nucleotide). This result is
consistent with guanine nucleotide binding data for purified septins
from Drosophila (9). A competition assay using
cold GTP or GDP showed that the septin dimers do not discriminate
between dinucleotides and trinucleotides (data not shown). Remarkably,
neither the binding nor release of guanine nucleotide was affected by
the addition of excess EDTA to complex the magnesium ions. This
behavior is very different from that of most other GTP-binding
proteins, which require magnesium ions to stabilize the association of
the nucleotide with the protein.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 3.
Guanine nucleotide binding and hydrolysis by
septins. Proteins were produced as described in the Fig. 1 legend
and under "Materials and Methods." A, binding was
performed at 30 °C, with 32P-labeled guanine nucleotide
(3000 Ci/mmol). B, off-rates were determined after dilution
into buffer containing 1 mM GTP plus 1 mM GDP.
Data are means of duplicate assays. Curves were fit assuming a single
exponential decay. C, hydrolysis of bound
[ -32P]GTP was measured after the removal of free
nucleotides from the septins by passage through a Centrisep spin
column. At intervals, aliquots of the labeled protein were bound to
nitrocellulose filters and denatured with SDS and EDTA. Released
nucleotides were separated by thin layer chromatography and quantified
on a PhosphorImager. Data are presented as percentage of total bound
nucleotide and are means of duplicate assays. D, binding was
performed as described for panel A, but using septin
monomers mixed in the presence of the nucleotide.
|
|
To test for GTPase activity, we incubated the septins with
[
-32P]GTP for 2 h and then passed the complex
through a size exclusion column to remove unincorporated nucleotide. At
intervals the septins were then denatured with SDS, and the bound
nucleotides were separated by thin layer chromatography. Under these
conditions, we observed a time-dependent fall in the level
of bound [
-32P]GTP coupled to a rise in the level of
bound [
-32P]GDP (Fig. 3C). This reciprocal
change strongly supports the hypothesis that the septin complex does
possess a GTPase activity, although it is extremely inefficient.
Finally, we wished to determine whether GTP hydrolysis might play a
role in the assembly of septin heterodimers or filaments from monomers.
Because the monomers are unstable, we were unable to detect efficient
association into dimers when they were mixed and could not directly
test the effects of slowly hydrolyzable analogs of GTP on dimerization.
Therefore, to address this issue, we prepared small quantities of pure
monomers and rapidly mixed them in the presence of either
[
-32P]GTP or [
-32P]GTP. We reasoned
that if GTP hydrolysis occurs on dimerization, then we would not detect
bound 32P when using the [
-32P]GTP; but
with [
-32P]GTP, 32P would be retained on
the septin heterodimer as [
-32P]GDP. On the other
hand, if GTPase activity was not involved in dimerization, both the
[
-32P]GTP and [
-32P]GTP should bind
equally well. The appearance of bound 32P in the experiment
would also provide an indirect measure of dimerization, because the
monomers bind nucleotide very inefficiently. The results of this type
of experiment are shown in Fig. 3D. When [
-32P]GTP was added to Sept6 or Sept7 alone, only a
small amount of binding to the Sept7 monomer was detected and none to
Sept6. However, when the Sept6 and Sept7 were mixed at the same time as
the nucleotide was added, bound counts accumulated over time,
suggesting that a fraction of the monomers was combining into stable
dimers that could bind nucleotide. When the same experiment was
performed but in the presence of an equal amount of
[
-32P]GTP, significantly less protein-bound
32P accumulated. This result suggests that GTP hydrolysis
is occurring at a rate that is equal to or greater than the rate of
dimer formation.
Septins Interact through Their Coiled-coil Domains--
Most
septins consist of a variable N-terminal region followed by a
GTP-binding domain and terminate in a C-terminal coiled-coil domain. To
determine which regions of the septins are required for dimerization,
we co-expressed either the isolated C-terminal regions or the
N-terminal regions (variable domain plus GTP-binding domain) in
bacteria, with an HS tag on only one of the components and asked if the
untagged component co-purified on Ni2+-agarose beads. The
isolated N terminus of Sept6 was mostly insoluble, and its solubility
was not increased by the co-expression of Sept7 (data not shown; also
see below and Fig. 5D). However, when the C-terminal
coiled-coil domains of each were expressed, the HS-tagged Sept7
co-purified with the untagged C terminus of Sept6 (Fig. 4). Note also that the stability of the
Sept7 fragment appears to be increased by the presence of
Sept6-(283-429). We conclude that at least two of the septins, Sept6
and Sept7, likely associate into a heterodimer through their
coiled-coil domains.

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 4.
Septin dimerization occurs through the
C-terminal coiled-coil regions of the subunits. Fragments of Sept6
and HS-tagged Sept7 were expressed either individually or together in
E. coli and purified from lysates on
Ni2+-nitrilotriacetic acid-agarose beads and then analyzed
by SDS-PAGE (12% gel) and stained with Coomassie Brilliant Blue.
Sept6-(283-429) and Sept7-(274-418) contain the putative coiled-coil
domain but lack the GTP-binding domain.
|
|
Borg3 Binds Specifically to the Sept6-Sept7
Heterodimer--
Affinity purification of cell lysates, using
GST-Borg3 attached to beads, can isolate a complex of at least three
septins from cell lysates. We have shown previously that a small domain of about 28 amino acids (residues 83-110), which is conserved within
the Borg family, is both necessary and sufficient to bind septins (19).
We named this sequence the BD3 domain. To identify the minimum
requirement for binding of septins to this BD3 domain, we tested three
septin monomers, their various heterodimers, and the trimer for
association with a GST-BD3. As shown in Fig.
5A, none of the monomers were
capable of binding to the BD3 domain. Additionally the Sept2-Sept6
heterodimer did not bind the BD3 domain. However, the Sept6-Sept7
complex and the septin trimer were efficiently captured by the BD3
domain. In a second experiment, a GST fusion of the full-length Borg3
was used, and all three heterodimers were tested using detection of the
S-peptide tag on one of the septins as a more sensitive assay for bound
protein (Fig. 5B). Again, only the Sept6-Sept7 heterodimer
bound to the GST-BD3 beads. This result supports the idea that
individual septin polypeptides are unable to recognize the BD3 domain
of Borg3 and that epitopes provided by both Sept6 and Sept7 are
required. As a further test of this hypothesis, we used a yeast
three-hybrid conjugation assay. Yeast (yDW-12a) was produced that
expresses Sept7 and/or a fusion protein comprising Sept6 attached to
the LexA-DNA binding domain (the bait). These strains were mated to yeast (W303) that expresses a fusion protein of Borg3 plus the activation domain of VP16 (the prey). If the Borg3 binds to Sept6, it
induces the expression of the His3 gene and allows survival of diploid
yeast on selective medium lacking histidine and containing a low
concentration of an inhibitor of histidine synthesis (3-aminotriazole). As shown in Fig. 5C, Sept6 alone did not allow growth of the
diploid yeast in the selective medium. Only yeast that expressed both Sept6 and Sept7 was able to grow. This result strongly supports the
hypothesis that binding of Borg3 occurs to a Sept6-Sept7 heterodimer and not to isolated monomers.

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 5.
A, Borg3 binds specifically to the
trimer and Sept6-Sept7 heterodimer. Septins were prepared as
described in the Fig. 1 legend and under "Materials and Methods."
GST-BD3 and GST alone (10 µg) were attached to glutathione beads and
incubated for 45 min with the septins (20 µg) at 4 °C. The beads
were then washed, and bound proteins were eluted with glutathione and
analyzed by SDS-PAGE and staining with Coomassie Brilliant Blue.
B, binding was performed as described for panel A
but to GST or full-length GST-Borg3. Bound HS-tagged protein was
detected by immunoblotting with horseradish peroxidase-coupled
S-protein. C, yeast three-hybrid conjugation assay. Yeast
that expressed LexA-DBD-Sept6 and/or untagged Sept7 (or vector alone)
were mated with yeast that expressed VP16-Borg3. Successful mating was
tested on a selective medium lacking Leu, Trp, and Ura. Interaction of
the Borg3 with the Sept6 was tested on a selective medium also lacking
His and containing a histidine synthesis inhibitor, 3-aminotriazole.
D, the C-terminal coiled-coil region of Sept6 is sufficient
for binding of Borg3 to the Sept6/7 dimer. HS-tagged Sept7 was
expressed together with either the N-terminal region (1-282) or the C
terminus (283-429) of Sept6, purified on Ni2+-agarose
beads, and then incubated with GST-BD3 or GST on glutathione beads.
After washing, the bound proteins were eluted with glutathione,
analyzed by SDS-PAGE, and stained with Coomassie Brilliant Blue.
|
|
To begin to identify the regions of these septins that are required for
the interaction with Borg3, we used the isolated C-terminal coiled-coil
region of Sept6 and full-length Sept7. Although Borg3 did not bind to
either of these proteins alone, it did bind to the paired proteins
(Fig. 5D). Note that this result supports the observation
(Fig. 4) that the C-terminal domain of Sept6, which lacks the
GTP-binding domain, is sufficient to bind Sept7. The N-terminal region
of Sept6 is mostly insoluble and was not captured in a complex with
Sept7 by GST-BD3 on beads (Fig. 5D). We conclude, therefore,
that Borg3 binding to septins requires the Sept6-Sept7 heterodimer and
that it does not require both GTP binding domains. We have been unable
thus far to detect Borg3 binding to the dimerized coiled-coil domains
of Sept6-Sept7, however, suggesting that the BD3 binding domain may
extend further into the N-terminal region than do the dimerization motifs.
To ascertain whether Borg3 modulates septin dimerization, we mixed
recombinant Sept6 and Sept7 monomers in the presence of [
-32P]GTP with or without GST-Borg3, but no difference
in nucleotide binding was observed. Similarly, when a preformed
Sept6-Sept7 dimer was loaded with [
-32P]GTP and
exposed to Borg3, no change in the release of 32P from the
protein was observed (data not shown). Therefore, Borg3 does not
increase the GTPase activity during or after dimerization.
 |
DISCUSSION |
Our understanding of septin function depends on knowledge of the
biochemistry of these proteins. However, their study has been hampered
by difficulties associated with their expression as recombinant
proteins in bacteria and the complexity of their interactions with one
another. We have developed systems to express multiple septins in
E. coli and have demonstrated that three septins can
together form filaments with dimensions similar to those purified from
cells. Understanding their biochemistry remains a challenge, however,
because the septin oligomers bind guanine nucleotides and hydrolyze
GTP, only very slowly. Moreover, the instability of the monomers
complicates investigations of oligomer assembly.
Nonetheless, we can begin to understand the assembly process. First, we
conclude that any pair of septins (at least Sept2, Sept6, and Sept7)
can form heterodimers, although the Sept6/7 dimer is more stable;
second, at least the Sept6/7 dimer associates through the coiled-coil
regions in the C terminus of each septin rather than through their
GTP-binding domains; and third, GTP hydrolysis accompanies dimer
formation (although we cannot prove that hydrolysis is required for
dimerization). Finally, we have found that the septin binding protein,
Borg3, associates not with individual monomeric septins, but rather
that it specifically recognizes the Sept6/7 heterodimer. We speculate
that the BD3 domain binds at the coiled-coil interface between the
subunits of the dimer.
The mechanism by which Borg3 binding triggers septin reorganization in
cells remains obscure, however, because we have been unable to detect
any effect on dimerization or on recombinant filament bundling in
vitro. One interesting possibility is that it displaces another
protein, perhaps another septin such as Sept9, which normally
associates with Sept6/7 in the cell via an overlapping binding site. We
are currently investigating the effects of Borg3 on septin dynamics,
both in vivo and in vitro. The ability to express
recombinant septin filaments and heterodimers will provide powerful new
tools in this endeavor, because these proteins can be tagged with
fluorescent markers and injected into cells. Speckle microscopy can
then be used to analyze the filament dynamics. The ability to express
septin dimers and trimers will also be useful for production of
affinity ligands, to aid in the identification of septin binding
proteins from mammalian cell extracts, and to search for regulatory
factors that may modulate GTP binding or hydrolysis. Finally, the
ability to produce septin oligomers is an essential step toward the
goal of obtaining high resolution x-ray crystallographic structures of
the septins.