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INTRODUCTION |
In many eukaryotes, including Schizosaccharomyces
pombe, cell division results from the constriction of an
actomyosin ring that is perpendicular to the mitotic spindle (reviewed
in Ref. 1). In fission yeast, a medial ring composed of F-actin and other proteins forms as cells enter mitosis. This ring is formed at the
future site of cell division (2). Following anaphase, this actomyosin
ring constricts (2-7), a primary septum is deposited, and secondary
septa are formed on both sides of the primary septum (8). The primary
septum is then degraded to yield two cells. Characterization of fission
yeast mutants that affect these processes suggests they represent the
following four categories: 1) division site selection mutants, 2)
actomyosin ring assembly mutants, 3) actomyosin ring constriction
and septation initiation mutants, and 4) cell separation mutants
(reviewed in Ref. 1).
cdc7
and spg1
mutants
form a medial ring but do not constrict this ring or deposit a septum,
leading to elongated, multinucleate cells (9-11). In contrast,
cdc16
and byr4
mutants undergo repeated rounds of septation (12, 13). These phenotypes
suggest that Cdc7 and Spg1 are positive regulators of septation,
whereas Cdc16 and Byr4 are negative regulators of septation. Consistent
with this notion, Spg1 and Cdc7 overexpression causes arrest of cells
with multiple septa (9, 10), whereas Byr4 overexpression causes arrest
of cells with multiple nuclei (13).
Spg1 is a GTPase of the Ras superfamily that is constitutively
localized to spindle-pole bodies where it presumably regulates septation (9, 14). In its GTP-bound form, Spg1 binds the Cdc7 protein
kinase and causes it to translocate to spindle poles (10, 14).
Consistent with their role as negative regulators of septation, Byr4
and Cdc16 form a two-component GTPase-activating protein
(GAP)1 (1) for the Spg1
GTPase (15). Although a role for Cdc16 in Spg1GAP activity is not
surprising because Cdc16 has sequence similarity to proteins with
YptGAP activity (9, 16), the requirement for two proteins for GAP
activity is unexpected because GAPs for other Ras family
GTPases contain one component (17). In addition to its role in
Spg1GAP activity, Byr4 binds Spg1, and this binding inhibits GTP
dissociation and hydrolysis but does not affect GDP dissociation (15).
Cdc16 binds Byr4 but does not bind Spg1 or affect the nucleotide bound
to Spg1 in the absence of Byr4. Byr4-Spg1 binding and Byr4-Cdc16
Spg1GAP activity are specific for Spg1 because Byr4 and Cdc16 do not
interact with Ypt family GTPases, which are the most similar to Spg1 by
primary sequence comparison. Genetic experiments support the
biochemical interactions between Spg1, Byr4, and Cdc16 (15, 18). Mild Byr4 overexpression suppresses the temperature-sensitive growth of
cdc16-116 mutants. This suppression is allele-specific
because Byr4 does not suppress the growth defect of
cdc16
mutants. Whereas higher Byr4
overexpression is lethal, Spg1 overexpression suppresses this lethality.
A Byr4-like regulator and a two-component GAP are unique to the Spg1
GTPase. Novel regulators for Spg1 are consistent with classification of
Spg1 in a separate subfamily of the Ras superfamily (9) because
regulators for different subfamilies are usually distinct (17). The
Saccharomyces cerevisiae Tem1 GTPase is currently the only
other member of the Spg1 subfamily (9). Unlike
spg1
mutants, tem1
mutants arrest in late mitosis perhaps because of an inability to
degrade mitotic cyclins (19, 20). Whereas tem1
mutants do not undergo septation or cytokinesis, it is unclear whether
this failure results from their cell cycle arrest or reflects a
requirement for Tem1 to regulate septation and cytokinesis. The
S. cerevisiae Bub2 protein is the likely homologue of Cdc16 because these proteins are globally similar, and BUB2
suppresses the temperature-sensitive growth defect of
cdc16-116 mutants (21). cdc16
mutants and bub2
mutants are similar in that
they are defective in the spindle-assembly checkpoint, but differ in
that BUB2 is not essential for viability (21, 22).
To further understand how Byr4 regulates septation, Byr4 mutants
were assayed for binding to Cdc16 or Spg1 and for Spg1GAP activity with
Cdc16. A single Cdc16-binding site (CBS) and four Spg1-binding sites
(SBS) were identified. A C-terminal fragment of Byr4, which contained
SBS4 and CBS, was necessary and sufficient for Spg1GAP activity with Cdc16.
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MATERIALS AND METHODS |
pbyr4/ET14b, pcdc16/GEX, and pspg1/GEX were previously described
(13, 15). byr4 mutants were constructed by amplification of
the byr4 cDNA using 5'-oligonucleotides with an
NdeI site and 3'-oligonucleotides with a BamHI
site. Amplified products were digested with NdeI and
BamHI and ligated with pET14b that was similarly digested.
Byr4 mutants, Gst-Cdc16, and Gst-Spg1 were expressed in
BL21(DE3)pLysE, NM522, and XL1-Blue bacteria, respectively. Methods
for purification of Spg1, Cdc16, and Byr4, Cdc16-Byr4 and Spg1-Byr4
binding assays, and Spg1GAP assays were previously described (13,
15).
The affinity of Byr4 mutants for Spg1 was determined by measuring the
effect of Byr4 on Spg1 GTPase properties. Radioactivity is lost from
Spg1-[
-32P]GTP by GTP dissociation and hydrolysis. The
fraction of the initial radioactivity bound to Spg1 is described in
terms of the GTP dissociation rate, kt, the GTP
hydrolysis rate, kh, and time, t, by
the following equation.
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(Eq. 1)
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Because the Byr4-Spg1-GTP complex had negligible rates of GTP
dissociation and hydrolysis (15), the effect of Byr4 on the observed
rate of radioactivity lost from Spg1-GTP, ko,
was used to measure the Byr4-Spg1-GTP equilibrium binding constant,
Kb. Kb is related to
ko by the following equation.
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(Eq. 2)
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If Byr4 mutants inhibited only GTP dissociation, then the
observed rate of radioactivity lost from Spg1-GTP would be as
follows.
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(Eq. 3)
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In either case, 50% maximal inhibition occurs when the Byr4
concentration equals Kb. Ras-Raf-1 binding was
measured similarly (23). Our experience with this method revealed that
the measured equilibrium constants varied by less than 50% between
different Byr4 preparations.
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RESULTS |
Byr4 C Terminus Is Necessary and Sufficient for Cdc16
Binding--
Byr4 contains 665 amino acids, and the only motifs
detected by primary sequence inspection were two imperfect direct
repeats of 43 amino acids (13) (Fig.
1A). To identify Byr4 regions
that bind Cdc16, Byr4 deletion mutants were expressed in
Escherichia coli, purified, and assayed for binding to
Gst-Cdc16 (Fig. 1A). Byr4-D2, a mutant with residues
533-665, bound Gst-Cdc16, although Byr4-D6, a mutant with residues
1-535, did not bind Gst-Cdc16 (Fig. 1B). To further limit
the CBS, mutants containing the N- and C-terminal portions of Byr4-D2
were analyzed. These assays showed that Byr4-D10, a mutant with
residues 595-665, bound Gst-Cdc16, although Byr4-D14 did not bind
Gst-Cdc16 (Fig. 1B). As with wild-type Byr4 (15), Byr4-D2
and Byr4-D10 degradation products that copurified with the full-length
proteins did not bind Gst-Cdc16 (Fig. 1B). This result was
consistent with a requirement for the Byr4 C terminus to bind Cdc16
because these degradation products were missing C-terminal residues
(data not shown). We conclude that the Byr4 C terminus is necessary and
sufficient for Byr4-Cdc16 binding.

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Fig. 1.
Ability of Byr4 mutant proteins to bind
Cdc16. A, a schematic diagram illustrates the location
of the direct repeats (arrows), mutant boundaries
(numbers), and mutant designation (left column).
Byr4 mutants that bound (+) or did not bind ( )
Gst-Cdc16 are indicated. WT, wild type. B,
purified Byr4-D6, Byr4-D2, Byr4-D10, or Byr4-D14 were incubated with
beads containing Gst (Gst) or Gst-Cdc16 (Cdc16),
and Byr4 mutant proteins bound to the beads after washing were detected
by Western blot analysis with anti-Byr4 antibodies (15). 5% of the
input protein (In) is shown. Byr4-D2 and Byr4-D10 bound to
Gst-Cdc16 due to an interaction with Cdc16 because Byr4-D2 and Byr4-D10
did not bind Gst. Technical limitations of this assay prevented
measurement of the binding affinity.
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Byr4 Contains Multiple SBSs--
The ability of Byr4 to inhibit
the dissociation and hydrolysis of GTP that is bound to Spg1 was used
to identify SBS in Byr4 (15). This assay was advantageous because it
was quantitative, and binding was measured under equilibrium
conditions. The equilibrium binding constant,
Kb, is the Byr4 concentration where 50% of the maximal inhibition of Spg1 GTPase properties occurs (15, 23). Four
SBSs, designated SBS1, SBS2, SBS3, and SBS4, were identified using this
assay (Fig. 2A). SBS1 was
contained within Byr4-D3, which had residues 1-200. Byr4-D3 inhibited
loss of radioactivity from Spg1-[
-32P]GTP about 50%
at saturating concentrations with a 140 nM
Kb (Fig. 2, A and B) and
from Spg1-[
-35S]GTP (Fig. 2C), showing that
SBS1 inhibited GTP dissociation. Because Byr4-D3 only partially
inhibited loss of radioactivity from Spg1-[
-32P]GTP,
Byr4-D3 may not affect GTP hydrolysis. Mutants that deleted sequences
in Byr4-D3, such as Byr4-D17 and Byr4-D18, did not bind Spg1 (Fig.
2A). SBS2 was contained within Byr4-D27, which had residues
200-475. Byr4-D27 inhibited loss of radioactivity from Spg1-[
-32P]GTP with a 175 nM
Kb (Fig. 2, A and B) and
from Spg1-[
-35S]GTP (Fig. 2C), showing that
Byr4-D27 inhibited both GTP hydrolysis and dissociation. Mutants that
deleted sequences in Byr4-D27, such as Byr4-D22 and Byr4-D21,
significantly weakened binding to Spg1 (Fig. 2A). SBS3 and
SBS4 were contained within Byr4-D13 and Byr4-D14, respectively (Fig.
2A). Byr4-D13 and Byr4-D14 inhibited loss of radioactivity
from Spg1-[
-32P]GTP with 1400 and 300 nM
Kb, respectively (Fig. 2B), and from Spg1-[
-35S]GTP (Fig. 2C), showing that
Byr4-D13 and Byr4-D14 inhibited both GTP hydrolysis and dissociation.
The boundaries of these mutants corresponded to the imperfect, direct
repeats identified by primary sequence analysis (13). Byr4-D10, which
contained CBS, did not affect Spg1 GTPase properties (Fig.
2B). Hence, Byr4 contained four SBSs with equilibrium
binding constants from 140 to 1400 nM. These binding
affinities were much weaker than that of Byr4, which had a 5 nM Kb (15).

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Fig. 2.
Identification of Spg1-binding sites in
Byr4. A, Byr4 mutant proteins were incubated with
Spg1-[ -32P]GTP, and the decrease in filter-bound
radioactivity was measured. The 50% maximal inhibition value,
Kb, for each mutant is indicated in the
right column. B, the fraction of the initial
Spg1-[ -32P]GTP following a 6-min incubation with the
indicated amount of Byr4-D3 and Byr4-D10 (upper graph) and
Byr4-D27, Byr4-D13, and Byr4-D14 (lower graph) is shown.
Data points are the average of at least two assays, which typically
differed by less than 5%. C, the fraction of the initial
Spg1-[ -35S]GTP following a 6-min incubation without
Byr4 ( ) or with Byr4-D3, Byr4-D27, Byr4-D13, and Byr4-D14
is shown. D, the Spg1 binding affinity,
Kb, for Byr4 mutants with multiple SBSs were
determined as described for B. E, purified Byr4
mutants were incubated with beads containing Gst (Gst),
Gst-Spg1-GDP (GDP), or Gst-Spg1-Gpp(NH)p (GTP),
and bound proteins were used for Western analysis with anti-Byr4
antibodies. 5% of the input protein (In) is shown.
Arrows indicate the location of the direct repeats.
S1, SBS1; S2, SBS2; S3, SBS3;
S4, SBS4; WT, wild type.
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The affinities of Byr4 mutants with multiple SBSs were measured to
determine which Byr4 regions were required for maximal binding to Spg1.
Two mutants with two sequential SBSs bound tighter than the
corresponding mutants with a single SBS. Byr4-D4, a mutant with SBS1
and SBS2, and Byr4-D7, a mutant with SBS2 and SBS3, had 50-60
nM Kb, which were about 3-fold lower
than mutants with the corresponding single SBS (Fig. 2D).
Interestingly, Byr4-D2, a mutant with SBS4 and CBS, had a 6-fold lower
Kb than a mutant with only SBS4. Whether the
higher affinity of Byr4-D4, Byr4-D7, and Byr4-D2 resulted from
simultaneous binding of multiple SBSs or increased binding of a single
SBS is unknown. In contrast, Byr4-D11, a mutant with SBS3 and SBS4, had
a 300 nM Kb, which was similar to
that of mutants with only SBS4 (Fig. 2D). The lack of
cooperativity between SBS3 and SBS4 was not surprising because these
domains share primary sequence similarity (13). All mutants tested with
three or four sequential SBSs and/or CBS had 30-50 nM
Kb (Fig. 2D), showing that these
combinations did not significantly increase Spg1 binding. Because a 5 nM Kb for Byr4-Spg1 was measured (15), we conclude that SBS1, CBS, and perhaps SBS2, SBS3, or SBS4 are
required for maximal binding affinity.
Byr4 binding to Spg1-GDP could not be measured using changes in GDP
dissociation because Byr4 did not affect this GTPase property (15).
Instead, the ability of Byr4 mutants to bind immobilized Spg1-GDP or
Spg1-Gpp(NH)p was used to test the effect of the Spg1 nucleotide state
on Byr4 binding. These assays showed that Byr4-D3 and Byr4-D27, mutants
with only SBS1 or SBS2, respectively, bound Spg1-Gpp(NH)p slightly
better than Spg1-GDP (Fig. 2E). Byr4-D13 and Byr4-D14,
mutants with only SBS3 or SBS4, respectively, bound Spg1-Gpp(NH)p much
better than Spg1-GDP (Fig. 2E). These binding preferences
were unexpected because Byr4 bound equally to Spg1-Gpp(NH)p and
Spg1-GDP (15). To understand the basis for the nucleotide specificity
difference between the individual SBSs and Byr4, Byr4 mutants with
multiple SBSs were assayed. Byr4-D1, a mutant with SBS2, SBS3, and
SBS4, bound Spg1-Gpp(NH)p slightly better than Spg1-GDP (Fig.
2E). Because the nucleotide specificity of Byr4-D1 binding
was similar to that of Byr4-D27, SBS2 appeared to dominate the
nucleotide specificity of Byr4-D27. Byr4-D4, a mutant with SBS1 and
SBS2, bound equally to Spg1-Gpp(NH)p and Spg1-GDP, suggesting that SBS1
and SBS2 interact to affect both binding specificity and affinity (Fig.
2E). The nucleotide-independent binding of Byr4-D4 to Spg1
probably explains the binding behavior of Byr4. Hence, Byr4 contains
four SBSs and some of these SBSs interact to cause high affinity,
nucleotide-independent binding to Spg1.
SBS4 and CBS Are Necessary and Sufficient to Form Spg1GAP Activity
with Cdc16--
Because a two-component GAP is unique to Byr4 and
Cdc16, we determined what regions of Byr4 were required for this
reaction. This analysis showed that Byr4-D2, which contained SBS4 and
CBS, was the minimal mutant that had Spg1GAP activity with Cdc16 (Fig. 3A). Neither Byr4-D16, which
lacked CBS, nor Byr4-D10, which contained only CBS, had detectable
Spg1GAP activity. Byr4 mutants without CBS had at least 100-fold less
Spg1GAP activity than Byr4 because Spg1GAP activity was detected with 2 nM Byr4 but not with 200 nM Byr4-D16 (Fig.
3B and data not shown). Spg1GAP assays with a range of
Byr4-D2 and Byr4 concentrations revealed that Byr4 and Byr4-D2 had
similar Spg1GAP activity (Fig. 3B). Hence, a region of Byr4
that binds Spg1 (SBS4) and a region that binds Cdc16 (CBS) are
necessary and sufficient to form Cdc16-dependent Spg1GAP
activity.

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Fig. 3.
Ability of Byr4 mutant proteins and Cdc16 to
form Spg1GAP activity. A, Byr4 mutants that formed
(+) or did not form Spg1GAP activity ( ) are
indicated. Arrows indicate the location of the direct
repeats. WT, wild type. B, the indicated
concentrations of Byr4 or Byr4-D2 were added to reactions with Cdc16
and Spg1-[ -32P]GTP, and the fraction of the initial
Spg1-[ -32P]GTP following a 6-min incubation at
30 °C was measured using a filter binding assay as before.
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 |
DISCUSSION |
To better understand how Byr4 regulates septation, Byr4 mutants
were assayed for binding to Spg1 or Cdc16 and for formation of Spg1GAP
activity with Cdc16. This analysis revealed that Byr4 contained one CBS
and four SBSs, designated SBS1-SBS4. SBS4 and CBS were necessary and
sufficient to form Spg1GAP activity with Cdc16, which was very similar
to that of Byr4.
Biochemical and sequence analyses suggest that the four SBSs in Byr4
represent three classes of SBSs. One SBS class includes SBS3 and SBS4,
which correspond to the imperfect direct repeats of 43 amino acids
identified by primary sequence analysis (13). Like Byr4, SBS3 and SBS4
inhibited GTP dissociation and hydrolysis by Spg1. Unlike Byr4, SBS3
and SBS4 bound Spg1-GTP with a 280- and 60-fold, respectively, weaker
affinity than Byr4 and bound Spg1-GTP better than Spg1-GDP. Although
unrelated in primary sequence, SBS3 and SBS4 have some similarities to
the Cdc42/Rac interactive binding (CRIB) motif, a sequence that binds
the Cdc42 and Rac1 GTPases (24) and is found in several proteins (25).
Like CRIB motifs, SBS3 and SBS4 are relatively short in length and bind the GTP-bound form of the GTPase better than the GDP-bound form of the
GTPase. Strengthening of SBS4 binding to Spg1-GTP by CBS is similar to
results found with the CRIB motif from the Wiskott-Aldrich syndrome
protein. In the case of Byr4, the relatively weak binding of SBS4 to
Spg1-GTP, characterized by an equilibrium binding affinity of 300 nM, was strengthened 5-fold by sequences containing CBS, which did not detectably interact with Spg1 without SBS4. In a similar
manner, the relatively weak binding of Cdc42-GTP to the CRIB peptide
from Wiskott-Aldrich syndrome protein, characterized by an equilibrium
binding affinity of 480 nM, was strengthened about 6-fold
by adjacent sequences that did not bind Cdc42-GTP in isolation (26).
Perhaps there will be additional similarities between SBS3/SBS4 and
CRIB motifs. It will be particularly interesting to test whether SBS3
and SBS4, like the Wiskott-Aldrich syndrome protein-CRIB motif (26),
lack a defined domain structure in the absence of the GTPase.
Cdc7, a protein kinase that is likely an effector of Spg1 (14), may use
a motif similar to SBS3 and SBS4 to bind Spg1 because Cdc7 contains a
sequence similar to SBS3 and SBS4 (13). If this is the case, then Byr4
and Cdc7 may bind competitively to Spg1, and Byr4 might negatively
regulate Spg1 by inhibiting effector binding as well as by forming
Spg1GAP activity with Cdc16.
SBS1 and SBS2 likely define distinct classes of SBSs. SBS1 and SBS2
differ from SBS3/SBS4 in that they lack the conserved sequence motifs
found in SBS3 and SBS4. Furthermore, SBS1 and SBS2 bind similarly to
Spg1-GTP and Spg1-GDP. SBS1 and SBS2 differ in that SBS1 inhibits GTP
hydrolysis weakly, if at all, whereas SBS2 inhibits GTP hydrolysis.
Consistent with SBS1, SBS2, and SBS3/SBS4 representing different
classes of SBSs, mutants with SBS1/SBS2 or SBS2/SBS3 bind Spg1 better
than the corresponding mutants with only one SBS.
Multiple SBSs were unexpected because of the other
GTPase-binding proteins; only Ran-binding protein 2 contains
multiple binding sites for the same GTPase (27, 28). Multiple SBSs may
be needed to increase Byr4-Spg1 affinity because all deletion mutants
had at least 10-fold weaker affinity than wild-type Byr4. In this case,
different SBSs may interact with different regions of Spg1 to form the
high affinity binding observed between Byr4 and Spg1. The increased
binding affinity of mutants with more than one SBS is consistent with
this notion. Multiple SBSs may also allow Byr4 to bind more than one
Spg1 molecule in vivo. Multivalent binding could
dramatically increase the effective Byr4-Spg1 affinity and allow
different SBSs to perform distinct functions. For instance, some SBSs
may target Byr4 to spindle poles, whereas other SBSs may form Spg1GAP
activity with Cdc16. In vivo analysis of Byr4 mutants will
be needed to understand the physiologic roles of the different SBSs.
Sequences containing SBS4 and CBS were necessary and sufficient for
Spg1GAP activity with Cdc16. GAPs are unusual enzymes in that two
proteins, the GTPase and the GAP, interact transiently and contain
residues important for catalysis. GAPs are thought to stimulate GTP
hydrolysis by stabilizing switch regions in the GTPase that orient the
GTPase catalytic machinery and by supplying an external arginine
residue that stabilizes the charge in the transition state (reviewed in
Ref. 29). Byr4 and Cdc16 may further divide these GAP functions. SBS4,
one part of Byr4 required for Spg1GAP activity, binds Spg1-GTP, affects
its GTPase properties, and may stabilize the switch regions of Spg1.
CBS, another part of Byr4 required for Spg1GAP activity, binds Cdc16
and is presumably required for Cdc16 to interact with Spg1. Once near
Spg1, Cdc16 may supply the arginine residue presumably required for
Spg1GAP activity. Byr4 would thus provide both binding and catalytic
functions required for Spg1GAP activity in this model. The division of
traditional GAP activities into two proteins may allow additional
regulation of Spg1GAP activity.