Regions of Byr4, a Regulator of Septation in Fission Yeast, That Bind Spg1 or Cdc16 and Form a Two-component GTPase-activating Protein with Cdc16*

Kyle A. FurgeDagger §, Qiu-chen ChengDagger , Mira Jwa, Sejeong Shin, Kiwon Song, and Charles F. AlbrightDagger parallel

From the Dagger  Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and the  Department of Biochemistry, College of Science, Yonsei University, Seoul 120-749, Korea

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the fission yeast Schizosaccharomyces pombe, septation and constriction of the actomyosin ring for cell division are positively regulated by the Spg1 GTPase, a member of the Ras superfamily. Spg1 is negatively regulated by Byr4 and Cdc16, which together form a two-component GTPase-activating protein for the Spg1 GTPase. To better understand how Byr4 regulates septation, Byr4 mutants were tested for in vitro functions. This analysis revealed that Byr4 contained one Cdc16-binding site and four Spg1-binding sites (SBS), designated SBS1-SBS4. Although mutants with a single SBS bound Spg1 and inhibited GTP dissociation, the equilibrium binding affinity of these mutants was 28-280-fold weaker than Byr4. Because some Byr4 mutants with multiple SBSs bound Spg1 tighter than the corresponding mutants with a single SBS, multiple SBSs probably interact to cause the high affinity binding of Byr4 to Spg1. A region of Byr4 that bound Spg1, SBS4, and the region that bound Cdc16, Cdc16-binding site, was necessary and sufficient to form Cdc16-dependent Spg1GAP activity that was similar to that of wild-type Byr4 with Cdc16.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-[gamma -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.
<FR><NU><UP>Spg1</UP>−[&ggr;-<SUP>32</SUP><UP>P</UP>]<UP>GTP</UP></NU><DE>(<UP>Spg1</UP>−[&ggr;-<SUP>32</SUP><UP>P</UP>]<UP>GTP</UP>)<SUB>t<UP>=</UP>0</SUB></DE></FR>=<UP>exp</UP>−<FR><NU>k<SUB>h</SUB>+k<SUB>t</SUB></NU><DE>t</DE></FR> (Eq. 1)
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.
k<SUB>o</SUB>=<FR><NU>k<SUB>h</SUB>+k<SUB>t</SUB></NU><DE>1+<FR><NU><UP>Byr4</UP></NU><DE>K<SUB>b</SUB></DE></FR></DE></FR> (Eq. 2)
If Byr4 mutants inhibited only GTP dissociation, then the observed rate of radioactivity lost from Spg1-GTP would be as follows.
k<SUB>o</SUB>=k<SUB>h</SUB>+<FR><NU>k<SUB>t</SUB></NU><DE>1+<FR><NU><UP>Byr4</UP></NU><DE>K<SUB>b</SUB></DE></FR></DE></FR> (Eq. 3)
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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (11K):
[in this window]
[in a new window]
 
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.

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-[gamma -32P]GTP about 50% at saturating concentrations with a 140 nM Kb (Fig. 2, A and B) and from Spg1-[gamma -35S]GTP (Fig. 2C), showing that SBS1 inhibited GTP dissociation. Because Byr4-D3 only partially inhibited loss of radioactivity from Spg1-[gamma -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-[gamma -32P]GTP with a 175 nM Kb (Fig. 2, A and B) and from Spg1-[gamma -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-[gamma -32P]GTP with 1400 and 300 nM Kb, respectively (Fig. 2B), and from Spg1-[gamma -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).


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 2.   Identification of Spg1-binding sites in Byr4. A, Byr4 mutant proteins were incubated with Spg1-[gamma -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-[gamma -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-[gamma -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.

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.


View larger version (12K):
[in this window]
[in a new window]
 
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-[gamma -32P]GTP, and the fraction of the initial Spg1-[gamma -32P]GTP following a 6-min incubation at 30 °C was measured using a filter binding assay as before.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    ACKNOWLEDGEMENT

We thank Kathleen Mach for helpful comments on the manuscript.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant GM-51952 (to C. F. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Partially supported by National Institutes of Health Grant T32-CA09582 (to G. Carpenter).

parallel To whom correspondence should be addressed: Dept. of Biochemistry, 655 Light Hall, Vanderbilt University School of Medicine, Nashville, TN 37232-0146. Tel.: 615-343-2174; Fax: 615-343-0704; E-mail: albright{at}lhmrba.hh.vanderbilt.edu.

    ABBREVIATIONS

The abbreviations used are: GAP, GTPase-activating protein; CBS, Cdc16-binding site; SBS, Spg1-binding site; CRIB, Cdc42/Rac interactive binding; Gpp(NH)p, guanosine 5'-(beta ,gamma -imido)triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
  1. Gould, K., and Simanis, V. (1997) Genes Dev. 11, 2939-2951[Free Full Text]
  2. Marks, J., Hagan, I., and Hyams, J. (1987) Spec. Publ. Soc. Gen. Microbiol. 23, 119-135
  3. Jochova, J., Rupes, I., and Streiblova, E. (1991) Cell Biol. Int. Rep. 15, 607-610[Medline] [Order article via Infotrieve]
  4. Fankhauser, C., Reymond, A., Cerutti, L., Utzig, S., Hofmann, K., and Simanis, V. (1995) Cell 82, 435-444[Medline] [Order article via Infotrieve]
  5. McCollum, D., Balasubramanian, M., Pelcher, L., Hemmingsen, S., and Gould, K. (1995) J. Cell Biol. 130, 1-11[Abstract]
  6. Chang, F., Drubin, D., and Nurse, P. (1997) J. Cell Biol. 137, 169-182[Abstract/Free Full Text]
  7. Kitayama, C., Sugimoto, A., and Yamamoto, M. (1997) J. Cell Biol. 137, 1309-1319[Abstract/Free Full Text]
  8. Johnson, B., Yoo, B., and Calleja, G. (1973) J. Bacteriol. 115, 358-366[Medline] [Order article via Infotrieve]
  9. Schmidt, S., Sohrmann, M., Hofmann, K., Woollard, A., and Simanis, V. (1997) Genes Dev. 11, 1519-1534[Abstract]
  10. Fankhauser, C., and Simanis, V. (1994) EMBO J. 13, 3011-3019[Abstract]
  11. Balasubramanian, M., McCollum, D., Chang, L., Wong, K., Naqvi, N., He, X., Sazer, S., and Gould, K. (1998) Genetics 149, 1265-1275[Abstract/Free Full Text]
  12. Minet, M., Nurse, P., Thuriaux, P., and Mitchison, J. (1979) J. Bacteriol. 137, 440-446[Medline] [Order article via Infotrieve]
  13. Song, K., Mach, K. E., Chen, C. Y., Reynolds, T., and Albright, C. F. (1996) J. Cell Biol. 133, 1307-1319[Abstract]
  14. Sohrman, M., Schmidt, S., Hagan, I., and Simanis, V. (1998) Genes Dev. 12, 84-94[Abstract/Free Full Text]
  15. Furge, K., Wong, K., Armstrong, J., Balasubramanian, M., and Albright, C. F. (1998) Curr. Biol. 8, 947-954[Medline] [Order article via Infotrieve]
  16. Neuwald, A. (1997) Trends Biochem. Sci. 22, 243-244[CrossRef][Medline] [Order article via Infotrieve]
  17. Boguski, M. S., and McCormick, F. (1993) Nature 366, 643-654[CrossRef][Medline] [Order article via Infotrieve]
  18. Jwa, M., and Song, K. (1998) Molecules Cells 8, 240-245
  19. Shirayama, M., Matsui, Y., and Toh-e, A. (1994) Mol. Cell. Biol. 14, 7476-7482[Abstract]
  20. Jaspersen, S., Charles, J., Tinker-Kulberg, R., and Morgan, D. (1998) Mol. Biol. Cell 9, 2803-2817[Abstract/Free Full Text]
  21. Fankhauser, C., Marks, J., Reymond, A., and Simanis, V. (1993) EMBO J. 12, 2697-2704[Abstract]
  22. Hoyt, M. A., Totis, L., and Roberts, B. (1991) Cell 66, 507-517[Medline] [Order article via Infotrieve]
  23. Herrmann, C., Martin, G., and Wittinghofer, A. (1995) J. Biol. Chem. 270, 2901-2905[Abstract/Free Full Text]
  24. Manser, E., Leung, T., Salihuddin, H., Zhao, Z., and Lim, L. (1994) Nature 367, 40-46[CrossRef][Medline] [Order article via Infotrieve]
  25. Burbelo, P., Drechel, D., and Hall, A. (1995) J. Biol. Chem. 270, 29071-29074[Abstract/Free Full Text]
  26. Rudolph, M., Bayer, P., Abo, A., Kuhlmann, J., Vetter, I., and Wittinghofer, A. (1998) J. Biol. Chem. 273, 18067-18076[Abstract/Free Full Text]
  27. Wu, J., Matunis, M., Kraemer, D., Blobel, G., and Coutavas, E. (1995) J. Biol. Chem. 270, 14209-14213[Abstract/Free Full Text]
  28. Yokoyama, N., Hayashi, N., Seki, T., Pante, N., Ohba, T., Nishii, K., Kuma, K., Hayashida, T., Miyata, T., Aebi, U., Fukui, M., and Nishimoto, T. (1995) Nature 376, 184-188[CrossRef][Medline] [Order article via Infotrieve]
  29. Scheffzek, K., Ahmadian, M., and Wittinghofer, A. (1998) Trends Biochem. Sci. 23, 257-262[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.