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Septin ring assembly involves cycles of GTP loading and hydrolysis by Cdc42p.

Gladfelter AS, Bose I, Zyla TR, Bardes ES, Lew DJ - J. Cell Biol. (2002)

Bottom Line: In performing its roles in actin polarization and transcriptional activation, GTP-Cdc42p is thought to function by activating and/or recruiting effectors to the site of polarization.Excess accumulation of GTP-Cdc42p due to a defect in GTP hydrolysis by the septin-specific alleles might cause unphysiological activation of effectors, interfering with septin assembly.These results suggest that a single GTPase, Cdc42p, can act either as a ras-like GTP-dependent "switch" to turn on effectors or as an EF-Tu-like "assembly factor" using the GTPase cycle to assemble a macromolecular structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.

ABSTRACT
At the beginning of the budding yeast cell cycle, the GTPase Cdc42p promotes the assembly of a ring of septins at the site of future bud emergence. Here, we present an analysis of cdc42 mutants that display specific defects in septin organization, which identifies an important role for GTP hydrolysis by Cdc42p in the assembly of the septin ring. The mutants show defects in basal or stimulated GTP hydrolysis, and the septin misorganization is suppressed by overexpression of a Cdc42p GTPase-activating protein (GAP). Other mutants known to affect GTP hydrolysis by Cdc42p also caused septin misorganization, as did deletion of Cdc42p GAPs. In performing its roles in actin polarization and transcriptional activation, GTP-Cdc42p is thought to function by activating and/or recruiting effectors to the site of polarization. Excess accumulation of GTP-Cdc42p due to a defect in GTP hydrolysis by the septin-specific alleles might cause unphysiological activation of effectors, interfering with septin assembly. However, the recessive and dose-sensitive genetic behavior of the septin-specific cdc42 mutants is inconsistent with the septin defect stemming from a dominant interference of this type. Instead, we suggest that assembly of the septin ring involves repeated cycles of GTP loading and GTP hydrolysis by Cdc42p. These results suggest that a single GTPase, Cdc42p, can act either as a ras-like GTP-dependent "switch" to turn on effectors or as an EF-Tu-like "assembly factor" using the GTPase cycle to assemble a macromolecular structure.

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GTP hydrolysis by Cdc42pV36T,K94E and Cdc42pY32H. GST-Cdc42p (•), GST-Cdc42pY32H (▵), and GST-Cdc42pV36T,K94E (□) were prebound to [γ-32P]GTP and incubated with buffer alone (A) or with a 1:10 molar ratio of recombinant GST-Rga1p GAP domain (B), and radioactivity remaining bound to Cdc42p is plotted against time of incubation. Control experiments indicated that the Rga1p GAP domain did not stimulate release of 32P from Cdc42p prebound with [α-32P]GTP, indicating that Rga1p stimulates GTP hydrolysis and not release.
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fig4: GTP hydrolysis by Cdc42pV36T,K94E and Cdc42pY32H. GST-Cdc42p (•), GST-Cdc42pY32H (▵), and GST-Cdc42pV36T,K94E (□) were prebound to [γ-32P]GTP and incubated with buffer alone (A) or with a 1:10 molar ratio of recombinant GST-Rga1p GAP domain (B), and radioactivity remaining bound to Cdc42p is plotted against time of incubation. Control experiments indicated that the Rga1p GAP domain did not stimulate release of 32P from Cdc42p prebound with [α-32P]GTP, indicating that Rga1p stimulates GTP hydrolysis and not release.

Mentions: The finding that overproduction of a Cdc42p GAP could ameliorate the septin defects of cdc42V36T,K94E/cdc42Δ and cdc42Y32H/cdc42Δ mutants raised the possibility that the septin misorganization arose due to a defect in the ability of Cdc42pV36T,K94E and Cdc42pY32H to hydrolyze GTP. To test directly whether such a defect existed, mutant and wild-type versions of Cdc42p were produced as recombinant GST fusion proteins in E. coli, purified using glutathione-sepharose, and loaded with [γ-32P]GTP. GTP hydrolysis was followed by monitoring loss of the 32P associated with Cdc42p using a filtration assay. As shown in Fig. 4 A, GTP hydrolysis by Cdc42pV36T,K94E was ∼40% slower than GTP hydrolysis by wild-type Cdc42p, whereas GTP hydrolysis by Cdc42pY32H was indistinguishable from wild-type.


Septin ring assembly involves cycles of GTP loading and hydrolysis by Cdc42p.

Gladfelter AS, Bose I, Zyla TR, Bardes ES, Lew DJ - J. Cell Biol. (2002)

GTP hydrolysis by Cdc42pV36T,K94E and Cdc42pY32H. GST-Cdc42p (•), GST-Cdc42pY32H (▵), and GST-Cdc42pV36T,K94E (□) were prebound to [γ-32P]GTP and incubated with buffer alone (A) or with a 1:10 molar ratio of recombinant GST-Rga1p GAP domain (B), and radioactivity remaining bound to Cdc42p is plotted against time of incubation. Control experiments indicated that the Rga1p GAP domain did not stimulate release of 32P from Cdc42p prebound with [α-32P]GTP, indicating that Rga1p stimulates GTP hydrolysis and not release.
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Related In: Results  -  Collection

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fig4: GTP hydrolysis by Cdc42pV36T,K94E and Cdc42pY32H. GST-Cdc42p (•), GST-Cdc42pY32H (▵), and GST-Cdc42pV36T,K94E (□) were prebound to [γ-32P]GTP and incubated with buffer alone (A) or with a 1:10 molar ratio of recombinant GST-Rga1p GAP domain (B), and radioactivity remaining bound to Cdc42p is plotted against time of incubation. Control experiments indicated that the Rga1p GAP domain did not stimulate release of 32P from Cdc42p prebound with [α-32P]GTP, indicating that Rga1p stimulates GTP hydrolysis and not release.
Mentions: The finding that overproduction of a Cdc42p GAP could ameliorate the septin defects of cdc42V36T,K94E/cdc42Δ and cdc42Y32H/cdc42Δ mutants raised the possibility that the septin misorganization arose due to a defect in the ability of Cdc42pV36T,K94E and Cdc42pY32H to hydrolyze GTP. To test directly whether such a defect existed, mutant and wild-type versions of Cdc42p were produced as recombinant GST fusion proteins in E. coli, purified using glutathione-sepharose, and loaded with [γ-32P]GTP. GTP hydrolysis was followed by monitoring loss of the 32P associated with Cdc42p using a filtration assay. As shown in Fig. 4 A, GTP hydrolysis by Cdc42pV36T,K94E was ∼40% slower than GTP hydrolysis by wild-type Cdc42p, whereas GTP hydrolysis by Cdc42pY32H was indistinguishable from wild-type.

Bottom Line: In performing its roles in actin polarization and transcriptional activation, GTP-Cdc42p is thought to function by activating and/or recruiting effectors to the site of polarization.Excess accumulation of GTP-Cdc42p due to a defect in GTP hydrolysis by the septin-specific alleles might cause unphysiological activation of effectors, interfering with septin assembly.These results suggest that a single GTPase, Cdc42p, can act either as a ras-like GTP-dependent "switch" to turn on effectors or as an EF-Tu-like "assembly factor" using the GTPase cycle to assemble a macromolecular structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.

ABSTRACT
At the beginning of the budding yeast cell cycle, the GTPase Cdc42p promotes the assembly of a ring of septins at the site of future bud emergence. Here, we present an analysis of cdc42 mutants that display specific defects in septin organization, which identifies an important role for GTP hydrolysis by Cdc42p in the assembly of the septin ring. The mutants show defects in basal or stimulated GTP hydrolysis, and the septin misorganization is suppressed by overexpression of a Cdc42p GTPase-activating protein (GAP). Other mutants known to affect GTP hydrolysis by Cdc42p also caused septin misorganization, as did deletion of Cdc42p GAPs. In performing its roles in actin polarization and transcriptional activation, GTP-Cdc42p is thought to function by activating and/or recruiting effectors to the site of polarization. Excess accumulation of GTP-Cdc42p due to a defect in GTP hydrolysis by the septin-specific alleles might cause unphysiological activation of effectors, interfering with septin assembly. However, the recessive and dose-sensitive genetic behavior of the septin-specific cdc42 mutants is inconsistent with the septin defect stemming from a dominant interference of this type. Instead, we suggest that assembly of the septin ring involves repeated cycles of GTP loading and GTP hydrolysis by Cdc42p. These results suggest that a single GTPase, Cdc42p, can act either as a ras-like GTP-dependent "switch" to turn on effectors or as an EF-Tu-like "assembly factor" using the GTPase cycle to assemble a macromolecular structure.

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