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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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Cdc42p GAPs and septin organization. (A) Strains DLY1 (WT), DLY3344 (rga1Δ), DLY3353 (rga2Δ), DLY3346 (bem3Δ), DLY3341 (rga1Δ bem3Δ), DLY3361 (rga2Δ bem3Δ), DLY3347 (rga1Δ rga2Δ), and DLY2723 (rga1Δ rga2Δ bem3Δ) were grown to exponential phase in YEPD at 30°C and processed to visualize septin distribution. (B) Photographs of strains DLY1 (WT) and DLY2723 (rga1Δ rga2Δ bem3Δ) above. Bar, 10 μm.
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fig7: Cdc42p GAPs and septin organization. (A) Strains DLY1 (WT), DLY3344 (rga1Δ), DLY3353 (rga2Δ), DLY3346 (bem3Δ), DLY3341 (rga1Δ bem3Δ), DLY3361 (rga2Δ bem3Δ), DLY3347 (rga1Δ rga2Δ), and DLY2723 (rga1Δ rga2Δ bem3Δ) were grown to exponential phase in YEPD at 30°C and processed to visualize septin distribution. (B) Photographs of strains DLY1 (WT) and DLY2723 (rga1Δ rga2Δ bem3Δ) above. Bar, 10 μm.

Mentions: The association between defects in Cdc42p GTP hydrolysis and defects in septin organization suggests that proper assembly of the septin ring requires proper regulation of GTP hydrolysis. If this is true, then mutational inactivation of Cdc42p GAPs Rga1p, Rga2p, and Bem3p might be expected to perturb septin organization also. Although none of the single mutants displayed striking septin defects, double mutants (particularly rga1Δ rga2Δ) were somewhat defective, and the triple mutants showed a strong defect comparable to that of cdc42V36T,K94E/cdc42Δ mutants (Fig. 7) , indicating that these proteins share a role in septin organization.


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)

Cdc42p GAPs and septin organization. (A) Strains DLY1 (WT), DLY3344 (rga1Δ), DLY3353 (rga2Δ), DLY3346 (bem3Δ), DLY3341 (rga1Δ bem3Δ), DLY3361 (rga2Δ bem3Δ), DLY3347 (rga1Δ rga2Δ), and DLY2723 (rga1Δ rga2Δ bem3Δ) were grown to exponential phase in YEPD at 30°C and processed to visualize septin distribution. (B) Photographs of strains DLY1 (WT) and DLY2723 (rga1Δ rga2Δ bem3Δ) above. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199227&req=5

fig7: Cdc42p GAPs and septin organization. (A) Strains DLY1 (WT), DLY3344 (rga1Δ), DLY3353 (rga2Δ), DLY3346 (bem3Δ), DLY3341 (rga1Δ bem3Δ), DLY3361 (rga2Δ bem3Δ), DLY3347 (rga1Δ rga2Δ), and DLY2723 (rga1Δ rga2Δ bem3Δ) were grown to exponential phase in YEPD at 30°C and processed to visualize septin distribution. (B) Photographs of strains DLY1 (WT) and DLY2723 (rga1Δ rga2Δ bem3Δ) above. Bar, 10 μm.
Mentions: The association between defects in Cdc42p GTP hydrolysis and defects in septin organization suggests that proper assembly of the septin ring requires proper regulation of GTP hydrolysis. If this is true, then mutational inactivation of Cdc42p GAPs Rga1p, Rga2p, and Bem3p might be expected to perturb septin organization also. Although none of the single mutants displayed striking septin defects, double mutants (particularly rga1Δ rga2Δ) were somewhat defective, and the triple mutants showed a strong defect comparable to that of cdc42V36T,K94E/cdc42Δ mutants (Fig. 7) , indicating that these proteins share a role in septin organization.

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.

Show MeSH