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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 is required for maintenance of actin polarization but not septin organization in budded cells. Strains RSY136 (GAL1p-SWE1), DLY5079 (cdc42–6 GAL1p-SWE1), DLY4849 (cdc42–17 GAL1p-SWE1), or DLY5078 (cdc24–4 GAL1p-SWE1) were grown to exponential phase in sucrose-containing medium at 24°C, galactose was added to 2% to induce Swe1p expression, and after 2 h cells were shifted to 37°C to inactivate Cdc24p/Cdc42p. After a further 4 h, cells were processed to visualize septin (top row) or actin (bottom row) distribution. Bar, 10 μm.
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fig2: Cdc42p is required for maintenance of actin polarization but not septin organization in budded cells. Strains RSY136 (GAL1p-SWE1), DLY5079 (cdc42–6 GAL1p-SWE1), DLY4849 (cdc42–17 GAL1p-SWE1), or DLY5078 (cdc24–4 GAL1p-SWE1) were grown to exponential phase in sucrose-containing medium at 24°C, galactose was added to 2% to induce Swe1p expression, and after 2 h cells were shifted to 37°C to inactivate Cdc24p/Cdc42p. After a further 4 h, cells were processed to visualize septin (top row) or actin (bottom row) distribution. Bar, 10 μm.

Mentions: To ask directly whether Cdc42p and its exchange factor, Cdc24p, were required to maintain septin localization after bud emergence, we inactivated conditional cdc24 and cdc42 alleles in budded cells. The Ts alleles of these genes analyzed to date were identified based on their homogeneous unbudded terminal phenotype, which may have biased the screen in favor of alleles that were still capable of contributing to septin maintenance in budded cells even at restrictive temperature. To avoid this problem, we used two new Ts alleles (cdc42-6 and cdc42-27) that were selected only for lethality rather than for any particular terminal phenotype (see Materials and methods). To provide as much time as possible for the inactivation of mutant gene products after bud formation, we arrested mutant cells in G2 by overexpressing Swe1p at the permissive temperature. We then shifted the cells to 37°C and maintained the G2 arrest for 4 h to allow ample time for Cdc42p inactivation. In both cdc42 and cdc24 mutants, septins remained localized to the neck with no observable diminishment in the intensity of staining (Fig. 2 A). In contrast, the mutants failed to maintain a polarized actin distribution under these conditions (Fig. 2 B), indicating that long-term maintenance of actin polarity does in fact require continued Cdc42p function. These results suggest that Cdc42p was effectively inactivated after shift to the restrictive temperature but that this did not affect maintenance of septins at the neck. Thus, Cdc42p appears not to be required for maintenance of 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 is required for maintenance of actin polarization but not septin organization in budded cells. Strains RSY136 (GAL1p-SWE1), DLY5079 (cdc42–6 GAL1p-SWE1), DLY4849 (cdc42–17 GAL1p-SWE1), or DLY5078 (cdc24–4 GAL1p-SWE1) were grown to exponential phase in sucrose-containing medium at 24°C, galactose was added to 2% to induce Swe1p expression, and after 2 h cells were shifted to 37°C to inactivate Cdc24p/Cdc42p. After a further 4 h, cells were processed to visualize septin (top row) or actin (bottom row) distribution. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199227&req=5

fig2: Cdc42p is required for maintenance of actin polarization but not septin organization in budded cells. Strains RSY136 (GAL1p-SWE1), DLY5079 (cdc42–6 GAL1p-SWE1), DLY4849 (cdc42–17 GAL1p-SWE1), or DLY5078 (cdc24–4 GAL1p-SWE1) were grown to exponential phase in sucrose-containing medium at 24°C, galactose was added to 2% to induce Swe1p expression, and after 2 h cells were shifted to 37°C to inactivate Cdc24p/Cdc42p. After a further 4 h, cells were processed to visualize septin (top row) or actin (bottom row) distribution. Bar, 10 μm.
Mentions: To ask directly whether Cdc42p and its exchange factor, Cdc24p, were required to maintain septin localization after bud emergence, we inactivated conditional cdc24 and cdc42 alleles in budded cells. The Ts alleles of these genes analyzed to date were identified based on their homogeneous unbudded terminal phenotype, which may have biased the screen in favor of alleles that were still capable of contributing to septin maintenance in budded cells even at restrictive temperature. To avoid this problem, we used two new Ts alleles (cdc42-6 and cdc42-27) that were selected only for lethality rather than for any particular terminal phenotype (see Materials and methods). To provide as much time as possible for the inactivation of mutant gene products after bud formation, we arrested mutant cells in G2 by overexpressing Swe1p at the permissive temperature. We then shifted the cells to 37°C and maintained the G2 arrest for 4 h to allow ample time for Cdc42p inactivation. In both cdc42 and cdc24 mutants, septins remained localized to the neck with no observable diminishment in the intensity of staining (Fig. 2 A). In contrast, the mutants failed to maintain a polarized actin distribution under these conditions (Fig. 2 B), indicating that long-term maintenance of actin polarity does in fact require continued Cdc42p function. These results suggest that Cdc42p was effectively inactivated after shift to the restrictive temperature but that this did not affect maintenance of septins at the neck. Thus, Cdc42p appears not to be required for maintenance of 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