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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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Septin defects in cdc42 mutants. (A) Strains DLY5461 (CDC42/GAL1p-CDC42), DLY4223 (cdc42V36T,K94E/GAL1p-CDC42), DLY4224 (cdc42Y32H/GAL1p-CDC42), and DLY5470 (cdc42Y32H/cdc42Y32H) were processed to visualize septins. Control Western blots confirmed that hemizygous strains contained approximately twofold less Cdc42p than homozygous strains in all cases. (B) Strains DLY5 (CDC42/CDC42), DLY5080 (cdc42V36T,K94E/cdc42V36T,K94E), DLY5082 (cdc42V36T,K94E/CDC42), DLY4223 (cdc42V36T,K94E/GAL1p-CDC42), DLY5471 (cdc42V36T,K94E/cdc42Y32H), DLY5470 (cdc42Y32H/cdc42Y32H), DLY5461 (cdc42Y32H/CDC42), and DLY4224 (cdc42Y32H/GAL1p-CDC42) were visualized by DIC microscopy to evaluate cell morphology. (C) Strains DLY5461 (CDC42/GAL1p-CDC42), DLY4223 (cdc42V36T,K94E/GAL1p-CDC42), and DLY4224 (cdc42Y32H/GAL1p-CDC42) were processed to visualize Cdc42p. In all cases, cells were grown to exponential phase in YEPD at 30°C and documented after >30 h of growth to allow complete depletion of GAL1p-regulated Cdc42p. Bars, 10 μm.
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fig1: Septin defects in cdc42 mutants. (A) Strains DLY5461 (CDC42/GAL1p-CDC42), DLY4223 (cdc42V36T,K94E/GAL1p-CDC42), DLY4224 (cdc42Y32H/GAL1p-CDC42), and DLY5470 (cdc42Y32H/cdc42Y32H) were processed to visualize septins. Control Western blots confirmed that hemizygous strains contained approximately twofold less Cdc42p than homozygous strains in all cases. (B) Strains DLY5 (CDC42/CDC42), DLY5080 (cdc42V36T,K94E/cdc42V36T,K94E), DLY5082 (cdc42V36T,K94E/CDC42), DLY4223 (cdc42V36T,K94E/GAL1p-CDC42), DLY5471 (cdc42V36T,K94E/cdc42Y32H), DLY5470 (cdc42Y32H/cdc42Y32H), DLY5461 (cdc42Y32H/CDC42), and DLY4224 (cdc42Y32H/GAL1p-CDC42) were visualized by DIC microscopy to evaluate cell morphology. (C) Strains DLY5461 (CDC42/GAL1p-CDC42), DLY4223 (cdc42V36T,K94E/GAL1p-CDC42), and DLY4224 (cdc42Y32H/GAL1p-CDC42) were processed to visualize Cdc42p. In all cases, cells were grown to exponential phase in YEPD at 30°C and documented after >30 h of growth to allow complete depletion of GAL1p-regulated Cdc42p. Bars, 10 μm.

Mentions: To address the role of Cdc42p in septin ring assembly, we have focused on two cdc42 mutants that cause defects in septin localization without overt effects on actin organization. In an earlier study, we described a mutant, cdc42V36T,K94E, that displayed relatively mild septin and cell morphology phenotypes (Gladfelter et al., 2001a). We also noted that the severity of many other cdc42 mutants was greatly affected by gene dosage so that elevated copy number could partially rescue mutant phenotypes (Gladfelter et al., 2001a). Conversely, we reasoned that lowering the gene copy number might reveal more severe phenotypes, which could be useful for characterizing mutants with mild defects. To that end, we examined the effects of reducing mutant gene dosage by generating hemizygous cdc42V36T,K94E/cdc42Δ mutant diploids. This reduction of gene copy number by a factor of two made the mutant phenotype significantly more penetrant and more severe (Fig. 1 B and Table I). In contrast, control hemizygous CDC42/cdc42Δ diploids were indistinguishable from homozygous CDC42/CDC42 wild-type diploids.


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)

Septin defects in cdc42 mutants. (A) Strains DLY5461 (CDC42/GAL1p-CDC42), DLY4223 (cdc42V36T,K94E/GAL1p-CDC42), DLY4224 (cdc42Y32H/GAL1p-CDC42), and DLY5470 (cdc42Y32H/cdc42Y32H) were processed to visualize septins. Control Western blots confirmed that hemizygous strains contained approximately twofold less Cdc42p than homozygous strains in all cases. (B) Strains DLY5 (CDC42/CDC42), DLY5080 (cdc42V36T,K94E/cdc42V36T,K94E), DLY5082 (cdc42V36T,K94E/CDC42), DLY4223 (cdc42V36T,K94E/GAL1p-CDC42), DLY5471 (cdc42V36T,K94E/cdc42Y32H), DLY5470 (cdc42Y32H/cdc42Y32H), DLY5461 (cdc42Y32H/CDC42), and DLY4224 (cdc42Y32H/GAL1p-CDC42) were visualized by DIC microscopy to evaluate cell morphology. (C) Strains DLY5461 (CDC42/GAL1p-CDC42), DLY4223 (cdc42V36T,K94E/GAL1p-CDC42), and DLY4224 (cdc42Y32H/GAL1p-CDC42) were processed to visualize Cdc42p. In all cases, cells were grown to exponential phase in YEPD at 30°C and documented after >30 h of growth to allow complete depletion of GAL1p-regulated Cdc42p. Bars, 10 μm.
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Related In: Results  -  Collection

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fig1: Septin defects in cdc42 mutants. (A) Strains DLY5461 (CDC42/GAL1p-CDC42), DLY4223 (cdc42V36T,K94E/GAL1p-CDC42), DLY4224 (cdc42Y32H/GAL1p-CDC42), and DLY5470 (cdc42Y32H/cdc42Y32H) were processed to visualize septins. Control Western blots confirmed that hemizygous strains contained approximately twofold less Cdc42p than homozygous strains in all cases. (B) Strains DLY5 (CDC42/CDC42), DLY5080 (cdc42V36T,K94E/cdc42V36T,K94E), DLY5082 (cdc42V36T,K94E/CDC42), DLY4223 (cdc42V36T,K94E/GAL1p-CDC42), DLY5471 (cdc42V36T,K94E/cdc42Y32H), DLY5470 (cdc42Y32H/cdc42Y32H), DLY5461 (cdc42Y32H/CDC42), and DLY4224 (cdc42Y32H/GAL1p-CDC42) were visualized by DIC microscopy to evaluate cell morphology. (C) Strains DLY5461 (CDC42/GAL1p-CDC42), DLY4223 (cdc42V36T,K94E/GAL1p-CDC42), and DLY4224 (cdc42Y32H/GAL1p-CDC42) were processed to visualize Cdc42p. In all cases, cells were grown to exponential phase in YEPD at 30°C and documented after >30 h of growth to allow complete depletion of GAL1p-regulated Cdc42p. Bars, 10 μm.
Mentions: To address the role of Cdc42p in septin ring assembly, we have focused on two cdc42 mutants that cause defects in septin localization without overt effects on actin organization. In an earlier study, we described a mutant, cdc42V36T,K94E, that displayed relatively mild septin and cell morphology phenotypes (Gladfelter et al., 2001a). We also noted that the severity of many other cdc42 mutants was greatly affected by gene dosage so that elevated copy number could partially rescue mutant phenotypes (Gladfelter et al., 2001a). Conversely, we reasoned that lowering the gene copy number might reveal more severe phenotypes, which could be useful for characterizing mutants with mild defects. To that end, we examined the effects of reducing mutant gene dosage by generating hemizygous cdc42V36T,K94E/cdc42Δ mutant diploids. This reduction of gene copy number by a factor of two made the mutant phenotype significantly more penetrant and more severe (Fig. 1 B and Table I). In contrast, control hemizygous CDC42/cdc42Δ diploids were indistinguishable from homozygous CDC42/CDC42 wild-type diploids.

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