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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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Effect of overexpressing Cdc42p effectors and GAPs on the septin defects of cdc42 mutants. Strains DLY4223 (cdc42V36T,K94E/GAL1p-CDC42) and DLY4224 (cdc42Y32H/GAL1p-CDC42) were transformed with pDLB722 (CLA4), pDLB723 (STE20), pDLB678 (BEM1), pMOSB229 (GIC1), pDLB1537 (RGA1), pDLB1981 (RGA2), pPB547 (BEM3), or an empty vector. Transformants were grown to exponential phase in dextrose-containing medium at 30°C, and cell morphology and septin localization were documented after >30 h of growth to allow depletion of GAL1p-regulated Cdc42p.
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fig3: Effect of overexpressing Cdc42p effectors and GAPs on the septin defects of cdc42 mutants. Strains DLY4223 (cdc42V36T,K94E/GAL1p-CDC42) and DLY4224 (cdc42Y32H/GAL1p-CDC42) were transformed with pDLB722 (CLA4), pDLB723 (STE20), pDLB678 (BEM1), pMOSB229 (GIC1), pDLB1537 (RGA1), pDLB1981 (RGA2), pPB547 (BEM3), or an empty vector. Transformants were grown to exponential phase in dextrose-containing medium at 30°C, and cell morphology and septin localization were documented after >30 h of growth to allow depletion of GAL1p-regulated Cdc42p.

Mentions: One way to identify the molecular pathways that are impaired in the septin-defective cdc42 mutants is to identify suppressors that restore septin organization. We began by asking whether overexpression of known Cdc42p effectors could rectify the defect in our mutants. As reported previously for haploid strains (Gladfelter et al., 2001a), overexpression of the Cdc42p-activated kinase Cla4p or the scaffold protein Bem1p but not of Ste20p or other effectors suppressed the septin misorganization phenotype of hemizygous cdc42V36T,K94E/cdc42Δ mutants, and we observed a similar pattern for cdc42Y32H/cdc42Δ mutants (Fig. 3) . Cla4p and Bem1p participate in a feedback loop that regulates the phosphorylation state of Cdc24p (Gulli et al., 2000; Bose et al., 2001), and moderate overexpression of these proteins suppresses many cdc42 mutants with varied defects (Gladfelter et al., 2001a). Thus, suppression in these cases may reflect a global enhancement of Cdc42p function rather than a specific compensation of impaired septin organization pathways.


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)

Effect of overexpressing Cdc42p effectors and GAPs on the septin defects of cdc42 mutants. Strains DLY4223 (cdc42V36T,K94E/GAL1p-CDC42) and DLY4224 (cdc42Y32H/GAL1p-CDC42) were transformed with pDLB722 (CLA4), pDLB723 (STE20), pDLB678 (BEM1), pMOSB229 (GIC1), pDLB1537 (RGA1), pDLB1981 (RGA2), pPB547 (BEM3), or an empty vector. Transformants were grown to exponential phase in dextrose-containing medium at 30°C, and cell morphology and septin localization were documented after >30 h of growth to allow depletion of GAL1p-regulated Cdc42p.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Effect of overexpressing Cdc42p effectors and GAPs on the septin defects of cdc42 mutants. Strains DLY4223 (cdc42V36T,K94E/GAL1p-CDC42) and DLY4224 (cdc42Y32H/GAL1p-CDC42) were transformed with pDLB722 (CLA4), pDLB723 (STE20), pDLB678 (BEM1), pMOSB229 (GIC1), pDLB1537 (RGA1), pDLB1981 (RGA2), pPB547 (BEM3), or an empty vector. Transformants were grown to exponential phase in dextrose-containing medium at 30°C, and cell morphology and septin localization were documented after >30 h of growth to allow depletion of GAL1p-regulated Cdc42p.
Mentions: One way to identify the molecular pathways that are impaired in the septin-defective cdc42 mutants is to identify suppressors that restore septin organization. We began by asking whether overexpression of known Cdc42p effectors could rectify the defect in our mutants. As reported previously for haploid strains (Gladfelter et al., 2001a), overexpression of the Cdc42p-activated kinase Cla4p or the scaffold protein Bem1p but not of Ste20p or other effectors suppressed the septin misorganization phenotype of hemizygous cdc42V36T,K94E/cdc42Δ mutants, and we observed a similar pattern for cdc42Y32H/cdc42Δ mutants (Fig. 3) . Cla4p and Bem1p participate in a feedback loop that regulates the phosphorylation state of Cdc24p (Gulli et al., 2000; Bose et al., 2001), and moderate overexpression of these proteins suppresses many cdc42 mutants with varied defects (Gladfelter et al., 2001a). Thus, suppression in these cases may reflect a global enhancement of Cdc42p function rather than a specific compensation of impaired septin organization pathways.

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
Related in: MedlinePlus