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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 blocking or accelerating Cdc42p GTP hydrolysis on septin organization. (A) Strains DLY5237 (CDC42 EG43p-CDC42) and DLY5240 (CDC42 EG43p-CDC42Q61L) were grown to exponential phase in either YEPD or YEP with galactose to induce CDC42 or CDC42Q61L expression and processed to visualize septins. (B) Anti-Cdc42p Western blot of strains shown in A. Lane 1 contains lysate from DLY1 (WT control), lane 2 from DLY5237, and lane 3 from DLY5240, all grown in galactose-containing medium. The same blot was probed with anti-PSTAIRE (which detects Cdc28p and Pho85p) as a control for loading. (C) Strain CCY3-3B (cdc42K186R) was grown to exponential phase in YEPD at 30°C and then processed to visualize septins. Bars, 10 μm.
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fig6: Effect of blocking or accelerating Cdc42p GTP hydrolysis on septin organization. (A) Strains DLY5237 (CDC42 EG43p-CDC42) and DLY5240 (CDC42 EG43p-CDC42Q61L) were grown to exponential phase in either YEPD or YEP with galactose to induce CDC42 or CDC42Q61L expression and processed to visualize septins. (B) Anti-Cdc42p Western blot of strains shown in A. Lane 1 contains lysate from DLY1 (WT control), lane 2 from DLY5237, and lane 3 from DLY5240, all grown in galactose-containing medium. The same blot was probed with anti-PSTAIRE (which detects Cdc28p and Pho85p) as a control for loading. (C) Strain CCY3-3B (cdc42K186R) was grown to exponential phase in YEPD at 30°C and then processed to visualize septins. Bars, 10 μm.

Mentions: The results described above establish a correlation between the effects of certain cdc42 alleles on septin organization and alterations in GTP hydrolysis by the encoded mutant proteins. However, it remained possible that the correlation was entirely coincidental and that the septin organization defects of these mutants were unrelated to their altered GTP hydrolysis. To test whether preventing Cdc42p GTP hydrolysis itself would affect septin organization, we turned to the CDC42Q61L allele that was generated by homology to the corresponding oncogenic allele of ras and has been characterized extensively as showing an essentially complete defect in GTP hydrolysis. Previous studies showed that moderate or high level expression of CDC42Q61L in yeast is lethal (Ziman et al., 1991), whereas low level expression can be tolerated (Mosch et al., 1996). We constructed strains expressing CDC42Q61L from a crippled version of the GAL1 promoter in addition to wild-type CDC42 expressed from its own promoter. These cells were able to proliferate well on galactose-containing medium, and the level of Cdc42pQ61L expression was roughly comparable to that of endogenous wild-type Cdc42p (Fig. 6 B). However, there were striking defects in septin organization in these cells: unbudded cells displayed large and faint initial rings similar to those observed in cdc42Y32H/cdc42Δ mutants, and budded cells displayed misorganized and diffuse septin staining at the neck (Fig. 6 A and Table I). In addition, cells expressing Cdc42pQ61L frequently had broad necks similar to those observed in cdc42V36T,K94E/cdc42Δ mutants. Thus, preventing GTP hydrolysis by Cdc42p leads to dominant effects on septin localization and neck morphology.


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 blocking or accelerating Cdc42p GTP hydrolysis on septin organization. (A) Strains DLY5237 (CDC42 EG43p-CDC42) and DLY5240 (CDC42 EG43p-CDC42Q61L) were grown to exponential phase in either YEPD or YEP with galactose to induce CDC42 or CDC42Q61L expression and processed to visualize septins. (B) Anti-Cdc42p Western blot of strains shown in A. Lane 1 contains lysate from DLY1 (WT control), lane 2 from DLY5237, and lane 3 from DLY5240, all grown in galactose-containing medium. The same blot was probed with anti-PSTAIRE (which detects Cdc28p and Pho85p) as a control for loading. (C) Strain CCY3-3B (cdc42K186R) was grown to exponential phase in YEPD at 30°C and then processed to visualize septins. Bars, 10 μm.
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fig6: Effect of blocking or accelerating Cdc42p GTP hydrolysis on septin organization. (A) Strains DLY5237 (CDC42 EG43p-CDC42) and DLY5240 (CDC42 EG43p-CDC42Q61L) were grown to exponential phase in either YEPD or YEP with galactose to induce CDC42 or CDC42Q61L expression and processed to visualize septins. (B) Anti-Cdc42p Western blot of strains shown in A. Lane 1 contains lysate from DLY1 (WT control), lane 2 from DLY5237, and lane 3 from DLY5240, all grown in galactose-containing medium. The same blot was probed with anti-PSTAIRE (which detects Cdc28p and Pho85p) as a control for loading. (C) Strain CCY3-3B (cdc42K186R) was grown to exponential phase in YEPD at 30°C and then processed to visualize septins. Bars, 10 μm.
Mentions: The results described above establish a correlation between the effects of certain cdc42 alleles on septin organization and alterations in GTP hydrolysis by the encoded mutant proteins. However, it remained possible that the correlation was entirely coincidental and that the septin organization defects of these mutants were unrelated to their altered GTP hydrolysis. To test whether preventing Cdc42p GTP hydrolysis itself would affect septin organization, we turned to the CDC42Q61L allele that was generated by homology to the corresponding oncogenic allele of ras and has been characterized extensively as showing an essentially complete defect in GTP hydrolysis. Previous studies showed that moderate or high level expression of CDC42Q61L in yeast is lethal (Ziman et al., 1991), whereas low level expression can be tolerated (Mosch et al., 1996). We constructed strains expressing CDC42Q61L from a crippled version of the GAL1 promoter in addition to wild-type CDC42 expressed from its own promoter. These cells were able to proliferate well on galactose-containing medium, and the level of Cdc42pQ61L expression was roughly comparable to that of endogenous wild-type Cdc42p (Fig. 6 B). However, there were striking defects in septin organization in these cells: unbudded cells displayed large and faint initial rings similar to those observed in cdc42Y32H/cdc42Δ mutants, and budded cells displayed misorganized and diffuse septin staining at the neck (Fig. 6 A and Table I). In addition, cells expressing Cdc42pQ61L frequently had broad necks similar to those observed in cdc42V36T,K94E/cdc42Δ mutants. Thus, preventing GTP hydrolysis by Cdc42p leads to dominant effects on septin localization and neck morphology.

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