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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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Rga1p GAP activity and cdc42 suppression. (A) Cdc42p prebound to [γ-32P]GTP was incubated with GST (•), GST-Rga1p GAP domain (○), or the same domain containing the K872A change (□), and radioactivity remaining bound to Cdc42p is plotted against time of incubation. The inset shows that somewhat less wild-type than mutant GAP domains were used in the assay. (B) Recombinant myc-tagged Rga1p or Rga1pK872A GAP domains were incubated with bead-bound recombinant GST-Cdc42pQ61L (GTP-bound) or GST-Cdc42pT17N (GDP-bound) to assess binding. (C) Strains DLY4223 (cdc42V36T,K94E/GAL1p-CDC42) and DLY4224 (cdc42Y32H/GAL1p-CDC42) were transformed with YEplac195 (vector), pDLB1537 (RGA1), or pDLB1580 (rga1K872A). Transformants were grown for >30 h in selective dextrose-containing medium at 30°C and processed to visualize septin distribution. Bar, 10 μm.
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fig5: Rga1p GAP activity and cdc42 suppression. (A) Cdc42p prebound to [γ-32P]GTP was incubated with GST (•), GST-Rga1p GAP domain (○), or the same domain containing the K872A change (□), and radioactivity remaining bound to Cdc42p is plotted against time of incubation. The inset shows that somewhat less wild-type than mutant GAP domains were used in the assay. (B) Recombinant myc-tagged Rga1p or Rga1pK872A GAP domains were incubated with bead-bound recombinant GST-Cdc42pQ61L (GTP-bound) or GST-Cdc42pT17N (GDP-bound) to assess binding. (C) Strains DLY4223 (cdc42V36T,K94E/GAL1p-CDC42) and DLY4224 (cdc42Y32H/GAL1p-CDC42) were transformed with YEplac195 (vector), pDLB1537 (RGA1), or pDLB1580 (rga1K872A). Transformants were grown for >30 h in selective dextrose-containing medium at 30°C and processed to visualize septin distribution. Bar, 10 μm.

Mentions: Like effectors, GTPase-activating proteins (GAPs) recognize specifically the GTP-bound form of small G proteins. There are three proteins currently thought to act as Cdc42p-directed GAPs in yeast: Rga1p, Rga2p, and Bem3p (Zheng et al., 1993; Stevenson et al., 1995). Strikingly, we found that overexpression of Rga1p (though not of Rga2p or Bem3p) effectively suppressed the septin defects of both cdc42V36T,K94E/cdc42Δ and cdc42Y32H/cdc42Δ mutants (Fig. 3; see Fig. 5 C).


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)

Rga1p GAP activity and cdc42 suppression. (A) Cdc42p prebound to [γ-32P]GTP was incubated with GST (•), GST-Rga1p GAP domain (○), or the same domain containing the K872A change (□), and radioactivity remaining bound to Cdc42p is plotted against time of incubation. The inset shows that somewhat less wild-type than mutant GAP domains were used in the assay. (B) Recombinant myc-tagged Rga1p or Rga1pK872A GAP domains were incubated with bead-bound recombinant GST-Cdc42pQ61L (GTP-bound) or GST-Cdc42pT17N (GDP-bound) to assess binding. (C) Strains DLY4223 (cdc42V36T,K94E/GAL1p-CDC42) and DLY4224 (cdc42Y32H/GAL1p-CDC42) were transformed with YEplac195 (vector), pDLB1537 (RGA1), or pDLB1580 (rga1K872A). Transformants were grown for >30 h in selective dextrose-containing medium at 30°C and processed to visualize septin distribution. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199227&req=5

fig5: Rga1p GAP activity and cdc42 suppression. (A) Cdc42p prebound to [γ-32P]GTP was incubated with GST (•), GST-Rga1p GAP domain (○), or the same domain containing the K872A change (□), and radioactivity remaining bound to Cdc42p is plotted against time of incubation. The inset shows that somewhat less wild-type than mutant GAP domains were used in the assay. (B) Recombinant myc-tagged Rga1p or Rga1pK872A GAP domains were incubated with bead-bound recombinant GST-Cdc42pQ61L (GTP-bound) or GST-Cdc42pT17N (GDP-bound) to assess binding. (C) Strains DLY4223 (cdc42V36T,K94E/GAL1p-CDC42) and DLY4224 (cdc42Y32H/GAL1p-CDC42) were transformed with YEplac195 (vector), pDLB1537 (RGA1), or pDLB1580 (rga1K872A). Transformants were grown for >30 h in selective dextrose-containing medium at 30°C and processed to visualize septin distribution. Bar, 10 μm.
Mentions: Like effectors, GTPase-activating proteins (GAPs) recognize specifically the GTP-bound form of small G proteins. There are three proteins currently thought to act as Cdc42p-directed GAPs in yeast: Rga1p, Rga2p, and Bem3p (Zheng et al., 1993; Stevenson et al., 1995). Strikingly, we found that overexpression of Rga1p (though not of Rga2p or Bem3p) effectively suppressed the septin defects of both cdc42V36T,K94E/cdc42Δ and cdc42Y32H/cdc42Δ mutants (Fig. 3; see Fig. 5 C).

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