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Tubulin cofactors and Arl2 are cage-like chaperones that regulate the soluble αβ-tubulin pool for microtubule dynamics.

Nithianantham S, Le S, Seto E, Jia W, Leary J, Corbett KD, Moore JK, Al-Bassam J - Elife (2015)

Bottom Line: Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown.A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo.Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αβ-tubulin assembly and maintenance to support microtubule dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cellular Biology, University of California, Davis, Davis, United States.

ABSTRACT
Microtubule dynamics and polarity stem from the polymerization of αβ-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown. Here, we reconstitute a core tubulin chaperone consisting of tubulin cofactors TBCD, TBCE, and Arl2, and reveal a cage-like structure for regulating αβ-tubulin. Biochemical assays and electron microscopy structures of multiple intermediates show the sequential binding of αβ-tubulin dimer followed by tubulin cofactor TBCC onto this chaperone, forming a ternary complex in which Arl2 GTP hydrolysis is activated to alter αβ-tubulin conformation. A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo. Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αβ-tubulin assembly and maintenance to support microtubule dynamics.

No MeSH data available.


Related in: MedlinePlus

Tubulin cofactor-Arl2 co-expression and biochemical studies onTBC-DEG constructs.(A) Domain structures of tubulin cofactors and Arl2. Top,TBCD (gray) composed of HEAT repeats. Second, TBCE, composed of Cap-Gly(blue), LRR (cyan), and ubiqutin-like (light blue) domains. Third, theArl2-GTPase composed of ARF-like G protein fold (red) and outer uniquetermini (orange). Fourth, TBCC composed of an N-terminal spectrinhomology domain (light green), and a β-sheet domain (dark green).(B) Summary of co-expression experiments, TBC, and Arl2proteins. The masses of each of the proteins are shown on the left andcorrespond to the order shown in A. The effects of deletion(Δ) or addition of GFP (GFP) or 6Xhis-tags (his) at the N-terminiand C-termini of each of the TBC and Arl2 proteins on TBC-DEG complexesare described, where check marks describe no effect on TBC-DEGexpression, while a cross mark describes loss of TBC-DEG expression.(C) Size exclusion chromatography (SEC) intensity tracesof TBC-DEG:αβ-tubulin (cyan),TBC-DEG:αβ-tubulin 1:2 molar ratio (blue),αβ-tubulin (red), and TBCC (purple). (D)Composition of SEC fractions shown in C using SDS-PAGE.Panel I, αβ-tubulin; panel II, TBCC; panel III,TBC-DEG+αβ-tubulin 2:1 molar ratio; and panel IV,TBC-DEG+αβ-tubulin+TBCC+GTPγS.The protein standard is shown on the left and proteins are marked on theright. (E) Size exclusion chromatography (SEC) intensitytraces of TBC-DE(N-GFP)G (black). (F) Composition of SECfractions shown in E using SDS-PAGE for TBC-DE(N-GFP)G. Theprotein standard is shown on the left and protein positions are marked onthe right.DOI:http://dx.doi.org/10.7554/eLife.08811.005
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fig2s1: Tubulin cofactor-Arl2 co-expression and biochemical studies onTBC-DEG constructs.(A) Domain structures of tubulin cofactors and Arl2. Top,TBCD (gray) composed of HEAT repeats. Second, TBCE, composed of Cap-Gly(blue), LRR (cyan), and ubiqutin-like (light blue) domains. Third, theArl2-GTPase composed of ARF-like G protein fold (red) and outer uniquetermini (orange). Fourth, TBCC composed of an N-terminal spectrinhomology domain (light green), and a β-sheet domain (dark green).(B) Summary of co-expression experiments, TBC, and Arl2proteins. The masses of each of the proteins are shown on the left andcorrespond to the order shown in A. The effects of deletion(Δ) or addition of GFP (GFP) or 6Xhis-tags (his) at the N-terminiand C-termini of each of the TBC and Arl2 proteins on TBC-DEG complexesare described, where check marks describe no effect on TBC-DEGexpression, while a cross mark describes loss of TBC-DEG expression.(C) Size exclusion chromatography (SEC) intensity tracesof TBC-DEG:αβ-tubulin (cyan),TBC-DEG:αβ-tubulin 1:2 molar ratio (blue),αβ-tubulin (red), and TBCC (purple). (D)Composition of SEC fractions shown in C using SDS-PAGE.Panel I, αβ-tubulin; panel II, TBCC; panel III,TBC-DEG+αβ-tubulin 2:1 molar ratio; and panel IV,TBC-DEG+αβ-tubulin+TBCC+GTPγS.The protein standard is shown on the left and proteins are marked on theright. (E) Size exclusion chromatography (SEC) intensitytraces of TBC-DE(N-GFP)G (black). (F) Composition of SECfractions shown in E using SDS-PAGE for TBC-DE(N-GFP)G. Theprotein standard is shown on the left and protein positions are marked onthe right.DOI:http://dx.doi.org/10.7554/eLife.08811.005

Mentions: (A) Size exclusion chromatography (SEC) intensity traces ofTBC-DEG (black), TBC-DEG:αβ-tubulin (cyan),αβ-tubulin (red), and TBCC (purple). (B) SECintensity traces ofTBC-DEG+TBCC+αβ-tubulin-GDP·ALFx(green), TBC-DEG+TBCC+αβ-tubulin-GTP (gray),TBC-DEG+TBCC-GTP-ALFx (black),TBCC+αβ-tubulin (blue), andαβ-tubulin+TBCC (blue). Additional states aredescribed in Figure 2—figuresupplement 1C,D. (C) Composition of SEC fractionsshown in A and B using SDS-PAGE. Panel I,TBC-DEG; panel II, TBC-DEG:αβ-tubulin; panel III,TBC-DEG+TBCC-GDP·ALFx; panel IV,TBCC+αβ-tubulin; panel V,TBC-DEG+TBCC+αβ-tubulin-GTP; and panel VI,TBC-DEG+TBCC+αβ-tubulin-GDP·ALFx.TBC-DEG forms an active heterotrimeric complex, and TBCC forms a complexthat co-migrates with TBC-DEG upon αβ-tubulin binding inthe presence of GDP·ALFx (panel IV). The proteinstandard is shown on the left and proteins are marked on the right.TBC-DEG complexes interact weakly with the resin media leading to wideelution SEC profiles in most conditions. (D) Molecularmasses of TBC-DEG, αβ-tubulin, TBCC, and their complexesmeasured using size exclusion chromatography with multi-angle lightscattering (SEC-MALS). Solid lines represent SEC intensity traces on anintensity scale shown on the right y-axis, and dotted lines representmasses calculated on the mass scale shown on the left y-axis; TBC-DEG(black), αβ-tubulin (red), TBCC (purple),TBC-DEG:αβ-tubulin (cyan), andTBC-DEG:αβ-tubulin:TBCC-GDP·ALFx(green). Masses and elution volumes are detailed in Table 2. (E) Schemefor the hierarchical assembly of TBC-DEG with TBCC andαβ-tubulin and the role of nucleotide. TBCD, TBCE, and Arl2form TBC-DEG complexes (TBC-DEG) and bind a singleαβ-tubulin dimer (αβ-tub) to formTBC-DEG:αβ-tubulin (TBC-DEG:αβ-tub), whichrecruits TBCC in the GTP-like state to formTBC-DEG:αβ-tubulin:TBCC(TBC-DEG:αβ-tub:TBCC).


Tubulin cofactors and Arl2 are cage-like chaperones that regulate the soluble αβ-tubulin pool for microtubule dynamics.

Nithianantham S, Le S, Seto E, Jia W, Leary J, Corbett KD, Moore JK, Al-Bassam J - Elife (2015)

Tubulin cofactor-Arl2 co-expression and biochemical studies onTBC-DEG constructs.(A) Domain structures of tubulin cofactors and Arl2. Top,TBCD (gray) composed of HEAT repeats. Second, TBCE, composed of Cap-Gly(blue), LRR (cyan), and ubiqutin-like (light blue) domains. Third, theArl2-GTPase composed of ARF-like G protein fold (red) and outer uniquetermini (orange). Fourth, TBCC composed of an N-terminal spectrinhomology domain (light green), and a β-sheet domain (dark green).(B) Summary of co-expression experiments, TBC, and Arl2proteins. The masses of each of the proteins are shown on the left andcorrespond to the order shown in A. The effects of deletion(Δ) or addition of GFP (GFP) or 6Xhis-tags (his) at the N-terminiand C-termini of each of the TBC and Arl2 proteins on TBC-DEG complexesare described, where check marks describe no effect on TBC-DEGexpression, while a cross mark describes loss of TBC-DEG expression.(C) Size exclusion chromatography (SEC) intensity tracesof TBC-DEG:αβ-tubulin (cyan),TBC-DEG:αβ-tubulin 1:2 molar ratio (blue),αβ-tubulin (red), and TBCC (purple). (D)Composition of SEC fractions shown in C using SDS-PAGE.Panel I, αβ-tubulin; panel II, TBCC; panel III,TBC-DEG+αβ-tubulin 2:1 molar ratio; and panel IV,TBC-DEG+αβ-tubulin+TBCC+GTPγS.The protein standard is shown on the left and proteins are marked on theright. (E) Size exclusion chromatography (SEC) intensitytraces of TBC-DE(N-GFP)G (black). (F) Composition of SECfractions shown in E using SDS-PAGE for TBC-DE(N-GFP)G. Theprotein standard is shown on the left and protein positions are marked onthe right.DOI:http://dx.doi.org/10.7554/eLife.08811.005
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4574351&req=5

fig2s1: Tubulin cofactor-Arl2 co-expression and biochemical studies onTBC-DEG constructs.(A) Domain structures of tubulin cofactors and Arl2. Top,TBCD (gray) composed of HEAT repeats. Second, TBCE, composed of Cap-Gly(blue), LRR (cyan), and ubiqutin-like (light blue) domains. Third, theArl2-GTPase composed of ARF-like G protein fold (red) and outer uniquetermini (orange). Fourth, TBCC composed of an N-terminal spectrinhomology domain (light green), and a β-sheet domain (dark green).(B) Summary of co-expression experiments, TBC, and Arl2proteins. The masses of each of the proteins are shown on the left andcorrespond to the order shown in A. The effects of deletion(Δ) or addition of GFP (GFP) or 6Xhis-tags (his) at the N-terminiand C-termini of each of the TBC and Arl2 proteins on TBC-DEG complexesare described, where check marks describe no effect on TBC-DEGexpression, while a cross mark describes loss of TBC-DEG expression.(C) Size exclusion chromatography (SEC) intensity tracesof TBC-DEG:αβ-tubulin (cyan),TBC-DEG:αβ-tubulin 1:2 molar ratio (blue),αβ-tubulin (red), and TBCC (purple). (D)Composition of SEC fractions shown in C using SDS-PAGE.Panel I, αβ-tubulin; panel II, TBCC; panel III,TBC-DEG+αβ-tubulin 2:1 molar ratio; and panel IV,TBC-DEG+αβ-tubulin+TBCC+GTPγS.The protein standard is shown on the left and proteins are marked on theright. (E) Size exclusion chromatography (SEC) intensitytraces of TBC-DE(N-GFP)G (black). (F) Composition of SECfractions shown in E using SDS-PAGE for TBC-DE(N-GFP)G. Theprotein standard is shown on the left and protein positions are marked onthe right.DOI:http://dx.doi.org/10.7554/eLife.08811.005
Mentions: (A) Size exclusion chromatography (SEC) intensity traces ofTBC-DEG (black), TBC-DEG:αβ-tubulin (cyan),αβ-tubulin (red), and TBCC (purple). (B) SECintensity traces ofTBC-DEG+TBCC+αβ-tubulin-GDP·ALFx(green), TBC-DEG+TBCC+αβ-tubulin-GTP (gray),TBC-DEG+TBCC-GTP-ALFx (black),TBCC+αβ-tubulin (blue), andαβ-tubulin+TBCC (blue). Additional states aredescribed in Figure 2—figuresupplement 1C,D. (C) Composition of SEC fractionsshown in A and B using SDS-PAGE. Panel I,TBC-DEG; panel II, TBC-DEG:αβ-tubulin; panel III,TBC-DEG+TBCC-GDP·ALFx; panel IV,TBCC+αβ-tubulin; panel V,TBC-DEG+TBCC+αβ-tubulin-GTP; and panel VI,TBC-DEG+TBCC+αβ-tubulin-GDP·ALFx.TBC-DEG forms an active heterotrimeric complex, and TBCC forms a complexthat co-migrates with TBC-DEG upon αβ-tubulin binding inthe presence of GDP·ALFx (panel IV). The proteinstandard is shown on the left and proteins are marked on the right.TBC-DEG complexes interact weakly with the resin media leading to wideelution SEC profiles in most conditions. (D) Molecularmasses of TBC-DEG, αβ-tubulin, TBCC, and their complexesmeasured using size exclusion chromatography with multi-angle lightscattering (SEC-MALS). Solid lines represent SEC intensity traces on anintensity scale shown on the right y-axis, and dotted lines representmasses calculated on the mass scale shown on the left y-axis; TBC-DEG(black), αβ-tubulin (red), TBCC (purple),TBC-DEG:αβ-tubulin (cyan), andTBC-DEG:αβ-tubulin:TBCC-GDP·ALFx(green). Masses and elution volumes are detailed in Table 2. (E) Schemefor the hierarchical assembly of TBC-DEG with TBCC andαβ-tubulin and the role of nucleotide. TBCD, TBCE, and Arl2form TBC-DEG complexes (TBC-DEG) and bind a singleαβ-tubulin dimer (αβ-tub) to formTBC-DEG:αβ-tubulin (TBC-DEG:αβ-tub), whichrecruits TBCC in the GTP-like state to formTBC-DEG:αβ-tubulin:TBCC(TBC-DEG:αβ-tub:TBCC).

Bottom Line: Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown.A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo.Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αβ-tubulin assembly and maintenance to support microtubule dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cellular Biology, University of California, Davis, Davis, United States.

ABSTRACT
Microtubule dynamics and polarity stem from the polymerization of αβ-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown. Here, we reconstitute a core tubulin chaperone consisting of tubulin cofactors TBCD, TBCE, and Arl2, and reveal a cage-like structure for regulating αβ-tubulin. Biochemical assays and electron microscopy structures of multiple intermediates show the sequential binding of αβ-tubulin dimer followed by tubulin cofactor TBCC onto this chaperone, forming a ternary complex in which Arl2 GTP hydrolysis is activated to alter αβ-tubulin conformation. A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo. Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αβ-tubulin assembly and maintenance to support microtubule dynamics.

No MeSH data available.


Related in: MedlinePlus