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Microtubule nucleating gamma-TuSC assembles structures with 13-fold microtubule-like symmetry.

Kollman JM, Polka JK, Zelter A, Davis TN, Agard DA - Nature (2010)

Bottom Line: The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template.The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins.The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, and Keck Advanced Microscopy Center, University of California, San Francisco, San Francisco, California 94158, USA.

ABSTRACT
Microtubules are nucleated in vivo by gamma-tubulin complexes. The 300-kDa gamma-tubulin small complex (gamma-TuSC), consisting of two molecules of gamma-tubulin and one copy each of the accessory proteins Spc97 and Spc98, is the conserved, essential core of the microtubule nucleating machinery. In metazoa multiple gamma-TuSCs assemble with other proteins into gamma-tubulin ring complexes (gamma-TuRCs). The structure of gamma-TuRC indicated that it functions as a microtubule template. Because each gamma-TuSC contains two molecules of gamma-tubulin, it was assumed that the gamma-TuRC-specific proteins are required to organize gamma-TuSCs to match 13-fold microtubule symmetry. Here we show that Saccharomyces cerevisiae gamma-TuSC forms rings even in the absence of other gamma-TuRC components. The yeast adaptor protein Spc110 stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins. The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

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γ-tubulin in the γTuSC filament has a geometry similar to 13-protofilament microtubulesa) The EM density of γ-tubulin (transparent) and the Spc97p/Spc98p binding domains (opaque) are shown with the γ-tubulin crystal structure fit in the density. The density is tilted relative to the helical axis so that in each case the plus end is vertical. An α/β-tubulin heterodimer is shown with simulated EM density for comparison. b) Thirteen γ-tubulins fit into one turn of the filament. Neighbouring γ-tubulins in the same γTuSC are the same colour. c) The γ-tubulin symmetry is similar to the symmetry of a 13-protofilament microtubule (d), but separated intra-γTuSC γ-tubulins alternate with contacting inter-γTuSC γ-tubulins. e) To illustrate the mismatch between geometries laterally-interacting α/β-tubulin heterodimers were aligned to the filament so that the central α/β-tubulin makes longitudinal contacts to γ-tubulin.
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Figure 3: γ-tubulin in the γTuSC filament has a geometry similar to 13-protofilament microtubulesa) The EM density of γ-tubulin (transparent) and the Spc97p/Spc98p binding domains (opaque) are shown with the γ-tubulin crystal structure fit in the density. The density is tilted relative to the helical axis so that in each case the plus end is vertical. An α/β-tubulin heterodimer is shown with simulated EM density for comparison. b) Thirteen γ-tubulins fit into one turn of the filament. Neighbouring γ-tubulins in the same γTuSC are the same colour. c) The γ-tubulin symmetry is similar to the symmetry of a 13-protofilament microtubule (d), but separated intra-γTuSC γ-tubulins alternate with contacting inter-γTuSC γ-tubulins. e) To illustrate the mismatch between geometries laterally-interacting α/β-tubulin heterodimers were aligned to the filament so that the central α/β-tubulin makes longitudinal contacts to γ-tubulin.

Mentions: The human γ-tubulin crystal structure20 was fit into the density in the regions previously assigned to γ-tubulin13 (Fig. 3a,b). The minus end longitudinal surface of γ-tubulin is completely buried in the interface with Spc97p/Spc98p. The lateral contacts between γ-tubulins of neighbouring γTuSCs are nearly identical to microtubule lateral contacts. The two γ-tubulins within each γTuSC are skewed slightly apart, in a configuration incompatible with the microtubule lattice (Fig 3b,c), as observed in the free γTuSC structure13. This symmetry gives rise to an alternating pattern of γ-tubulin pairs with microtubule-like lateral spacing separated by gaps, generating a staggered mismatch with the microtubule lattice (Fig. 3c–e). The only microtubule lattice surface of γ-tubulin fully exposed in the filament is the plus end face, favouring a model in which γ-tubulin makes longitudinal contacts with α/β-tubulin. This, combined with the thirteen-fold γ-tubulin symmetry, provides the strongest evidence to date to support a γ-tubulin template mechanism for microtubule nucleation.


Microtubule nucleating gamma-TuSC assembles structures with 13-fold microtubule-like symmetry.

Kollman JM, Polka JK, Zelter A, Davis TN, Agard DA - Nature (2010)

γ-tubulin in the γTuSC filament has a geometry similar to 13-protofilament microtubulesa) The EM density of γ-tubulin (transparent) and the Spc97p/Spc98p binding domains (opaque) are shown with the γ-tubulin crystal structure fit in the density. The density is tilted relative to the helical axis so that in each case the plus end is vertical. An α/β-tubulin heterodimer is shown with simulated EM density for comparison. b) Thirteen γ-tubulins fit into one turn of the filament. Neighbouring γ-tubulins in the same γTuSC are the same colour. c) The γ-tubulin symmetry is similar to the symmetry of a 13-protofilament microtubule (d), but separated intra-γTuSC γ-tubulins alternate with contacting inter-γTuSC γ-tubulins. e) To illustrate the mismatch between geometries laterally-interacting α/β-tubulin heterodimers were aligned to the filament so that the central α/β-tubulin makes longitudinal contacts to γ-tubulin.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2921000&req=5

Figure 3: γ-tubulin in the γTuSC filament has a geometry similar to 13-protofilament microtubulesa) The EM density of γ-tubulin (transparent) and the Spc97p/Spc98p binding domains (opaque) are shown with the γ-tubulin crystal structure fit in the density. The density is tilted relative to the helical axis so that in each case the plus end is vertical. An α/β-tubulin heterodimer is shown with simulated EM density for comparison. b) Thirteen γ-tubulins fit into one turn of the filament. Neighbouring γ-tubulins in the same γTuSC are the same colour. c) The γ-tubulin symmetry is similar to the symmetry of a 13-protofilament microtubule (d), but separated intra-γTuSC γ-tubulins alternate with contacting inter-γTuSC γ-tubulins. e) To illustrate the mismatch between geometries laterally-interacting α/β-tubulin heterodimers were aligned to the filament so that the central α/β-tubulin makes longitudinal contacts to γ-tubulin.
Mentions: The human γ-tubulin crystal structure20 was fit into the density in the regions previously assigned to γ-tubulin13 (Fig. 3a,b). The minus end longitudinal surface of γ-tubulin is completely buried in the interface with Spc97p/Spc98p. The lateral contacts between γ-tubulins of neighbouring γTuSCs are nearly identical to microtubule lateral contacts. The two γ-tubulins within each γTuSC are skewed slightly apart, in a configuration incompatible with the microtubule lattice (Fig 3b,c), as observed in the free γTuSC structure13. This symmetry gives rise to an alternating pattern of γ-tubulin pairs with microtubule-like lateral spacing separated by gaps, generating a staggered mismatch with the microtubule lattice (Fig. 3c–e). The only microtubule lattice surface of γ-tubulin fully exposed in the filament is the plus end face, favouring a model in which γ-tubulin makes longitudinal contacts with α/β-tubulin. This, combined with the thirteen-fold γ-tubulin symmetry, provides the strongest evidence to date to support a γ-tubulin template mechanism for microtubule nucleation.

Bottom Line: The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template.The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins.The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, and Keck Advanced Microscopy Center, University of California, San Francisco, San Francisco, California 94158, USA.

ABSTRACT
Microtubules are nucleated in vivo by gamma-tubulin complexes. The 300-kDa gamma-tubulin small complex (gamma-TuSC), consisting of two molecules of gamma-tubulin and one copy each of the accessory proteins Spc97 and Spc98, is the conserved, essential core of the microtubule nucleating machinery. In metazoa multiple gamma-TuSCs assemble with other proteins into gamma-tubulin ring complexes (gamma-TuRCs). The structure of gamma-TuRC indicated that it functions as a microtubule template. Because each gamma-TuSC contains two molecules of gamma-tubulin, it was assumed that the gamma-TuRC-specific proteins are required to organize gamma-TuSCs to match 13-fold microtubule symmetry. Here we show that Saccharomyces cerevisiae gamma-TuSC forms rings even in the absence of other gamma-TuRC components. The yeast adaptor protein Spc110 stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins. The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

Show MeSH
Related in: MedlinePlus