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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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γTuSC oligomers form spontaneously and are stabilized by Spc110pa) Ring-like structures were observed in negative stain electron micrographs of Saccharomyces γTuSC at pH 6.9. b) γTuRC purified from Drosophila embryos is similar in shape and size to the γTuSC rings. c) Capped ends were observed on microtubules grown in the presence of pre-formed γTuSC rings. Scalebar for a, b, and c, 25 nm. d) Negative stain images of γTuSC filaments formed upon copurification with Spc110p1-220. Scalebar, 50 nm.
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Figure 1: γTuSC oligomers form spontaneously and are stabilized by Spc110pa) Ring-like structures were observed in negative stain electron micrographs of Saccharomyces γTuSC at pH 6.9. b) γTuRC purified from Drosophila embryos is similar in shape and size to the γTuSC rings. c) Capped ends were observed on microtubules grown in the presence of pre-formed γTuSC rings. Scalebar for a, b, and c, 25 nm. d) Negative stain images of γTuSC filaments formed upon copurification with Spc110p1-220. Scalebar, 50 nm.

Mentions: Our previous 25 Å structure of Saccharomyces γTuSC was determined in buffer conditions that yielded predominantly monomeric complexes12,13. Here, we show that buffer conditions that promote microtubule growth (BRB80: low salt, pH 6.9) also promote spontaneous assembly of γTuSCs into rings similar to Drosophila γTuRCs2,4 (Fig. 1a,b). Ring formation was sensitive to both salt and pH (Supplementary Fig. 2a–c). The γTuSC rings bound microtubules, and many of these microtubule ends are capped (Fig. 1c), similar to microtubules nucleated in vivo14,15 or from γTuRCs in vitro3,4,16. Spontaneous assembly suggests that ring formation is an intrinsic property of γTuSC, and not dependent on γTuRC-specific proteins.


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

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

γTuSC oligomers form spontaneously and are stabilized by Spc110pa) Ring-like structures were observed in negative stain electron micrographs of Saccharomyces γTuSC at pH 6.9. b) γTuRC purified from Drosophila embryos is similar in shape and size to the γTuSC rings. c) Capped ends were observed on microtubules grown in the presence of pre-formed γTuSC rings. Scalebar for a, b, and c, 25 nm. d) Negative stain images of γTuSC filaments formed upon copurification with Spc110p1-220. Scalebar, 50 nm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: γTuSC oligomers form spontaneously and are stabilized by Spc110pa) Ring-like structures were observed in negative stain electron micrographs of Saccharomyces γTuSC at pH 6.9. b) γTuRC purified from Drosophila embryos is similar in shape and size to the γTuSC rings. c) Capped ends were observed on microtubules grown in the presence of pre-formed γTuSC rings. Scalebar for a, b, and c, 25 nm. d) Negative stain images of γTuSC filaments formed upon copurification with Spc110p1-220. Scalebar, 50 nm.
Mentions: Our previous 25 Å structure of Saccharomyces γTuSC was determined in buffer conditions that yielded predominantly monomeric complexes12,13. Here, we show that buffer conditions that promote microtubule growth (BRB80: low salt, pH 6.9) also promote spontaneous assembly of γTuSCs into rings similar to Drosophila γTuRCs2,4 (Fig. 1a,b). Ring formation was sensitive to both salt and pH (Supplementary Fig. 2a–c). The γTuSC rings bound microtubules, and many of these microtubule ends are capped (Fig. 1c), similar to microtubules nucleated in vivo14,15 or from γTuRCs in vitro3,4,16. Spontaneous assembly suggests that ring formation is an intrinsic property of γTuSC, and not dependent on γTuRC-specific proteins.

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