Limits...
Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules.

Oegema K, Wiese C, Martin OC, Milligan RA, Iwamatsu A, Mitchison TJ, Zheng Y - J. Cell Biol. (1999)

Bottom Line: Mitchison. 1995.The gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting that assembly into a larger complex enhances nucleating activity.Analysis of the nucleotide content of the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering gamma-tubulin an unusual member of the tubulin superfamily.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. Karen.Omega@EMBL-Heidelburg.DE

ABSTRACT
gamma-tubulin exists in two related complexes in Drosophila embryo extracts (Moritz, M., Y. Zheng, B.M. Alberts, and K. Oegema. 1998. J. Cell Biol. 142:1- 12). Here, we report the purification and characterization of both complexes that we name gamma-tubulin small complex (gammaTuSC; approximately 280,000 D) and Drosophila gammaTuRC ( approximately 2,200,000 D). In addition to gamma-tubulin, the gammaTuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The gammaTuSC is a structural subunit of the gammaTuRC, a larger complex containing about six additional polypeptides. Like the gammaTuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), the Drosophila gammaTuRC can nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron microscopy reveals a modular structure with approximately 13 radially arranged structural repeats. The gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering gamma-tubulin an unusual member of the tubulin superfamily.

Show MeSH

Related in: MedlinePlus

Structure of Drosophila γTuRC. (A and B) Negative  stain electron microscopy of the peptide-eluted complexes (A) or  of Drosophila γTuRC after isolation on a sucrose gradient (B).  Bar, 100 nm. (C) A gallery of cryo-electron microscopy images of  Drosophila γTuRC. Cryo-electron microscopy reveals a modular  structure with ∼13 structural repeats (arrows) in a radial pattern.  The ring has a diameter of ∼25 nm. Some internal structures are  also apparent (arrowhead). Bar, 25 nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132928&req=5

Figure 4: Structure of Drosophila γTuRC. (A and B) Negative stain electron microscopy of the peptide-eluted complexes (A) or of Drosophila γTuRC after isolation on a sucrose gradient (B). Bar, 100 nm. (C) A gallery of cryo-electron microscopy images of Drosophila γTuRC. Cryo-electron microscopy reveals a modular structure with ∼13 structural repeats (arrows) in a radial pattern. The ring has a diameter of ∼25 nm. Some internal structures are also apparent (arrowhead). Bar, 25 nm.

Mentions: Negative stain electron microscopy of the peptide-eluted complexes (Fig. 4 A), and of the γTuRC after sucrose gradient sedimentation (Fig. 4 B) reveals an open ring structure with a diameter of ∼25 nm. In side-by-side pictures of comparable preparations, the structure of the Drosophila γTuRC is indistinguishable from that of the Xenopus γTuRC (Wiese, C., and Y. Zheng, unpublished observations). To get a more detailed view of the γTuRC, we examined the structure of the purified Drosophila γTuRC by cryo-electron microscopy. A gallery of cryo-EM images reveals a modular structure (Fig. 4 C). The γTuRC appears to have ∼13 structural repeats arranged in a radial symmetric pattern with a diameter of 25 nm. Some internal structures are also visible. A more detailed view will require single particle reconstructions.


Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules.

Oegema K, Wiese C, Martin OC, Milligan RA, Iwamatsu A, Mitchison TJ, Zheng Y - J. Cell Biol. (1999)

Structure of Drosophila γTuRC. (A and B) Negative  stain electron microscopy of the peptide-eluted complexes (A) or  of Drosophila γTuRC after isolation on a sucrose gradient (B).  Bar, 100 nm. (C) A gallery of cryo-electron microscopy images of  Drosophila γTuRC. Cryo-electron microscopy reveals a modular  structure with ∼13 structural repeats (arrows) in a radial pattern.  The ring has a diameter of ∼25 nm. Some internal structures are  also apparent (arrowhead). Bar, 25 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132928&req=5

Figure 4: Structure of Drosophila γTuRC. (A and B) Negative stain electron microscopy of the peptide-eluted complexes (A) or of Drosophila γTuRC after isolation on a sucrose gradient (B). Bar, 100 nm. (C) A gallery of cryo-electron microscopy images of Drosophila γTuRC. Cryo-electron microscopy reveals a modular structure with ∼13 structural repeats (arrows) in a radial pattern. The ring has a diameter of ∼25 nm. Some internal structures are also apparent (arrowhead). Bar, 25 nm.
Mentions: Negative stain electron microscopy of the peptide-eluted complexes (Fig. 4 A), and of the γTuRC after sucrose gradient sedimentation (Fig. 4 B) reveals an open ring structure with a diameter of ∼25 nm. In side-by-side pictures of comparable preparations, the structure of the Drosophila γTuRC is indistinguishable from that of the Xenopus γTuRC (Wiese, C., and Y. Zheng, unpublished observations). To get a more detailed view of the γTuRC, we examined the structure of the purified Drosophila γTuRC by cryo-electron microscopy. A gallery of cryo-EM images reveals a modular structure (Fig. 4 C). The γTuRC appears to have ∼13 structural repeats arranged in a radial symmetric pattern with a diameter of 25 nm. Some internal structures are also visible. A more detailed view will require single particle reconstructions.

Bottom Line: Mitchison. 1995.The gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting that assembly into a larger complex enhances nucleating activity.Analysis of the nucleotide content of the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering gamma-tubulin an unusual member of the tubulin superfamily.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. Karen.Omega@EMBL-Heidelburg.DE

ABSTRACT
gamma-tubulin exists in two related complexes in Drosophila embryo extracts (Moritz, M., Y. Zheng, B.M. Alberts, and K. Oegema. 1998. J. Cell Biol. 142:1- 12). Here, we report the purification and characterization of both complexes that we name gamma-tubulin small complex (gammaTuSC; approximately 280,000 D) and Drosophila gammaTuRC ( approximately 2,200,000 D). In addition to gamma-tubulin, the gammaTuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The gammaTuSC is a structural subunit of the gammaTuRC, a larger complex containing about six additional polypeptides. Like the gammaTuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), the Drosophila gammaTuRC can nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron microscopy reveals a modular structure with approximately 13 radially arranged structural repeats. The gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering gamma-tubulin an unusual member of the tubulin superfamily.

Show MeSH
Related in: MedlinePlus