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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.

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Comparison of the nucleating activity of peptide-eluted  complexes and isolated γTuSC. (A) Representative fields from  solution nucleation assays. Bar, 20 μm. (B) Quantitation of solution nucleation assays. MTs in 10 microscope fields were counted  and the average number of MTs/field is plotted. Error bars represent the SEM.
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Figure 6: Comparison of the nucleating activity of peptide-eluted complexes and isolated γTuSC. (A) Representative fields from solution nucleation assays. Bar, 20 μm. (B) Quantitation of solution nucleation assays. MTs in 10 microscope fields were counted and the average number of MTs/field is plotted. Error bars represent the SEM.

Mentions: Concentrated γTuSC fractions were dialyzed to remove salt and tested for nucleating activity. In contrast to the robust activity of the peptide-eluted complexes, nucleation by isolated γTuSC in solution was weak and slightly variable between preparations. A direct comparison between the nucleating activity of the peptide-eluted complexes (containing 0.87 μM total γ-tubulin, 0.65 μM γ-tubulin in γTuRC) and isolated γTuSC (0.74 μM γ-tubulin) in a solution nucleation assay is shown in Fig. 6. In nucleation reactions containing between 350–450 nM γ-tubulin and 4 mg/ml tubulin, the nucleating activity of the peptide-eluted complexes was typically 80–100-fold above the level of spontaneous nucleation; under these conditions the level of nucleation for isolated γTuSC was only two- to threefold above background (see Fig. 6 B). Therefore, it is likely that the nucleating activity of the peptide-eluted complexes is due to primarily the activity of γ-tubulin in γTuRC. Based on those data, we estimate that per mole of γ-tubulin γTuRC is ∼25 times more active than γTuSC in promoting nucleation. Since there are ∼12 γ-tubulin molecules in γTuRC and only 2 in γTuSC (see below for stoichiometry estimates), γTuRC is ∼150 times more active than γTuSC per mole of complex. We also tested the nucleating activity of γTuSC in the coverslip nucleation assay. The concentrated γTuSC was also able to nucleate MTs in this assay (Fig. 5 B).


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)

Comparison of the nucleating activity of peptide-eluted  complexes and isolated γTuSC. (A) Representative fields from  solution nucleation assays. Bar, 20 μm. (B) Quantitation of solution nucleation assays. MTs in 10 microscope fields were counted  and the average number of MTs/field is plotted. Error bars represent the SEM.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Comparison of the nucleating activity of peptide-eluted complexes and isolated γTuSC. (A) Representative fields from solution nucleation assays. Bar, 20 μm. (B) Quantitation of solution nucleation assays. MTs in 10 microscope fields were counted and the average number of MTs/field is plotted. Error bars represent the SEM.
Mentions: Concentrated γTuSC fractions were dialyzed to remove salt and tested for nucleating activity. In contrast to the robust activity of the peptide-eluted complexes, nucleation by isolated γTuSC in solution was weak and slightly variable between preparations. A direct comparison between the nucleating activity of the peptide-eluted complexes (containing 0.87 μM total γ-tubulin, 0.65 μM γ-tubulin in γTuRC) and isolated γTuSC (0.74 μM γ-tubulin) in a solution nucleation assay is shown in Fig. 6. In nucleation reactions containing between 350–450 nM γ-tubulin and 4 mg/ml tubulin, the nucleating activity of the peptide-eluted complexes was typically 80–100-fold above the level of spontaneous nucleation; under these conditions the level of nucleation for isolated γTuSC was only two- to threefold above background (see Fig. 6 B). Therefore, it is likely that the nucleating activity of the peptide-eluted complexes is due to primarily the activity of γ-tubulin in γTuRC. Based on those data, we estimate that per mole of γ-tubulin γTuRC is ∼25 times more active than γTuSC in promoting nucleation. Since there are ∼12 γ-tubulin molecules in γTuRC and only 2 in γTuSC (see below for stoichiometry estimates), γTuRC is ∼150 times more active than γTuSC per mole of complex. We also tested the nucleating activity of γTuSC in the coverslip nucleation assay. The concentrated γTuSC was also able to nucleate MTs in this assay (Fig. 5 B).

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