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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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Photo cross-linking of radiolabeled GTP to γ-tubulin in  γTuSC and γTuRC. (A) GTP cross-linking of peptide-eluted  complexes. γ-tubulin is the only protein that cross-links to GTP.  (B) GTP cross-linking after sucrose gradient fractionation of the  material in A, demonstrating that γ-tubulin in both γTuSC and  γTuRC cross-links to GTP. (C) Competition of the GTP cross-link of the material in A with 200-fold excess of the indicated unlabeled nucleotides.
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Figure 8: Photo cross-linking of radiolabeled GTP to γ-tubulin in γTuSC and γTuRC. (A) GTP cross-linking of peptide-eluted complexes. γ-tubulin is the only protein that cross-links to GTP. (B) GTP cross-linking after sucrose gradient fractionation of the material in A, demonstrating that γ-tubulin in both γTuSC and γTuRC cross-links to GTP. (C) Competition of the GTP cross-link of the material in A with 200-fold excess of the indicated unlabeled nucleotides.

Mentions: The homology between γ-, α-, and β-tubulins extends into domains that are involved in GTP binding by α- and β-tubulin (Burns, 1995). Thus, it is tempting to speculate that γ-tubulin can bind, and possibly hydrolyze, GTP. To determine if γ-tubulin binds guanine nucleotide, we immunoisolated γ-tubulin–containing complexes in the absence of GTP. The isolated complexes, either before or after sucrose gradient sedimentation, were incubated with [α-32P]GTP and UV cross-linked. In the peptide-eluted complexes, γ-tubulin is the only protein that cross-links to GTP (Fig. 8 A). Furthermore, γ-tubulin in both the γTuRC and γTuSC cross-links to GTP (Fig. 8 B). Competition experiments showed that the cross-link can be competed by addition of excess cold GTP, GDP, and GTPγS but not GMP-PNP, ATP, or CTP (Fig. 8 C).


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)

Photo cross-linking of radiolabeled GTP to γ-tubulin in  γTuSC and γTuRC. (A) GTP cross-linking of peptide-eluted  complexes. γ-tubulin is the only protein that cross-links to GTP.  (B) GTP cross-linking after sucrose gradient fractionation of the  material in A, demonstrating that γ-tubulin in both γTuSC and  γTuRC cross-links to GTP. (C) Competition of the GTP cross-link of the material in A with 200-fold excess of the indicated unlabeled nucleotides.
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Related In: Results  -  Collection

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

Figure 8: Photo cross-linking of radiolabeled GTP to γ-tubulin in γTuSC and γTuRC. (A) GTP cross-linking of peptide-eluted complexes. γ-tubulin is the only protein that cross-links to GTP. (B) GTP cross-linking after sucrose gradient fractionation of the material in A, demonstrating that γ-tubulin in both γTuSC and γTuRC cross-links to GTP. (C) Competition of the GTP cross-link of the material in A with 200-fold excess of the indicated unlabeled nucleotides.
Mentions: The homology between γ-, α-, and β-tubulins extends into domains that are involved in GTP binding by α- and β-tubulin (Burns, 1995). Thus, it is tempting to speculate that γ-tubulin can bind, and possibly hydrolyze, GTP. To determine if γ-tubulin binds guanine nucleotide, we immunoisolated γ-tubulin–containing complexes in the absence of GTP. The isolated complexes, either before or after sucrose gradient sedimentation, were incubated with [α-32P]GTP and UV cross-linked. In the peptide-eluted complexes, γ-tubulin is the only protein that cross-links to GTP (Fig. 8 A). Furthermore, γ-tubulin in both the γTuRC and γTuSC cross-links to GTP (Fig. 8 B). Competition experiments showed that the cross-link can be competed by addition of excess cold GTP, GDP, and GTPγS but not GMP-PNP, ATP, or CTP (Fig. 8 C).

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