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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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Characterization of γ-tubulin–containing complexes isolated from Drosophila  embryo extracts. γ-tubulin–containing complexes were immunoisolated and fractionated  by sucrose gradient sedimentation in 100 mM  NaCl. (A) 75 μl of each sucrose gradient fraction was TCA precipitated and analyzed by  10% SDS-PAGE and Coomassie staining.  The sucrose gradient load is also shown. Peak  fractions for standards run on a parallel gradient were: BSA (4.3 S), fraction 2.7; aldolase  (7.35 S), fraction 4.0; catalase (11.3 S), fraction 5.6; and bovine thyroglobulin (19.4 S),  fraction 8.0. (B) Protein profiles of γTuRC  and γTuSC. γTuSC consists of the three most  prominent bands in γTuRC. The profile of  Drosophila γTuRC resembles that of Xenopus γTuRC (Zheng et al., 1995). (C) Schematic of the coverslip nucleation assay. The  coverslip is washed and blocked with a BSA-containing buffer, incubated with the sample  to be tested, rinsed to remove unbound protein, incubated with a mixture of unlabeled  and rhodamine-labeled tubulin, fixed, and  viewed using fluorescence microscopy. (D)  Analysis of sucrose gradient fractions in A  using the coverslip assay. The top two rows  are equivalent exposures for fractions 3–14.  The bottom row shows longer exposures (either 40× or 5× longer, as indicated) for some  fractions. Bar, 10 μm.
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Figure 3: Characterization of γ-tubulin–containing complexes isolated from Drosophila embryo extracts. γ-tubulin–containing complexes were immunoisolated and fractionated by sucrose gradient sedimentation in 100 mM NaCl. (A) 75 μl of each sucrose gradient fraction was TCA precipitated and analyzed by 10% SDS-PAGE and Coomassie staining. The sucrose gradient load is also shown. Peak fractions for standards run on a parallel gradient were: BSA (4.3 S), fraction 2.7; aldolase (7.35 S), fraction 4.0; catalase (11.3 S), fraction 5.6; and bovine thyroglobulin (19.4 S), fraction 8.0. (B) Protein profiles of γTuRC and γTuSC. γTuSC consists of the three most prominent bands in γTuRC. The profile of Drosophila γTuRC resembles that of Xenopus γTuRC (Zheng et al., 1995). (C) Schematic of the coverslip nucleation assay. The coverslip is washed and blocked with a BSA-containing buffer, incubated with the sample to be tested, rinsed to remove unbound protein, incubated with a mixture of unlabeled and rhodamine-labeled tubulin, fixed, and viewed using fluorescence microscopy. (D) Analysis of sucrose gradient fractions in A using the coverslip assay. The top two rows are equivalent exposures for fractions 3–14. The bottom row shows longer exposures (either 40× or 5× longer, as indicated) for some fractions. Bar, 10 μm.

Mentions: To determine the protein compositions of the γTuSC and γTuRC, we fractionated the peptide-eluted complexes on a 5–40% sucrose gradient (Fig. 3 A). For clarity, γTuSC and γTuRC are shown side by side in Fig. 3 B. The protein profile of the Drosophila γTuRC is reminiscent of the Xenopus γTuRC (Fig. 3 B). Therefore, by analogy to the Xgrips (Martin et al., 1998), we name Drosophila γTuRC proteins Dgrips and designate them by their apparent molecular weights. Like the Xenopus γTuRC, the Drosophila γTuRC is composed of two high molecular mass proteins (Dgrip163 and Dgrip128), two prominent proteins near 100 kD (Dgrip91 and Dgrip84), and a group of three or four proteins with molecular masses near 75 kD (Dgrip75s). The protein below γ-tubulin (between the 56- and 38.5-kD markers) has been identified as actin. It is not clear whether actin is a specific component of γTuRC, or if it fortuitously copurifies. Depending on the purification protocol, varying amounts of α- and β-tubulin copurify with Xenopus γTuRC (Zheng et al., 1995; Y. Zheng, unpublished results). In contrast, we have been unable to detect α- or β-tubulin copurifying with Drosophila γTuRC. Consistent with the idea that γTuSC is a structural subunit of γTuRC, γTuSC is composed of the three most prominent proteins in γTuRC: γ-tubulin, Dgrip84, and Dgrip91 (Fig. 3 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)

Characterization of γ-tubulin–containing complexes isolated from Drosophila  embryo extracts. γ-tubulin–containing complexes were immunoisolated and fractionated  by sucrose gradient sedimentation in 100 mM  NaCl. (A) 75 μl of each sucrose gradient fraction was TCA precipitated and analyzed by  10% SDS-PAGE and Coomassie staining.  The sucrose gradient load is also shown. Peak  fractions for standards run on a parallel gradient were: BSA (4.3 S), fraction 2.7; aldolase  (7.35 S), fraction 4.0; catalase (11.3 S), fraction 5.6; and bovine thyroglobulin (19.4 S),  fraction 8.0. (B) Protein profiles of γTuRC  and γTuSC. γTuSC consists of the three most  prominent bands in γTuRC. The profile of  Drosophila γTuRC resembles that of Xenopus γTuRC (Zheng et al., 1995). (C) Schematic of the coverslip nucleation assay. The  coverslip is washed and blocked with a BSA-containing buffer, incubated with the sample  to be tested, rinsed to remove unbound protein, incubated with a mixture of unlabeled  and rhodamine-labeled tubulin, fixed, and  viewed using fluorescence microscopy. (D)  Analysis of sucrose gradient fractions in A  using the coverslip assay. The top two rows  are equivalent exposures for fractions 3–14.  The bottom row shows longer exposures (either 40× or 5× longer, as indicated) for some  fractions. Bar, 10 μm.
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Figure 3: Characterization of γ-tubulin–containing complexes isolated from Drosophila embryo extracts. γ-tubulin–containing complexes were immunoisolated and fractionated by sucrose gradient sedimentation in 100 mM NaCl. (A) 75 μl of each sucrose gradient fraction was TCA precipitated and analyzed by 10% SDS-PAGE and Coomassie staining. The sucrose gradient load is also shown. Peak fractions for standards run on a parallel gradient were: BSA (4.3 S), fraction 2.7; aldolase (7.35 S), fraction 4.0; catalase (11.3 S), fraction 5.6; and bovine thyroglobulin (19.4 S), fraction 8.0. (B) Protein profiles of γTuRC and γTuSC. γTuSC consists of the three most prominent bands in γTuRC. The profile of Drosophila γTuRC resembles that of Xenopus γTuRC (Zheng et al., 1995). (C) Schematic of the coverslip nucleation assay. The coverslip is washed and blocked with a BSA-containing buffer, incubated with the sample to be tested, rinsed to remove unbound protein, incubated with a mixture of unlabeled and rhodamine-labeled tubulin, fixed, and viewed using fluorescence microscopy. (D) Analysis of sucrose gradient fractions in A using the coverslip assay. The top two rows are equivalent exposures for fractions 3–14. The bottom row shows longer exposures (either 40× or 5× longer, as indicated) for some fractions. Bar, 10 μm.
Mentions: To determine the protein compositions of the γTuSC and γTuRC, we fractionated the peptide-eluted complexes on a 5–40% sucrose gradient (Fig. 3 A). For clarity, γTuSC and γTuRC are shown side by side in Fig. 3 B. The protein profile of the Drosophila γTuRC is reminiscent of the Xenopus γTuRC (Fig. 3 B). Therefore, by analogy to the Xgrips (Martin et al., 1998), we name Drosophila γTuRC proteins Dgrips and designate them by their apparent molecular weights. Like the Xenopus γTuRC, the Drosophila γTuRC is composed of two high molecular mass proteins (Dgrip163 and Dgrip128), two prominent proteins near 100 kD (Dgrip91 and Dgrip84), and a group of three or four proteins with molecular masses near 75 kD (Dgrip75s). The protein below γ-tubulin (between the 56- and 38.5-kD markers) has been identified as actin. It is not clear whether actin is a specific component of γTuRC, or if it fortuitously copurifies. Depending on the purification protocol, varying amounts of α- and β-tubulin copurify with Xenopus γTuRC (Zheng et al., 1995; Y. Zheng, unpublished results). In contrast, we have been unable to detect α- or β-tubulin copurifying with Drosophila γTuRC. Consistent with the idea that γTuSC is a structural subunit of γTuRC, γTuSC is composed of the three most prominent proteins in γTuRC: γ-tubulin, Dgrip84, and Dgrip91 (Fig. 3 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