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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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Purified Drosophila γ-tubulin complexes nucleate MTs  in vitro. (A) Protein profile of peptide-eluted immunoisolated  Drosophila γ-tubulin complexes after separation by 10% SDS-PAGE and Coomassie staining. (B) Nucleating activity is proportional to the concentration of γ-tubulin complexes in the reaction.  The results shown here are the average of three independent experiments performed using the same preparation of peptide  eluted complexes. At the highest concentrations tested, ∼370 nM  or ∼0.02 mg/ml γ-tubulin, there were 94 times more MTs than in  control reactions without γ-tubulin complexes. We estimate that  the maximal concentration of MTs was ∼0.30 nM. Error bars  represent the SEM.
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Figure 2: Purified Drosophila γ-tubulin complexes nucleate MTs in vitro. (A) Protein profile of peptide-eluted immunoisolated Drosophila γ-tubulin complexes after separation by 10% SDS-PAGE and Coomassie staining. (B) Nucleating activity is proportional to the concentration of γ-tubulin complexes in the reaction. The results shown here are the average of three independent experiments performed using the same preparation of peptide eluted complexes. At the highest concentrations tested, ∼370 nM or ∼0.02 mg/ml γ-tubulin, there were 94 times more MTs than in control reactions without γ-tubulin complexes. We estimate that the maximal concentration of MTs was ∼0.30 nM. Error bars represent the SEM.

Mentions: PEG (polyethylene glycol P-2139; average mol wt = 8,000; Sigma Chemical Co.) was added to a final concentration of 2% (from a 30% stock in HB100) to clarified Drosophila embryo extract from 20-g embryos. The mixture was incubated on ice for 20 min, spun at 17,000 rpm for 10 min in a SS34 rotor and the supernatant was discarded. The pellets were resuspended in 20 ml of HB200 plus 0.05% NP-40, and 100 μM GTP by gentle Dounce homogenization and clarified at 35,000 rpm for 30 min in a 50.2 Ti rotor. γ-tubulin complexes were immunoprecipitated from the supernatant by adding 190 μg of DrosC17 antibody and incubating at 4°C for 1 h with gentle rotation. The immunoprecipitate was collected by slowly (over 1 h) pumping the antibody-extract mixture over a 350-μl column of protein A–agarose (GIBCO/BRL) in a disposable Bio-spin column housing (Bio-Rad). The column was washed with 15 ml of HB200 plus 0.05% NP-40 and 100 μM GTP, and 15 ml of the same buffer without NP-40. 400 μl of EB200 was loaded onto the column and the column was sealed with parafilm and incubated for 16–18 h at 4°C. γ-tubulin complexes were collected by loading an additional 400 μl of EB200 onto the column and collecting the flow through. For the sucrose gradient fractionation described in Fig. 2, 150 μl of isolated complexes was loaded onto a 2.1-ml 5–40% sucrose gradient, poured in HB100 plus 100 μM GTP, and sedimented at 50,000 rpm for 4 h in an TLS55 rotor at 4°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)

Purified Drosophila γ-tubulin complexes nucleate MTs  in vitro. (A) Protein profile of peptide-eluted immunoisolated  Drosophila γ-tubulin complexes after separation by 10% SDS-PAGE and Coomassie staining. (B) Nucleating activity is proportional to the concentration of γ-tubulin complexes in the reaction.  The results shown here are the average of three independent experiments performed using the same preparation of peptide  eluted complexes. At the highest concentrations tested, ∼370 nM  or ∼0.02 mg/ml γ-tubulin, there were 94 times more MTs than in  control reactions without γ-tubulin complexes. We estimate that  the maximal concentration of MTs was ∼0.30 nM. Error bars  represent the SEM.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132928&req=5

Figure 2: Purified Drosophila γ-tubulin complexes nucleate MTs in vitro. (A) Protein profile of peptide-eluted immunoisolated Drosophila γ-tubulin complexes after separation by 10% SDS-PAGE and Coomassie staining. (B) Nucleating activity is proportional to the concentration of γ-tubulin complexes in the reaction. The results shown here are the average of three independent experiments performed using the same preparation of peptide eluted complexes. At the highest concentrations tested, ∼370 nM or ∼0.02 mg/ml γ-tubulin, there were 94 times more MTs than in control reactions without γ-tubulin complexes. We estimate that the maximal concentration of MTs was ∼0.30 nM. Error bars represent the SEM.
Mentions: PEG (polyethylene glycol P-2139; average mol wt = 8,000; Sigma Chemical Co.) was added to a final concentration of 2% (from a 30% stock in HB100) to clarified Drosophila embryo extract from 20-g embryos. The mixture was incubated on ice for 20 min, spun at 17,000 rpm for 10 min in a SS34 rotor and the supernatant was discarded. The pellets were resuspended in 20 ml of HB200 plus 0.05% NP-40, and 100 μM GTP by gentle Dounce homogenization and clarified at 35,000 rpm for 30 min in a 50.2 Ti rotor. γ-tubulin complexes were immunoprecipitated from the supernatant by adding 190 μg of DrosC17 antibody and incubating at 4°C for 1 h with gentle rotation. The immunoprecipitate was collected by slowly (over 1 h) pumping the antibody-extract mixture over a 350-μl column of protein A–agarose (GIBCO/BRL) in a disposable Bio-spin column housing (Bio-Rad). The column was washed with 15 ml of HB200 plus 0.05% NP-40 and 100 μM GTP, and 15 ml of the same buffer without NP-40. 400 μl of EB200 was loaded onto the column and the column was sealed with parafilm and incubated for 16–18 h at 4°C. γ-tubulin complexes were collected by loading an additional 400 μl of EB200 onto the column and collecting the flow through. For the sucrose gradient fractionation described in Fig. 2, 150 μl of isolated complexes was loaded onto a 2.1-ml 5–40% sucrose gradient, poured in HB100 plus 100 μM GTP, and sedimented at 50,000 rpm for 4 h in an TLS55 rotor at 4°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