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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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Analysis of nucleotide bound  to γ-tubulin in γTuSC, a comparison  with αβ-tubulin dimer. Desalting was  used to remove free nucleotide from  samples of purified γTuSC or αβ-tubulin. Bound nucleotide was released by  urea treatment and analyzed on a mono  Q column. Arrows indicate the elution  positions of GDP and GTP. For samples containing protein (B, C, E, and  F), a gel lane of the desalted sample is  shown to the right of the UV trace.  (A–C) Nucleotide analysis after isolation from buffer containing 20 μM  GDP. (A) After desalting of control  buffer no nucleotide is detected (the  broad peak just to the left of the GDP  arrow is a background peak). (B) Both  GDP and GTP are bound to αβ-tubulin  dimer (for quantitation see Table II: 20  μM GDP, experiment 3). (C) Exclusively, GDP binds to γ-tubulin in  γTuSC (for quantitation see Table II:  20 μM GDP, experiment 3). (D–F) Nucleotide analysis after isolation from  buffer containing 20 μM GTP. (D) Although GTP is desalted slightly less efficiently than GDP (compare with A),  >99.9% of free GTP is removed from  control buffer. (E) Exclusively, GTP binds αβ-tubulin dimer (for quantitation see Table II: 20 μM GTP, experiment 1). (F) Small  amounts of both GDP and GTP are detected bound to γ-tubulin in the γTuSC (for quantitation see Table II: 20 μM GTP, experiment  1). (G) Summary of nucleotide analysis from three independent experiments (raw data shown in Table II). Bar graphs indicate the ratio  of bound nucleotide per αβ-tubulin dimer, or γ-tubulin monomer in γTuSC after isolation from buffers containing 20 μM GDP or 20  μM GTP. Error bars represent the SEM. The ratio of GDP/GTP recovered when we desalt αβ-tubulin dimer from buffer containing  GDP is very reproducible (0.733 ± 0.015, n = 5) suggesting that the protein concentration determined by densitometry is the least accurate parameter in this analysis. This ratio also suggests that we are recovering ∼73% of the GDP bound to the β-tubulin E-site.
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Figure 9: Analysis of nucleotide bound to γ-tubulin in γTuSC, a comparison with αβ-tubulin dimer. Desalting was used to remove free nucleotide from samples of purified γTuSC or αβ-tubulin. Bound nucleotide was released by urea treatment and analyzed on a mono Q column. Arrows indicate the elution positions of GDP and GTP. For samples containing protein (B, C, E, and F), a gel lane of the desalted sample is shown to the right of the UV trace. (A–C) Nucleotide analysis after isolation from buffer containing 20 μM GDP. (A) After desalting of control buffer no nucleotide is detected (the broad peak just to the left of the GDP arrow is a background peak). (B) Both GDP and GTP are bound to αβ-tubulin dimer (for quantitation see Table II: 20 μM GDP, experiment 3). (C) Exclusively, GDP binds to γ-tubulin in γTuSC (for quantitation see Table II: 20 μM GDP, experiment 3). (D–F) Nucleotide analysis after isolation from buffer containing 20 μM GTP. (D) Although GTP is desalted slightly less efficiently than GDP (compare with A), >99.9% of free GTP is removed from control buffer. (E) Exclusively, GTP binds αβ-tubulin dimer (for quantitation see Table II: 20 μM GTP, experiment 1). (F) Small amounts of both GDP and GTP are detected bound to γ-tubulin in the γTuSC (for quantitation see Table II: 20 μM GTP, experiment 1). (G) Summary of nucleotide analysis from three independent experiments (raw data shown in Table II). Bar graphs indicate the ratio of bound nucleotide per αβ-tubulin dimer, or γ-tubulin monomer in γTuSC after isolation from buffers containing 20 μM GDP or 20 μM GTP. Error bars represent the SEM. The ratio of GDP/GTP recovered when we desalt αβ-tubulin dimer from buffer containing GDP is very reproducible (0.733 ± 0.015, n = 5) suggesting that the protein concentration determined by densitometry is the least accurate parameter in this analysis. This ratio also suggests that we are recovering ∼73% of the GDP bound to the β-tubulin E-site.

Mentions: Each αβ-tubulin dimer has two guanine nucleotide binding sites, one on each tubulin subunit. Exclusively GTP is bound to α-tubulin at the nonexchangeable or N-site; this nucleotide does not exchange with GTP/GDP in solution and does not undergo hydrolysis. In contrast, β-tubulin binds guanine nucleotide in an exchangeable fashion at the E-site. Both GTP and GDP bind to the E-site with GTP having a three- to fourfold higher affinity than GDP (Zeeberg and Caplow, 1979). GTP bound to the E-site does not undergo significant hydrolysis in the absence of polymerization but gets hydrolyzed soon after incorporation into the MT lattice, resulting in GDP that is locked in the lattice and can only exchange after depolymerization (reviewed in Desai and Mitchison, 1997). These properties predict that if αβ-tubulin dimer is isolated from buffers containing GDP, then there will be 1 mol GTP (N-site) and 1 mol GDP (E-site) per mole of αβ-tubulin dimer. In contrast, if αβ-tubulin dimer is isolated from buffers containing GTP, under conditions where there is no polymerization, then there will be 2 mol GTP (1 N-site GTP and 1 E-site GTP) per mole of αβ-tubulin dimer. Consistent with these predictions, we recovered 1.1 mol of GTP and 0.8 mol GDP per mole of αβ-tubulin dimer isolated from GDP buffer (Fig. 9, B and G, and Table II); in contrast, we recovered exclusively 2.0 mol of GTP per mole of αβ-tubulin dimer isolated from GTP buffer (Fig. 9, E and G, and Table II). These results establish the validity of our assay for comparing the nucleotide-binding properties of γTuSC to those of αβ-tubulin dimer.


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)

Analysis of nucleotide bound  to γ-tubulin in γTuSC, a comparison  with αβ-tubulin dimer. Desalting was  used to remove free nucleotide from  samples of purified γTuSC or αβ-tubulin. Bound nucleotide was released by  urea treatment and analyzed on a mono  Q column. Arrows indicate the elution  positions of GDP and GTP. For samples containing protein (B, C, E, and  F), a gel lane of the desalted sample is  shown to the right of the UV trace.  (A–C) Nucleotide analysis after isolation from buffer containing 20 μM  GDP. (A) After desalting of control  buffer no nucleotide is detected (the  broad peak just to the left of the GDP  arrow is a background peak). (B) Both  GDP and GTP are bound to αβ-tubulin  dimer (for quantitation see Table II: 20  μM GDP, experiment 3). (C) Exclusively, GDP binds to γ-tubulin in  γTuSC (for quantitation see Table II:  20 μM GDP, experiment 3). (D–F) Nucleotide analysis after isolation from  buffer containing 20 μM GTP. (D) Although GTP is desalted slightly less efficiently than GDP (compare with A),  >99.9% of free GTP is removed from  control buffer. (E) Exclusively, GTP binds αβ-tubulin dimer (for quantitation see Table II: 20 μM GTP, experiment 1). (F) Small  amounts of both GDP and GTP are detected bound to γ-tubulin in the γTuSC (for quantitation see Table II: 20 μM GTP, experiment  1). (G) Summary of nucleotide analysis from three independent experiments (raw data shown in Table II). Bar graphs indicate the ratio  of bound nucleotide per αβ-tubulin dimer, or γ-tubulin monomer in γTuSC after isolation from buffers containing 20 μM GDP or 20  μM GTP. Error bars represent the SEM. The ratio of GDP/GTP recovered when we desalt αβ-tubulin dimer from buffer containing  GDP is very reproducible (0.733 ± 0.015, n = 5) suggesting that the protein concentration determined by densitometry is the least accurate parameter in this analysis. This ratio also suggests that we are recovering ∼73% of the GDP bound to the β-tubulin E-site.
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Related In: Results  -  Collection

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Figure 9: Analysis of nucleotide bound to γ-tubulin in γTuSC, a comparison with αβ-tubulin dimer. Desalting was used to remove free nucleotide from samples of purified γTuSC or αβ-tubulin. Bound nucleotide was released by urea treatment and analyzed on a mono Q column. Arrows indicate the elution positions of GDP and GTP. For samples containing protein (B, C, E, and F), a gel lane of the desalted sample is shown to the right of the UV trace. (A–C) Nucleotide analysis after isolation from buffer containing 20 μM GDP. (A) After desalting of control buffer no nucleotide is detected (the broad peak just to the left of the GDP arrow is a background peak). (B) Both GDP and GTP are bound to αβ-tubulin dimer (for quantitation see Table II: 20 μM GDP, experiment 3). (C) Exclusively, GDP binds to γ-tubulin in γTuSC (for quantitation see Table II: 20 μM GDP, experiment 3). (D–F) Nucleotide analysis after isolation from buffer containing 20 μM GTP. (D) Although GTP is desalted slightly less efficiently than GDP (compare with A), >99.9% of free GTP is removed from control buffer. (E) Exclusively, GTP binds αβ-tubulin dimer (for quantitation see Table II: 20 μM GTP, experiment 1). (F) Small amounts of both GDP and GTP are detected bound to γ-tubulin in the γTuSC (for quantitation see Table II: 20 μM GTP, experiment 1). (G) Summary of nucleotide analysis from three independent experiments (raw data shown in Table II). Bar graphs indicate the ratio of bound nucleotide per αβ-tubulin dimer, or γ-tubulin monomer in γTuSC after isolation from buffers containing 20 μM GDP or 20 μM GTP. Error bars represent the SEM. The ratio of GDP/GTP recovered when we desalt αβ-tubulin dimer from buffer containing GDP is very reproducible (0.733 ± 0.015, n = 5) suggesting that the protein concentration determined by densitometry is the least accurate parameter in this analysis. This ratio also suggests that we are recovering ∼73% of the GDP bound to the β-tubulin E-site.
Mentions: Each αβ-tubulin dimer has two guanine nucleotide binding sites, one on each tubulin subunit. Exclusively GTP is bound to α-tubulin at the nonexchangeable or N-site; this nucleotide does not exchange with GTP/GDP in solution and does not undergo hydrolysis. In contrast, β-tubulin binds guanine nucleotide in an exchangeable fashion at the E-site. Both GTP and GDP bind to the E-site with GTP having a three- to fourfold higher affinity than GDP (Zeeberg and Caplow, 1979). GTP bound to the E-site does not undergo significant hydrolysis in the absence of polymerization but gets hydrolyzed soon after incorporation into the MT lattice, resulting in GDP that is locked in the lattice and can only exchange after depolymerization (reviewed in Desai and Mitchison, 1997). These properties predict that if αβ-tubulin dimer is isolated from buffers containing GDP, then there will be 1 mol GTP (N-site) and 1 mol GDP (E-site) per mole of αβ-tubulin dimer. In contrast, if αβ-tubulin dimer is isolated from buffers containing GTP, under conditions where there is no polymerization, then there will be 2 mol GTP (1 N-site GTP and 1 E-site GTP) per mole of αβ-tubulin dimer. Consistent with these predictions, we recovered 1.1 mol of GTP and 0.8 mol GDP per mole of αβ-tubulin dimer isolated from GDP buffer (Fig. 9, B and G, and Table II); in contrast, we recovered exclusively 2.0 mol of GTP per mole of αβ-tubulin dimer isolated from GTP buffer (Fig. 9, E and G, and Table II). These results establish the validity of our assay for comparing the nucleotide-binding properties of γTuSC to those of αβ-tubulin dimer.

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