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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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Hydrodynamic analysis of γ-tubulin in concentrated  Drosophila embryo extracts. (Top) γ-tubulin immunoblots of Superose 6 gel filtration column fractions in buffer containing 100 μM  GTP and 100 or 500 mM NaCl. Calibration standards for the Superose 6 column: bovine thyroglobulin (Stokes radius = 8.5 nm),  horse spleen ferritin (6.1 nm), bovine liver catalase (5.22 nm),  and bovine serum albumin (3.55 nm), as indicated with arrowheads. (Bottom) γ-tubulin immunoblots of 5–40% sucrose gradient fractions in buffer containing 100 μM GTP and 100 or 500 mM  NaCl. Gradients were sedimented at 50,000 rpm for 4 h in an  SW55 rotor at 4°C and fractionated from the top; gradient pellets  are also shown (P). The peak locations of standards run on parallel gradients are indicated with arrowheads. Sucrose gradient  standards: bovine serum albumin (4.3 S), rabbit muscle aldolase  (7.35 S), bovine liver catalase (11.3 S), and porcine thyroglobulin  (19.4S).
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Figure 1: Hydrodynamic analysis of γ-tubulin in concentrated Drosophila embryo extracts. (Top) γ-tubulin immunoblots of Superose 6 gel filtration column fractions in buffer containing 100 μM GTP and 100 or 500 mM NaCl. Calibration standards for the Superose 6 column: bovine thyroglobulin (Stokes radius = 8.5 nm), horse spleen ferritin (6.1 nm), bovine liver catalase (5.22 nm), and bovine serum albumin (3.55 nm), as indicated with arrowheads. (Bottom) γ-tubulin immunoblots of 5–40% sucrose gradient fractions in buffer containing 100 μM GTP and 100 or 500 mM NaCl. Gradients were sedimented at 50,000 rpm for 4 h in an SW55 rotor at 4°C and fractionated from the top; gradient pellets are also shown (P). The peak locations of standards run on parallel gradients are indicated with arrowheads. Sucrose gradient standards: bovine serum albumin (4.3 S), rabbit muscle aldolase (7.35 S), bovine liver catalase (11.3 S), and porcine thyroglobulin (19.4S).

Mentions: Drosophila embryo extracts contain two γ-tubulin–containing complexes that can be separated by gel filtration chromatography or sucrose gradient sedimentation. In the presence of 500 mM KCl or NaCl, γ-tubulin is found exclusively in the smaller complex, indicating that the larger complex has been disrupted, and that the smaller complex is likely to be a structural subunit of the larger complex (Moritz et al., 1998). We named the large complex, Drosophila γTuRC (see below), and the small complex the γTuSC. To obtain size estimates for each complex, we performed gel filtration and sucrose gradient sedimentation under low salt conditions in buffers that were supplemented with magnesium and GTP (Fig. 1) to reduce aggregation that occurs in nucleotide-free buffers. Under these conditions, the γTuSC has an S value of 9.8 and a Stokes radius of 7.0 nm, while the γTuRC has an S value of 35.5 S and a 15-nm Stokes radius. Based on these values, we estimate the molecular masses of the γTuSC and γTuRC to be 280,000 and 2,200,000 D (Table I), respectively.


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)

Hydrodynamic analysis of γ-tubulin in concentrated  Drosophila embryo extracts. (Top) γ-tubulin immunoblots of Superose 6 gel filtration column fractions in buffer containing 100 μM  GTP and 100 or 500 mM NaCl. Calibration standards for the Superose 6 column: bovine thyroglobulin (Stokes radius = 8.5 nm),  horse spleen ferritin (6.1 nm), bovine liver catalase (5.22 nm),  and bovine serum albumin (3.55 nm), as indicated with arrowheads. (Bottom) γ-tubulin immunoblots of 5–40% sucrose gradient fractions in buffer containing 100 μM GTP and 100 or 500 mM  NaCl. Gradients were sedimented at 50,000 rpm for 4 h in an  SW55 rotor at 4°C and fractionated from the top; gradient pellets  are also shown (P). The peak locations of standards run on parallel gradients are indicated with arrowheads. Sucrose gradient  standards: bovine serum albumin (4.3 S), rabbit muscle aldolase  (7.35 S), bovine liver catalase (11.3 S), and porcine thyroglobulin  (19.4S).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Hydrodynamic analysis of γ-tubulin in concentrated Drosophila embryo extracts. (Top) γ-tubulin immunoblots of Superose 6 gel filtration column fractions in buffer containing 100 μM GTP and 100 or 500 mM NaCl. Calibration standards for the Superose 6 column: bovine thyroglobulin (Stokes radius = 8.5 nm), horse spleen ferritin (6.1 nm), bovine liver catalase (5.22 nm), and bovine serum albumin (3.55 nm), as indicated with arrowheads. (Bottom) γ-tubulin immunoblots of 5–40% sucrose gradient fractions in buffer containing 100 μM GTP and 100 or 500 mM NaCl. Gradients were sedimented at 50,000 rpm for 4 h in an SW55 rotor at 4°C and fractionated from the top; gradient pellets are also shown (P). The peak locations of standards run on parallel gradients are indicated with arrowheads. Sucrose gradient standards: bovine serum albumin (4.3 S), rabbit muscle aldolase (7.35 S), bovine liver catalase (11.3 S), and porcine thyroglobulin (19.4S).
Mentions: Drosophila embryo extracts contain two γ-tubulin–containing complexes that can be separated by gel filtration chromatography or sucrose gradient sedimentation. In the presence of 500 mM KCl or NaCl, γ-tubulin is found exclusively in the smaller complex, indicating that the larger complex has been disrupted, and that the smaller complex is likely to be a structural subunit of the larger complex (Moritz et al., 1998). We named the large complex, Drosophila γTuRC (see below), and the small complex the γTuSC. To obtain size estimates for each complex, we performed gel filtration and sucrose gradient sedimentation under low salt conditions in buffers that were supplemented with magnesium and GTP (Fig. 1) to reduce aggregation that occurs in nucleotide-free buffers. Under these conditions, the γTuSC has an S value of 9.8 and a Stokes radius of 7.0 nm, while the γTuRC has an S value of 35.5 S and a 15-nm Stokes radius. Based on these values, we estimate the molecular masses of the γTuSC and γTuRC to be 280,000 and 2,200,000 D (Table I), respectively.

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