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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 the γTuSC. (A) Immunoisolated  γTuSC was fractionated on a 5–20% sucrose gradient in buffer  containing 500 mM NaCl. Fractions were separated by SDS-PAGE on a 10% gel and stained with Coomassie blue. A standards gradient was run in parallel. Peak fractions for standards  were: BSA (4.3 S), fraction 4.9; aldolase (7.35 S), fraction 7.5;  and catalase (11.3 S), fraction 10.8. (B) Fractions from the gradient in A were dialyzed against buffer containing 100 mM NaCl  and tested in the coverslip assay. Bar, 10 μm. (C) γTuSC from  the sucrose gradient in 500 mM NaCl was fractionated by Superose 6 gel filtration in 500 mM NaCl (top), or was first dialyzed  against buffer containing 100 mM NaCl and then fractionated by  Superose-6 gel filtration in buffer containing 100 mM NaCl (bottom). Standards of known Stokes radius were used to calibrate  the column. The peak fractions for the gel filtration standards  were: bovine thyroglobulin (8.5 nm), fraction 13.5; horse spleen  ferritin (6.1 nm), fraction 15.4; bovine liver catalase (5.22 nm),  fraction 16.9; aldolase (4.81 nm), fraction 17.1; ovalbumin (3.05  nm), fraction 18.0; and chymotrypsinogen (2.09 nm), fraction  20.0.
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Figure 5: Characterization of the γTuSC. (A) Immunoisolated γTuSC was fractionated on a 5–20% sucrose gradient in buffer containing 500 mM NaCl. Fractions were separated by SDS-PAGE on a 10% gel and stained with Coomassie blue. A standards gradient was run in parallel. Peak fractions for standards were: BSA (4.3 S), fraction 4.9; aldolase (7.35 S), fraction 7.5; and catalase (11.3 S), fraction 10.8. (B) Fractions from the gradient in A were dialyzed against buffer containing 100 mM NaCl and tested in the coverslip assay. Bar, 10 μm. (C) γTuSC from the sucrose gradient in 500 mM NaCl was fractionated by Superose 6 gel filtration in 500 mM NaCl (top), or was first dialyzed against buffer containing 100 mM NaCl and then fractionated by Superose-6 gel filtration in buffer containing 100 mM NaCl (bottom). Standards of known Stokes radius were used to calibrate the column. The peak fractions for the gel filtration standards were: bovine thyroglobulin (8.5 nm), fraction 13.5; horse spleen ferritin (6.1 nm), fraction 15.4; bovine liver catalase (5.22 nm), fraction 16.9; aldolase (4.81 nm), fraction 17.1; ovalbumin (3.05 nm), fraction 18.0; and chymotrypsinogen (2.09 nm), fraction 20.0.

Mentions: The absence of nucleation activity of sucrose gradient–isolated γTuSC in the coverslip assay (Fig. 3 D) can be explained in several ways: γTuSC (a) might not have nucleating activity; (b) might not bind to the coverslip under the assay conditions; (c) might become inactivated upon binding to the coverslip, or (d) might be too dilute to exhibit activity. To distinguish between these possibilities we developed a protocol to prepare more concentrated γTuSC, taking advantage of the disruption of γTuRC into γTuSC by high salt (Fig. 1). γTuRC was disrupted by isolating γ-tubulin–containing complexes in the presence of 500 mM NaCl. The resulting γTuSC was eluted with peptide containing buffer in 500 mM NaCl. The peptide-eluted material was further fractionated on a 5–20% sucrose gradient in 500 mM NaCl (Fig. 5 A). This gradient separated γTuSC from residual larger complexes and from non-γTuSC components of γTuRC. This resulted in highly concentrated, relatively pure γTuSC (15 μl of each fraction was loaded on the gel in Fig. 5 A compared with 50 μl in Fig. 3 A). Inclusion of 500 mM salt in the sucrose gradient was important to prevent any reassociation of γTuSC with nonγTuSC components of γTuRC. Typically, the peak γTuSC sucrose gradient fraction contained ∼700 nM γ-tubulin (as judged by densitometry of Coomassie-stained bands relative to αβ-tubulin standards). For comparison, after sucrose gradient fractionation, the peak γTuSC-containing sucrose gradient fraction in the mixed complex preparation (Fig. 3 A) contained ∼70 nM γ-tubulin.


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 the γTuSC. (A) Immunoisolated  γTuSC was fractionated on a 5–20% sucrose gradient in buffer  containing 500 mM NaCl. Fractions were separated by SDS-PAGE on a 10% gel and stained with Coomassie blue. A standards gradient was run in parallel. Peak fractions for standards  were: BSA (4.3 S), fraction 4.9; aldolase (7.35 S), fraction 7.5;  and catalase (11.3 S), fraction 10.8. (B) Fractions from the gradient in A were dialyzed against buffer containing 100 mM NaCl  and tested in the coverslip assay. Bar, 10 μm. (C) γTuSC from  the sucrose gradient in 500 mM NaCl was fractionated by Superose 6 gel filtration in 500 mM NaCl (top), or was first dialyzed  against buffer containing 100 mM NaCl and then fractionated by  Superose-6 gel filtration in buffer containing 100 mM NaCl (bottom). Standards of known Stokes radius were used to calibrate  the column. The peak fractions for the gel filtration standards  were: bovine thyroglobulin (8.5 nm), fraction 13.5; horse spleen  ferritin (6.1 nm), fraction 15.4; bovine liver catalase (5.22 nm),  fraction 16.9; aldolase (4.81 nm), fraction 17.1; ovalbumin (3.05  nm), fraction 18.0; and chymotrypsinogen (2.09 nm), fraction  20.0.
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Figure 5: Characterization of the γTuSC. (A) Immunoisolated γTuSC was fractionated on a 5–20% sucrose gradient in buffer containing 500 mM NaCl. Fractions were separated by SDS-PAGE on a 10% gel and stained with Coomassie blue. A standards gradient was run in parallel. Peak fractions for standards were: BSA (4.3 S), fraction 4.9; aldolase (7.35 S), fraction 7.5; and catalase (11.3 S), fraction 10.8. (B) Fractions from the gradient in A were dialyzed against buffer containing 100 mM NaCl and tested in the coverslip assay. Bar, 10 μm. (C) γTuSC from the sucrose gradient in 500 mM NaCl was fractionated by Superose 6 gel filtration in 500 mM NaCl (top), or was first dialyzed against buffer containing 100 mM NaCl and then fractionated by Superose-6 gel filtration in buffer containing 100 mM NaCl (bottom). Standards of known Stokes radius were used to calibrate the column. The peak fractions for the gel filtration standards were: bovine thyroglobulin (8.5 nm), fraction 13.5; horse spleen ferritin (6.1 nm), fraction 15.4; bovine liver catalase (5.22 nm), fraction 16.9; aldolase (4.81 nm), fraction 17.1; ovalbumin (3.05 nm), fraction 18.0; and chymotrypsinogen (2.09 nm), fraction 20.0.
Mentions: The absence of nucleation activity of sucrose gradient–isolated γTuSC in the coverslip assay (Fig. 3 D) can be explained in several ways: γTuSC (a) might not have nucleating activity; (b) might not bind to the coverslip under the assay conditions; (c) might become inactivated upon binding to the coverslip, or (d) might be too dilute to exhibit activity. To distinguish between these possibilities we developed a protocol to prepare more concentrated γTuSC, taking advantage of the disruption of γTuRC into γTuSC by high salt (Fig. 1). γTuRC was disrupted by isolating γ-tubulin–containing complexes in the presence of 500 mM NaCl. The resulting γTuSC was eluted with peptide containing buffer in 500 mM NaCl. The peptide-eluted material was further fractionated on a 5–20% sucrose gradient in 500 mM NaCl (Fig. 5 A). This gradient separated γTuSC from residual larger complexes and from non-γTuSC components of γTuRC. This resulted in highly concentrated, relatively pure γTuSC (15 μl of each fraction was loaded on the gel in Fig. 5 A compared with 50 μl in Fig. 3 A). Inclusion of 500 mM salt in the sucrose gradient was important to prevent any reassociation of γTuSC with nonγTuSC components of γTuRC. Typically, the peak γTuSC sucrose gradient fraction contained ∼700 nM γ-tubulin (as judged by densitometry of Coomassie-stained bands relative to αβ-tubulin standards). For comparison, after sucrose gradient fractionation, the peak γTuSC-containing sucrose gradient fraction in the mixed complex preparation (Fig. 3 A) contained ∼70 nM γ-tubulin.

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