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GTP regulates the microtubule nucleation activity of γ-tubulin

Nature Cell Biology volume 15, pages 1317–1327 (2013)Cite this article

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Abstract

Both subunits of αβ-tubulin that comprise the core components of microtubules bind GTP. GTP binding to α-tubulin has a structural role, whereas β-tubulin binds and hydrolyses GTP to regulate microtubule dynamics. γ-tubulin, another member of the tubulin superfamily that seeds microtubule nucleation at microtubule-organizing centres, also binds GTP; however, the importance of this association remains elusive. To address the role of GTP binding to γ-tubulin, we systematically mutagenized the GTP contact residues in the yeast γ-tubulin Tub4. Tub4GTP-mutant proteins that exhibited greatly reduced GTP affinity still assembled into the small γ-tubulin complex. However, tub4GTP mutants were no longer viable, and had defects in interaction between γ-tubulin and αβ-tubulin, decreased microtubule nucleation and defects in microtubule organization. In vitro and in vivo data show that only γ-tubulin loaded with GTP nucleates microtubules. Our results suggest that GTP recruitment to γ-tubulin enhances its interaction with αβ-tubulin similarly to GTP recruitment to β-tubulin.

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Figure 1: GTP binding to the yeast γ-tubulin Tub4.
Figure 2: Tub4GTP proteins assemble together with Spc97 and Spc98 into γ-TuSC and localize to spindle pole bodies (SPBs).
Figure 3: microtubule organization defects of tub4GTP strains.
Figure 4: microtubule dynamics of tub4GTP cells.
Figure 5: microtubule nucleation defects of tub4GTP mutants.
Figure 6: GTP hydrolysis by Tub4 may regulate the γ-tubulin–tubulin interaction.

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Acknowledgements

This work was supported by the grant DFG Schi 295/4-1/2. We acknowledge D. Botstein (Princeton University, USA) and M. Knop (ZMBH, Heidelberg University, Germany) for plasmids and J. Kilmartin (MRC, Cambridge, UK) for antibodies. We thank I. Hagan for critical reading of the manuscript and U. Jäkle for technical assistance. We thank the EMBL EM facility for assistance and helpful discussions.

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  1. Luca L. Fava

    Present address: Present address: Division of Developmental Immunology, Innsbruck Medical University, Innrain 80-82, 6020 Innsbruck, Austria,

Authors and Affiliations

  1. Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), ZMBH-DKFZ Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany

    Linda Gombos, Annett Neuner, Luca L. Fava, Rebecca C. Wade & Elmar Schiebel

  2. Heidelberg Institute for Theoretical Studies, Schloss-Wolfsbrunnenweg 35, 69118 Heidelberg, Germany

    Mykhaylo Berynskyy & Rebecca C. Wade

  3. Structural and Computational Biology, EMBL Heidelberg, Meyerhofstr. 1, 69117 Heidelberg, Germany

    Carsten Sachse

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Contributions

L.G. and E.S. designed experiments and wrote the manuscript; L.G. performed most experiments; A.N. performed all the electron microscopy; L.L.F. generated some yeast strains; L.G., M.B. and R.C.W. performed homology modelling; C.S. designed and evaluated single particle electron microscopy.

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Correspondence to Elmar Schiebel.

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Integrated supplementary information

Supplementary Figure 1 GTP binding to yeast γ-tubulin Tub4.

(a) Sequence alignment of Tub4 and human γ-tubulin from the 1Z5V crystal structure, which was used as a template for modelling. Conserved residues are marked with an asterisk. Residues contacting the bound GTPγS are highlighted in red. (b) γ-TuSC purified from insect cells contained 9.4% bound GTP as determined by RP-HPLC on a C18 column1. Elution profiles of 20 μl of a 100 μM GTP standard and of 73.3 μM γ-TuSC are shown. The protein concentration was determined using the calculated molar extinction coefficient. (c) Phenotypes of wild type TUB4 cells and nuclear migration defects of a representative ts tub4GTP strain (T146A). (d) Quantification of nuclear migration phenotypes of tub4GTP strains obtained from DAPI stained cultures grown overnight at 30 °C.

Supplementary Figure 2 tub4GTP levels and complex formation.

(a) Spc97-3HA was immunoprecipitated (IP) by anti-HA antibodies from cell extracts of ts tub4GTP strains. Before immunoprecipitation, cells were synchronized with α-factor at 23 °C and released at 37 °C for 2 h. (b) Quantification of a. Error bars represent s.e.m. (normalized Tub4 values in Spc97-3HA Co-IPs of n = 3 independent experiments). See Supplementary Table 3 for raw data. Two-tailed, unpaired Student’s t-test was used to obtain P values. Tub4 in the γ-TuSC is not significantly different between tub4GTP and wild type cells (P≥0.05). (c) TUB4-AID GFP-TUB1 SPC42-mCherry cells with pGalS (null), pGalS-TUB4 (wt) orpGalS−tub4GTP (N228A; C13S T146A; N206A L224A) were synchronised with α-factor, followed by both, galactose and IAA addition. Cells were released from the arrest in the presence of galactose and IAA and fixed at the time points indicated. Cell cycle progression of DAPI stained cells was analysed. (phenotypes of n = 100 cells for each time point per experiment). (d) MT defects of TUB4-AID pGalS−tub4GTP cells. Cells were treated as in c and analysed 2 h after release, when entering metaphase. Bar, 5 μm. (e) Quantification of d (phenotypes of n = 100 cells per strain per experiment). (f) Overexpression of ectopic pGalS−tub4GTP in the presence of the wild type TUB4 allele in rich medium. Extracts of cells grown overnight at 30 °C under repressive (glucose) or inducing (raffinose/galactose) conditions were analysed by immunostaining with the indicated antibodies. (g) Quantification of the nuclear migration phenotypes shows metaphase arrest of cells overexpressing ectopic pGalS−tub4GTP in the presence of the wild type TUB4 allele. Cells were grown as in f. DNA was stained with DAPI. (phenotypes of n = 100 cells per strain per experiment).

Supplementary Figure 3 γ-TuSCGTP localizes to SPBs.

(a) Calibration of anti-Tub4 immunostaining using a Δtub4 strain (TUB4-AID) in the presence of IAA), two haploid strains containing one ectopic copy of TUB4 (TUB4-AID/TUB4 in the presence of IAA) or the wild type TUB4 (YPH499) and a diploid strain carrying two copies of TUB4/TUB4 (YPH501). Cells were fixed 75 min after release from α-factor when entering metaphase. Bar, 2 μm. (b) Quantification of a shows linearity. Boxes represent upper and lower quartiles (25–75%) with a line at the median, whiskers extend from the minimum to the maximum. 5 independent experiments with each n = 50 cells per strain analyzed. (c) Tub4GTP (class 2: T146A; N206A, class 3: Q12G C13S T146A) localizes to SPBs by anti-Tub4 immunostaining. Conditional lethal tub4GTP cells were synchronized with α-factor at 23 °C and released at 37 °C for 2 h. Bar, 2 μm. (d) Quantification of c. Boxes represent upper and lower quartiles (25-75%) with a line at the median, whiskers extend from the minimum to the maximum. 5 independent experiments with n = 50 cells analyzed per experiment. (e) FRAP experiments of Spc97-yeGFP in TUB4-AID cells with TUB4 and a representative tub4GTP mutant (C13S T146A) in the presence of IAA. Cells were released from α-factor arrest into nocodazole containing medium for 2 h. Normalized fluorescence intensity of Spc97-yeGFP at SPBs (top) and images of photobleached cells (bottom) are shown. ROI means region of interest– in this experiment the SPB. As a control the whole cell was bleached. None of the strains showed fluorescence recovery of SPB-associated Spc97-yeGFP. Bars, 5 μm.

Supplementary Figure 4 Analysis γ-TuSCGTP complexes purified from insect cells.

(a) SDS-PAGE of purified γ-TuSC (wt) and γ-TuSCGTP. Tub4 gives two bands due to partial cleavage of Met1 as determined by mass spectrometry analysis. Bottom: band intensities of the Coomassie-stained gel were assessed by densitometry. (b) Size-exclusion chromatography of recombinant and purified GST-Spc1101−220 (top left), γ-TuSC (wt) and γ-TuSCGTP with (right) and without (left) the N-terminus of the receptor protein Spc110 (GST-Spc1101−220). γ-TuSC /γ-TuSCGTP and GST-Spc1101−220 were mixed at a 1:2 molar ratio and analysed by gel filtration. SDS-PAGE of the peak fractions shows that γ-TuSC/γ-TuSCGTP co-elutes with GST-Spc1101−220. (c) Electron micrographs of negatively stained γ-TuSCGTP show typical Y-shaped complexes resembling wild type γ-TuSC2. Bar, 10 nm. (d) Aligned and classified particles of negatively stained wt γ-TuSC and a representative TuSCGTP (C13S T146A). 1911 particles were analyzed for wild type and 1826 particles for the TuSCC13ST146A mutant and classified into 20 classes each. Bar, 10 nm.

Supplementary Figure 5 MT organization defects of tub4GTP strains.

(a) ts tub4GTP cells (class 2: T146A; N206A and class 3: Q12G C13G T146A) arrest in metaphase at the restrictive temperature. Cells were synchronized with α-factor at 23 °C, released at 37 °C and fixed at the time points indicated. Cell cycle progression of DAPI stained cells was analysed. (phenotypes of n = 100 cells per strain per experiment; 5 independent experiments). (b) MT defects of ts tub4GTP cells. Cells were treated as in a and analysed 2 h after release. Bar, 5 μm. (c) Quantification of b. (phenotype of n = 100 cells for each strain per experiment). (d) Electron micrographs of thin sections stained with uranyl-acetate showing MT phenotypes of ts tub4GTP cells. Cells were treated as in a. Cartoons on the right shows the SPB and MTs of the micrograph on the left. CP, cytoplasm; N, nucleus; NE, nuclear envelope; cMT, cytoplasmic MT; nMT, nuclear MT. Bar, 200 nm. (e) Quantification of d. MT phenotypes of n = 9 cells for wt, 12 for T146A, 6 for N206A, and 11 for Q12G C13G T146A. (f) Benomyl rescue of ts tub4GTP cells. Cells were grown at 23 °C and spotted onto benomyl containing plates or control plates and grown at the indicated temperatures.

Supplementary Figure 6 MT nucleation by tub4GTP.

(a) Anti-Tub4 immunostaining of TUB4-AID cells (“null”) with TUB4 (wt) or tub4GTP at SPBs in the presence of IAA and nocodazole. Cells were treated as in Fig. 5a. Bar, 2 μm. (b) Quantification of a. Boxes represent upper and lower quartiles (25-75%) with a line at the median, whiskers extend from the minimum to the maximum. n = 50 cells analyzed per strain per experiment. 5 independent experiments were performed. Two-tailed, unpaired Student’s t-test was used to compare samples and to obtain P values. There are no significant differences between TUB4 and tub4GTP cells (P ≥ 0.05), while the null mutant is different from the wild type with P 0.001. (c) The kinetics of MT re-nucleation in tub4GTP cells is not influenced by functional kinetochores. TUB4-AID NDC10-AID cells with additional TUB4, tub4N228A or NDC10 (as indicated in the figure) were grown and treated with nocodazole as in Fig. 5c. The formation of MTs was followed over time. Middle: For each time point n = 100 cells were analyzed per strain per experiment for MTs at the SPB. The right panel shows the quantification of cells for MT phenotypes (illustrated in right upper corner). n = 100 cells were analyzed for each time point and strain. (d) Electron micrographs of negatively stained rings of γ-TuSCGTP formed under low pH and low salt conditions. Bar, 25 nm. (e) Alignment of Tub1 and FtsZ by MODELLER. Note the long insertion in Tub1 between residues 261-319, which is not included in our model due to the lack of a homologous template. Conserved residues are marked with an asterisk. The two acidic residues that promote GTP hydrolysis are highlighted in red. (f) Anti-Tub4 immunostaining of ts tub4GTP cells at 37 °C in the presence of nocodazole. Bar, 2 nm. (g) Quantification of f. Boxes represent upper and lower quartiles (25-75%) with a line at the median, whiskers extend from the minimum to the maximum. 5 independent experiments with n = 50 cells analyzed per experiment per strain. Two-tailed, unpaired Student’s t-test was used to obtain P values. There are no significant differences between TUB4 and tub4GTP cells (P ≥ 0.05).

Supplementary Figure 7 Model for the GTP/MT nucleation cycle of γ-tubulin.

See Discussion for details.

Supplementary Figure 8 Full scans of blots.

The background on the α-Spc98 blot from Fig. 2a is due to residual α-Tub4 staining after stripping.

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Gombos, L., Neuner, A., Berynskyy, M. et al. GTP regulates the microtubule nucleation activity of γ-tubulin. Nat Cell Biol 15, 1317–1327 (2013). https://doi.org/10.1038/ncb2863

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