Ring closure activates yeast γTuRC for species-specific microtubule nucleation

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Abstract

The γ-tubulin ring complex (γTuRC) is the primary microtubule nucleator in cells. γγTuRC is assembled from repeating γγ-tubulin small complex (γTuSC) subunits and is thought to function as a template by presenting a γ-tubulin ring that mimics microtubule geometry. However, a previous yeast γTuRC structure showed γTuSC in an open conformation that prevents matching to microtubule symmetry. By contrast, we show here that γ-tubulin complexes are in a closed conformation when attached to microtubules. To confirm the functional importance of the closed γTuSC ring, we trapped the closed state and determined its structure, showing that the γ-tubulin ring precisely matches microtubule symmetry and providing detailed insight into γTuRC architecture. Importantly, the closed state is a stronger nucleator, thus suggesting that this conformational switch may allosterically control γTuRC activity. Finally, we demonstrate that γTuRCs have a strong preference for tubulin from the same species.

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Figure 1: The yeast γTuRC is formed from seven γTuSCs and is limited in size by Spc110.
Figure 2: Engineered disulfides alter γTuSC filament morphology.
Figure 3: In the closed state, γTuSC matches microtubule symmetry and has increased nucleation activity.
Figure 4: Pseudoatomic model of γTuSC in the closed conformation.
Figure 5: Pseudoatomic model of γTuRC and its interactions with microtubules.

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Acknowledgements

The authors are grateful to M. Braunfeld and X. Li for assistance with cryo-EM data collection and L. Rice for help with overexpression and purification of yeast tubulin. This work was funded by the Howard Hughes Medical Institute and by US National Institutes of Health grants R01 GM031627 to D.A.A., R01 GM040506 to T.N.D. and T32 GM007270 to K.K.F.

Author information

J.M.K. prepared samples, collected cryo-EM data, performed three-dimensional reconstructions, analyzed data and wrote the paper. C.H.G. performed molecular modeling, analyzed results and contributed to writing the paper. S.L. prepared samples, collected tomographic data, performed tomographic reconstructions and analyzed data. M.M. performed microtubule nucleation assays and analyzed data. A.Z. generated expression constructs and optimized protein expression. K.K.F. performed yeast viability assays and spindle morphology experiments and analyzed data. J.-J.F. assisted with subvolume averaging. A.S. analyzed data and contributed to writing the paper. J.K. provided samples and analyzed data. T.N.D. analyzed data and contributed to writing the paper. D.A.A. analyzed data and contributed to writing the paper.

Correspondence to David A Agard.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The structure of wild-type γTuSC oligomers does not perfectly match 13-protofilament-microtubule geometry.

a) Two laterally-associated γTuSC subunits from ring-like γTuSC assemblies are shown. The two γ-tubulins (gold) within each γTuSC are held separate from each other by GCP2 and GCP3, while the γ-tubulins at the inter-γTuSC interface are positioned with microtubule-like lateral contacts. b) The intra-γTuSC separation results in a staggered pattern of γ-tubulin in γTuSC assemblies, with a helical pitch of 147 Å. c) By contrast, lateral contacts between tubulin subunits in a microtubule are uniform around the filament, and have a helical pitch of 122 Å. d) To illustrate the mismatch between γTuSC oligomers and the microtubule, the upper γ-tubulin in the ring has been aligned to make longitudinal contacts with α-tubulin at the minus end of the microtubule. This would result in a large gap between α- and γ-tubulin at the last position in the ring.

Supplementary Figure 2 γTuSCCC does not affect cell viability.

a) Representative images of WT, glr1Δ, and γTuSCCC glr1Δ fluorescent strains. GLR1 encodes glutathione reductase; glr1Δ cells were used to maximize the chance that the γTuSCCC disulfides would be oxidized. Cells in the top row have the spindle pole bodies marked by Spc42-mCherry and the microtubules marked by GFP-Tub1. Cells in the bottom row have the spindle pole bodies marked by Spc42-mCherry and the kinetochores marked by Nuf2-GFP. b) The doubling times of WT and γTuSCCC strains. c) Average GFP-Tub1 fluorescence distributions show that γTuSCCC has no effect on tubulin distribution across three spindle length classifications. d) Average Nuf2-GFP fluorescence distributions show that γTuSCCC has no effect on kinetochore clustering in metaphase spindles (1.28-1.59 µm), while shorter spindles show kinetochores cluster slightly closer to the spindle pole bodies. Error bars represent the standard error of the mean.

Supplementary Figure 3 Cross-linking and structural analysis of γTuSCCC.

a) γTuSCCC in the presence of 5 mM DTT is predominantly monomeric complex, although γTuSC pairs were frequently observed. b) Under non-reducing conditions γTuSCs spontaneously assemble into oligomers, even in the presence of 500 mM KCl, which prevents association of wildtype γTuSC. c) SDS-PAGE of γTuSCCC under reducing conditions (5 mM DTT), and non-reducing conditions (0 mM DTT). In the non-reducing condition γ-tubulin forms disulfide crosslinks, resulting in a ladder of 1-5 γ-tubulin chains. Formation of γ-tubulin oligomers greater than two chains indicates that crosslinks were formed at both inter-γTuSC and intra-γTuSC interfaces. d) Co-purified γTuSCCC and Spc1101–220 was dialyzed for 72 h. against an oxidizing buffer containing 1mM oxidized glutathione. On SDS-PAGE the reduced sample has the expected bands for Spc1101–220, γ-tubulin, and GCP2/GCP3, while in the unreduced sample extensive γ-tubulin crosslinking prevents its migration into the gel. This highly-cross-linked sample was used for cryo-EM imaging. e) Cryo-EM image of cross-linked γTuSCCC-Spc1101–220 filaments (same sample as in d). f) Segments of γTuSCCC filaments were compared to projections of the γTuSC filament in the open state and a preliminary reconstruction of the closed state. The histogram plots the fractional difference in correlation coefficient, with negative values representing a better match to the open state. Segments matching better to the open state were omitted from the final refinement of the closed filament structure. g) Fourier shell correlation (FSC) of the final γTuSCCC-Spc1101–220 reconstruction indicates a resolution of 6.9 Å at the 0.5 cutoff. Scalebars, 100 nm.

Supplementary Figure 4 Yeast γTuSC nucleation activity is species specific.

a) Comparison of nucleating activity of γTuSC rings with yeast and pig brain tubulin. Nucleation assays were performed as in Fig. 3C (data for yeast tubulin is the same as Fig. 3C), except that the experiments with brain tubulin were at 12 μM tubulin and 37° C (n=4 independent experiments; error bars represent the s.e.m.). b) The predicted longitudinal interaction between α- and γ-tubulin is shown at left. Human γ-tubulin and pig α-tubulin are shown as these are the existing crystal structures. At right the interaction surfaces are highlighted as a function of the conservation between yeast and vertebrate tubulin: grey sites are identical, yellow have conservative amino acid substitutions, red have non-conservative substitutions, magenta is a two amino acid insertion in γ-tubulin, and GTP is shown in orange. c) Binary sequence alignment of yeast and vertebrate a-tubulin. d) Binary sequence alignment of yeast and vertebrate g-tubulin. Color in g (c) and (d) is the same as for (b).

Supplementary Figure 5 Subunit localization and conformation in the γTuSC closed-state structure.

Comparison of the open and closed states of γTuSC. The elongated GCP2 and GCP3 structures each appear slightly straightened. b) Overlay of GCP3 in the open (grey) and closed (blue) states shows that it straightens by about 9°. c) Overlay of GCP2 in the open (grey) and closed (blue) states shows that it straightens by about 8°. d) Superposition of GCP2 and GCP3 in the closed state shows that they are in very similar overall conformation. GCP4, which was used as the starting structure for homology modeling both GCP2 and GCP3, is in a similar overall conformation. e) Superposition of GCP2 and GCP3 in the open state. For B-E all structural alignments were done using the two N-terminal helical bundles. f) The density predicted to correspond to part of Spc1101–220 resembles a two-stranded coiled-coil. A generic two-stranded coiled coil was used to model Spc110, and flexibly fit into the EM density. The closest contacts with Spc110 are between residues 77-97 of GCP2 (which includes 9 residues not included in our model) near the very base of γTuSC, and further up the structure with residues 119-126 of GCP3. g) A cutaway view of the γTuSCCC filament in the closed state, low-pass filtered at 15 Å. The γTuSC and modeled Spc110 structure is colored grey, and disordered density that runs along the center of the filament is colored green. Arrows denote the connections between the ends of the modeled Spc110 structure and the disordered density, which suggest that the disordered density is composed of flexible regions of Spc1101–220.

Supplementary Figure 6 Mapping phosphorylation sites on the γTuSC structure.

a) A single γTuSC is shown with residues known to be phosphorylated highlighted as spheres in red, yellow, and orange for sites on γ-tubulin, GCP2, and GCP3 respectively. c) The model of yeast γTuRC with known phosphorylation sites rendered as spheres. Sites freely accessible on the outer surface of the ring are colored purple, sites at the plus end of γ-tubulin predicted to be involved in longitudinal contacts with the microtubule in blue, and sites on the ring interior that would be inaccessible when a microtubule is bound are coral.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 10072 kb)

Capped microtubule minus end

The subtomogram average of capped microtubule ends from spindle pole bodies (MOV 5195 kb)

Comparison of γTuSC ring with capped microtubule end

A single ring of γTuSCCC-Spc1101–220 in the closed state is fit to the subtomogram average of capped microtubule ends from spindle pole bodies (MOV 6890 kb)

Model of γTuRC-microtubule interactions

A single ring of γTuSCCC-Spc1101–220 in the closed state, colored by subunit, is shown followed by the model of longitudinal interactions with a microtubule. (MOV 14930 kb)

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Kollman, J., Greenberg, C., Li, S. et al. Ring closure activates yeast γTuRC for species-specific microtubule nucleation. Nat Struct Mol Biol 22, 132–137 (2015) doi:10.1038/nsmb.2953

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