The structure of the gamma-tubulin small complex: implications of its architecture and flexibility for microtubule nucleation

Abstract

The gamma-tubulin small complex (gamma-TuSC) is an evolutionarily conserved heterotetramer essential for microtubule nucleation. We have determined the structure of the Saccharomyces cerevisiae gamma-TuSC at 25-A resolution by electron microscopy. gamma-TuSC is Y-shaped, with an elongated body connected to two arms. Gold labeling showed that the two gamma-tubulins are located in lobes at the ends of the arms, and the relative orientations of the other gamma-TuSC components were determined by in vivo FRET. The structures of different subpopulations of gamma-TuSC indicate flexibility in the connection between a mobile arm and the rest of the complex, resulting in variation of the relative positions and orientations of the gamma-tubulins. In all of the structures, the gamma-tubulins are distinctly separated, a configuration incompatible with the microtubule lattice. The separation of the gamma-tubulins in isolated gamma-TuSC likely plays a role in suppressing its intrinsic microtubule-nucleating activity, which is relatively weak until the gamma-TuSC is incorporated into higher order complexes or localized to microtubule-organizing centers. We propose that further movement of the mobile arm is required to bring the gamma-tubulins together in microtubule-like interactions, and provide a template for microtubule growth.

Figure 1.

Negative stain images of γ-TuSC. (A) Section of an untilted micrograph. Y-shaped views (filled arrowheads) predominate, but most images also included i-shaped side views (empty arrowheads). Only Y-shaped views were selected for reconstruction. Bar, 50 nm. (B) Individual untilted particles from the reconstruction data set. (C) Averages of aligned and classified particles, with the indicated number of particles in each class average. Bar, 10 nm (B and C).

Figure 2.

FSC resolution measurements of the RCT reconstructions in Figure 3. The resolution of the reconstructions is taken as the spatial frequency at which the FSC drops below 0.5.

Figure 3.

Three-dimensional reconstructions of γ-TuSC. Volumes were generated with tilted particles corresponding to classes 1, 2, and 3 in Figure 1C. Four orthogonal views of each particle are shown, and the rotations between the views are indicated at left. The aligned structures are superimposed at right, with the constant region used for the alignment rendered semitransparent. The arm on the left in the top view adopts different orientations in the three structures, and it is rotated relative to the arm in class 3 by 8° in class 2 and by 15° in class 1.

Figure 4.

Average γ-TuSC map. Each of the reconstructed volumes was segmented into the constant region and the mobile arm, as indicated in Figure 3. An average was calculated for each segment, weighted for class size. In this view, the two average maps are aligned to the corresponding segments of structure 3. The dotted white lines indicate the boundaries used for calculating the approximate volumes of the three segments of the map—115 kDa for each arm and 70 kDa for the body.

Figure 5.

Labeling of the Tub4p N-terminus. (A) Individual particles of γ-TuSC with an N-terminal 6xHis-tag on Tub4p incubated with Ni²⁺-Nanogold. (B) Average of 68 labeled particles. (C) Average of 68 unlabeled particles. (D) Difference map generated by subtracting the labeled average from the unlabeled average. The prominent peak corresponding to the location of the gold label is 9.3σ above the mean. Bar, 10 nm.

Figure 6.

Docking γ-tubulin in the average γ-TuSC structure. (A) The crystal structure of γ-tubulin (gold), the human Tub4p homologue, was manually fit into the lobes at the ends of the arms. (B) Side views, rotated 90° relative to A in the directions indicated by the arrows.

Figure 7.

FRET relationships and the overall architecture of γ-TuSC. (A) The average FRET_R values for the CFP- and YFP-tagged proteins presented in Table 1. The lines connect the protein pairs that showed significant FRET, and they are not meant to correspond to distances. The dotted lines indicate that the 1.1 FRET_R value is just at the limit of detection, and it is considered tentative. (B) The arrangement of Tub4p, Spc97p, and Spc98p within γ-TuSC. The N-terminal regions of Spc97p and Spc98p constitute a dimerization interface in the body, whereas C-terminal regions form the bases of the arms and bind Tub4p.

Figure 8.

Model of the conformational changes required to bring Tub4p into a nucleation-competent orientation consistent with the interprotofilament spacing of the microtubule. (A) Two tubulin dimers (pink, α-tubulin; green, β-tubulin) making lateral contacts, and the average γ-TuSC map with the smallest observed distance between the arms are shown. The Tub4p density on the right is aligned at the base of one protofilament, but the separation of the two arms precludes the possibility of both Tub4p densities making protofilament-like contacts simultaneously. (B) A rotation of 14° about the base of the mobile arm (inset) brings the two Tub4p densities into orientations compatible with the microtubule lattice. As a result of this modeled conformation, two γ-tubulin crystal structures making microtubule-like lateral contacts fit remarkably well into the density. The crystal structures were manually fit in the density in B, and this fit was extrapolated in A.

Figure 9.

Model for the oligomerization of γ-TuSC. (A) Multiple γ-TuSCs with γ- and α/β-tubulin docked as in Figure 7B were aligned with the minus end of a microtubule to indicate a possible arrangement of a higher order, γ-TuRC-like structure that would be competent for microtubule nucleation. (B) The γ-TuSC assembly from A shown making longitudinal contacts with the minus end of a microtubule.