Ring closure activates yeast γTuRC for species-specific microtubule nucleation

Abstract

The γ-tubulin ring complex (γTuRC) is the primary microtubule nucleator in cells. γTuRC is assembled from repeating γ-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.

Figure 1. The yeast γTuRC is formed from seven γTuSCs and is limited in size by Spc110

a) A slice from a tomogram of isolated spindle pole bodies clearly shows the capped minus ends of microtubules. b) Subtomogram averaged structure of microtubule minus end. Red arrows indicate the position of the half-subunit overlap between the first and seventh γTuSC (outlined in yellow and orange, respectively). The 120 Å longitudinal rise of the γTuSC ring is indicated. c) γTuSC assembles extended filaments when bound to Spc110^1–220. Spc110 binds the outer surface of γTuSC, and fits within the groove of the filament (cartoon). d) Spc110^1–401 promotes assembly of γTuSC rings, but prevents extension beyond a single ring, suggesting that the longer predicted coiled-coil domain interferes with formation of oligomers greater than 7 γTuSC subunits.

Figure 2. Engineered disulfides alter γTuSC filament morphology

a) The lateral interface between β-tubulin subunits in the 13-protofilament microtubule, and the corresponding lateral interactions between γ-tubulins in the open state filament are shown. b) Negative stain electron micrograph of the double mutant S58C G288C (γTuSC^CC) in complex with Spc110^1–220 show two distinct filament morphologies were apparent (blue and orange arrows). c) Power spectra of individual filaments of different morphologies from (b) have different layer line spacing indicating different helical pitch.

Figure 3. In the closed state γTuSC matches microtubule symmetry and has increased nucleation activity

a) The open state γTuSC filament, closed state γTuSC^cc filament, and 13-protofilament microtubule structure. γ-tubulin is highlighted in gold in the γTuSC structures, and the pitch of the 3-start helix in the microtubule is highlighted in cyan. Refined helical pitch and rotation per subunit are indicated. b) Superposition of the open and closed γ-tubulin rings (gold) on the microtubule (cyan). The γ-tubulin indicated by the arrow was superimposed on a β-tubulin from the microtubule. c) Representative fluorescence images of solution microtubule nucleation experiments d) Microtubules were counted for five fields per experiment, and the fold increase over buffer/γTuSC alone controls is plotted for reduced (Red.) and oxidized (Oxid.) states (n=4 independent experiments; error bars represent the s.e.m.). Activity of γTuSCs alone was similar to buffer controls, with only a few microtubules on the entire coverslip (not shown). T-tests confirm significant differences between activity of ɣTuSC110^CC complexes under oxidizing and reducing conditions (p ≤ 0.013), or between mutant and wild-type: (p ≤ 0.055).

Figure 4. Pseudo-atomic model of γTuSC in the closed conformation

a) The pseduo-atomic model of γTuSC (ribbon diagram) fit into the cryo-EM structure (semi-transparent surface). b) Close up views of the interactions of γ-tubulin with the C-terminal domains of GCP2 and GCP3, compared to longitudinal interactions within the α/β-tubulin heterodimer, with the T7 loop highlighted in red. The top view is the view from inside the microtubule, and the bottom view is looking at lateral interaction surfaces. Contacts are made between the H1–S2 loop of γ-tubulin and residues 524–536 of GCP3; the corresponding region of GCP2 is shifted away from γ-tubulin in the GCP2 structure (arrows).

Figure 5. Pseudo-atomic model of γTuRC and its interactions with microtubules

a) The pseudo-atomic model of a complete yeast γTuRC with seven γTuSCs. b) A model of yeast γTuRC interacting with the minus end of a microtubule. c) A potential contact between the last γ-tubulin in the ring, which is not directly interacting with the microtubule, and Spc110 bound to the first γTuSC (arrow) is seen in the γTuSC^CC structure. d) Magnified view of interactions between the first γTuSC and the microtubule. Known phosphorylation sites on γTuSC that could potentially modulate lateral interactions with α/β-tubulin are indicated with red spheres.