ABSTRACT
Microtubule (MT) nucleation is regulated by the γ-tubulin ring complex (γTuRC), conserved from yeast to humans. In Saccharomyces cerevisiae, γTuRC is composed of seven identical γ-tubulin small complex (γTuSC) sub-assemblies which associate helically to template microtubule growth. γTuRC assembly provides a key point of regulation for the MT cytoskeleton. Here we combine cross-linking mass spectrometry (XL-MS), X-ray crystallography and cryo-EM structures of both monomeric and dimeric γTuSCs, and open and closed helical γTuRC assemblies in complex with Spc110p to elucidate the mechanisms of γTuRC assembly. γTuRC assembly is substantially aided by the evolutionarily conserved CM1 motif in Spc110p spanning a pair of adjacent γTuSCs. By providing the highest resolution and most complete views of any γTuSC assembly, our structures allow phosphorylation sites to be mapped, surprisingly suggesting that they are mostly inhibitory. A comparison of our structures with the CM1 binding site in the human γTuRC structure at the interface between GCP2 and GCP6 allows for the interpretation of significant structural changes arising from CM1 helix binding to metazoan γTuRC.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Rev 1. Fixed and updated figure captions. Rev 2. Added X-ray data table S1, fixed S17 caption. Rev3. Revisions in response to reviewer comments. Introductory and Model figure added. Minor modifications to text and figures for clarity and consistency. Minor modifications to atomic models presented in figures. Changed figure numbering to be consistent with elife format. Author affiliations updated.
Abbreviations Used
- 2D
- two-dimensional
- 3D
- three-dimensional
- γTuNA
- γTuRC nucleation activator
- γTuSC
- γ-Tubulin Small Complex
- γTuRC
- γ-Tubulin Ring Complex
- CDK5RAP2
- Cyclin-dependent kinase 5 regulatory subunit associated protein 2
- CK1δ
- Casein kinase 1δ
- CM1
- Centrosomin motif 1
- Cryo-EM
- Electron cryomicroscopy
- DNA
- deoxyribonucleic acid
- DSS
- disuccinimidyl suberate
- DTT
- Dithiothreitol
- EDC
- 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
- EDTA
- Ethylenediaminetetraacetic
- EGTA
- ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- EM
- electron microscopy
- FSC
- Fourier Shell correlation
- GCN4
- General Control Nonderepressible 4
- GCP
- γ-Tubulin complex protein
- grip domain
- γ-Tubulin ring protein domain
- HCl
- Hydrogen chloride
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- Hrr25
- Casein kinase I homolog HRR25
- KCl
- Potassium chloride
- MgCl2
- Magnesium chloride
- MT
- Microtubule
- NaCl
- Sodium Chloride
- NCC
- N-terminal coiled coil
- NiNTA
- Nickel-nitrilotriacetic acid
- PCM
- pericentriolar matrix
- PDB
- Protein Data Bank
- PEG
- Polyethylene glycol
- PSM
- peptide-spectrum match
- RT
- room temperature
- SPB
- Spindle Pole Body
- SPC42p
- Spindle pole body component 42
- Spc72p
- Spindle pole body component 72
- Spc97p
- Spindle pole body component 97
- Spc98p
- Spindle pole body component 98
- Spc110p
- Spindle pole body component 110
- Sulfo-NHS
- N-hydroxysulfosuccinimide
- Tub4p
- Yeast γ-tubulin
- WT
- Wild-type
- XL-MS
- Cross-linking mass spectrometry
- Xrcc4
- X-Ray Repair Cross Complementing 4