UCSF

The microtubule (MT) cytoskeleton is a dynamic, micrometer-scale network of polymeric filaments composed of αβ-tubulin heterodimers. The MT cytoskeleton dramatically reorganizes in response to cell cycle state, developmental transitions, and signaling events. This requires the coordinated activity of a variety of regulatory molecules. These processes ultimately depend on the creation of new MTs, which is regulated by molecules known as MT nucleators. The γ-tubulin ring complex (γTuRC), an approximately 2.1 megadalton protein complex, is one such nucleator that plays important roles in regulating the MT cytoskeleton in organisms ranging from unicellular fungi to humans. γTuRC contains multiple γ-tubulin molecules arranged in a helix which acts as a template from which αβ-tubulin may polymerize. In the budding yeast S. cerevisiae, γTuRC lacks several components present in metazoans which are thought to stabilize γTuRC assembly. Previous work indicates that the subcomplex that comprises γTuRC, γ-tubulin small complex (γTuSC), fail to form helical assemblies in isolation. However, the presence of the coiled-coil protein Spc110, characterized as receptor for γTuSC at the nuclear face of the spindle pole body (SPB), induces formation of helical γTuSC assemblies which are the S. cerevisiae counterpart of metazoan γTuRCs. This work aims to characterize in molecular detail the mechanisms underlying assembly of budding yeast γTuRC. I show through biochemical and live-cell imaging approaches that γTuRC assembly critically depends on the oligomerization state of Spc110, as γTuSCs self-interact weakly and must be stabilized by high-order oligomers of Spc110. By a variety of structural approaches, I show that the 44-residue N-terminal coiled-coil domain forms ordered contacts with γTuSC. The disordered N-terminal domain, which stabilizes γTuRC and is required in vivo, was not observed in previously determined cryo-electron microscopy reconstructions. Cross-linking mass spectrometry and biochemical experiments suggests that one N-terminal domain within an Spc110 dimer interacts with the same γTuSC bound by the dimer’s N-terminal coiled-coil domain, while the other N-terminal domain stabilizes contacts with an adjacent γTuSC. Together, this work provides a framework for understanding the spatial specificity of MT nucleation by coupling γTuRC assembly to its localization via dependence on Spc110.