Reconstitution and Characterization of Budding Yeast γ-Tubulin Complex

Abstract

Nucleation of microtubules is central to assembly of the mitotic spindle, which is required for each cell division. ␥-Tubulin is a universal component essential for microtubule nucleation from centrosomes. To elucidate the mechanism of microtubule nucleation in budding yeast we reconstituted and characterized the yeast ␥-tubulin complex (Tub4p complex) produced in insect cells. The recombinant complex has the same sedimentation coefficient (11.6 S) as the native complex in yeast cell extracts and contains one molecule of Spc97p, one molecule of Spc98p, and two molecules of Tub4p. The reconstituted Tub4p complex binds preformed microtubules and has a low nucleating activity, allowing us to begin a detailed analysis of conditions that enhance this nucleating activity. We tested whether binding of the recombinant Tub4p complex to the spindle pole body docking protein Spc110p affects its nucleating activity. The solubility of recombinant Spc110p in insect cells is improved by coexpression with yeast calmodulin (Cmd1p). The Spc110p/Cmd1p complex has a small sedimentation coefficient (4.2 S) and a large Stokes radius (14.3 nm), indicative of an elongated structure. The Tub4p complex binds Spc110p/Cmd1p via Spc98p and the K d for binding is 150 nM. The low nucleation activity of the Tub4p complex is not enhanced when it is bound to Spc110p/Cmd1p, suggesting that it requires additional components or modifications to achieve robust activity. Finally, we report the identification of a large 22 S Tub4p complex in yeast extract that contains multimers of Spc97p similar to ␥-tubulin ring complexes found in higher eukaryotic cells.

Microtubule assembly during mitosis – from distinct origins to distinct functions?

“…Interestingly, it has recently been shown that c-TuSCs are able to form a ring structure without the need of the other GCPs (Kollman et al, 2010). It is therefore tempting to speculate that the c-TuSCs could be sufficient to promote MT nucleation in metazoans, as it is the case in budding yeast (Kollman et al, 2011;Vinh et al, 2002). Taken together, these data suggest the possibility that different c-tubulin nucleation complexes might exist in the eukaryotic cell.…”

The where, when and how of microtubule nucleation – one ring to rule them all

“…However, sucrose gradient fractionation under more physiological buffer conditions revealed mostly small, cTuSCsized complexes (,8-9S) (Anders et al, 2006;Fujita et al, 2002;Venkatram et al, 2004). Saccharomyces cerevisiae: only cTuSC (,12S) (Vinh et al, 2002).…”

Identification and Characterization of Two Novel Proteins Affecting Fission Yeast γ-tubulin Complex Function

“…This suggests that the SPB docking of Gfh1p is independent of the presence of core ␥-TuC components. mbo1 ؉ Is Similar to pcp1 ؉ and SPC110 and Is Dispensable for Vegetative Growth Searches of the S. pombe and S. cerevisiae genome databases with Mbo1p, the protein encoded by the ORF SPCC417.07c, revealed that it shares significant sequence similarity to two known SPB components: S. pombe Pcp1p, a protein homologous to S. cerevisiae Spc110p (Flory et al, 2002), and S. cerevisiae Spc110p, which interacts with and anchors the ␥-TuC to the nuclear face of the S. cerevisiae SPB (Knop and Schiebel, 1998;Vinh et al, 2002). Like the aforementioned proteins, Mbo1p contains predicted coiled-coil regions and a region at its extreme C-terminus related to the C-termini of Pcp1p and Spc110p ( Figure 4A) that includes their calmodulin-binding sites.…”

“…The cytosolic ␥-tubulin small complex (␥-TuSC), contains just ␥-tubulin and two other proteins belonging to the Spc97/98p (hGCP2/3) family and has no microtubule nucleating capacity on its own (Oegema et al, 1999;Zhang et al, 2000). The ␥-TuSC is similar in size and composition to the Tub4p complex in Saccharomyces cerevisiae (Geissler et al, 1996;Vinh et al, 2002).…”