Abstract
Budding yeast Spc110, a member of γ-tubulin complex receptor family (γ-TuCR), recruits γ-tubulin complexes to microtubule (MT) organizing centers (MTOCs). Biochemical studies suggest that Spc110 facilitates higher-order γ-tubulin complex assembly (Kollman et al., 2010). Nevertheless the molecular basis for this activity and the regulation are unclear. Here we show that Spc110 phosphorylated by Mps1 and Cdk1 activates γ-TuSC oligomerization and MT nucleation in a cell cycle dependent manner. Interaction between the N-terminus of the γ-TuSC subunit Spc98 and Spc110 is important for this activity. Besides the conserved CM1 motif in γ-TuCRs (Sawin et al., 2004), a second motif that we named Spc110/Pcp1 motif (SPM) is also important for MT nucleation. The activating Mps1 and Cdk1 sites lie between SPM and CM1 motifs. Most organisms have both SPM-CM1 (Spc110/Pcp1/PCNT) and CM1-only (Spc72/Mto1/Cnn/CDK5RAP2/myomegalin) types of γ-TuCRs. The two types of γ-TuCRs contain distinct but conserved C-terminal MTOC targeting domains.DOI: http://dx.doi.org/10.7554/eLife.02208.001.
Highlights
The budding yeast spindle consists of ∼40 microtubules (MTs) that extend between the two opposed spindle pole bodies (SPBs)
Addition of Spc1101–220-P shifted γ-tubulin small complex (γ-TuSC) into fractions that eluted earlier than when γ-TuSC was run on the columns on its own (Figure 1B, shift from fraction 10 to fraction 8)
This result suggests that Mps1 and Cdk1 kinases regulate the interaction between Spc110 and γ-TuSC through phosphorylation of Spc110 N-terminal domain
Summary
The budding yeast spindle consists of ∼40 microtubules (MTs) that extend between the two opposed spindle pole bodies (SPBs). In budding yeast Saccharomyces cerevisiae, two molecules of γ-tubulin (Tub4) assemble together with one molecule of Spc (ortholog of human GCP2) and Spc (ortholog of human GCP3) into a tetrameric γ-tubulin small complex (γ-TuSC), which is conserved in all eukaryotes (Geissler et al, 1996; Knop et al, 1997; Guillet et al, 2011). The in vitro nucleation activity of the γ-TuSC assemblies remained poor, presumably because of the suboptimal spacing of every second Tub within the γ-TuSC ring that blocks interaction with tubulin in the MT cylinder (Kollman et al, 2008, 2010; Choy et al, 2009). The concept of γ-TuSC oligomerization is further supported by in vivo measurements of budding yeast γ-tubulin complex components on detached single MT nucleation sites. The γ-tubulin:Spc97:Spc ratio was 2.4:1.0:1.3 with a total of ∼17 γ-tubulin molecules per nucleation site (Erlemann et al, 2012), suggesting a slight excess of γ-tubulin and Spc molecules over Spc in the assembled MT nucleation site
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