A Mutational Analysis Identifies Three Functional Regions of the Spindle Pole Component Spc110p inSaccharomyces cerevisiae

Abstract

The central coiled coil of the essential spindle pole component Spc110p spans the distance between the central and inner plaques of the Saccharomyces cerevisiae spindle pole body (SPB). The carboxy terminus of Spc110p, which binds calmodulin, resides at the central plaque, and the amino terminus resides at the inner plaque from which nuclear microtubules originate. To dissect the functions of Spc110p, we created temperature-sensitive mutations in the amino and carboxy termini. Analysis of the temperature-sensitive spc110 mutations and intragenic complementation analysis of the spc110 alleles defined three functional regions of Spc110p. Region I is located at the amino terminus. Region II is located at the carboxy-terminal end of the coiled coil, and region III is the previously defined calmodulin-binding site. Overexpression of SPC98 suppresses the temperature sensitivity conferred by mutations in region I but not the phenotypes conferred by mutations in the other two regions, suggesting that the amino terminus of Spc110p is involved in an interaction with the ␥-tubulin complex composed of Spc97p, Spc98p, and Tub4p. Mutations in region II lead to loss of SPB integrity during mitosis, suggesting that this region is required for the stable attachment of Spc110p to the central plaque. Our results strongly argue that Spc110p links the ␥-tubulin complex to the central plaque of the SPB.

The Centrosome and Its Duplication Cycle

“…2). The nuclear receptor, Spc110 is a member of the pericentrin family of microtubule-nucleating proteins in which microtubule-nucleating motifs are separated from anchors by extended coiled-coil spacers (Kilmartin et al 1993;Kilmartin and Goh 1996;Sundberg and Davis 1997). These g-tubulin docking motifs are highly conserved from human pericentrin and kendrin through Drosophila centrosomin (CNN) to fission yeast Mto1 and Pcp1 (Flory et al 2002;Zhang and Megraw 2007;Fong et al 2008;Samejima et al 2008;Lin et al 2014).…”

“…These g-tubulin docking motifs are highly conserved from human pericentrin and kendrin through Drosophila centrosomin (CNN) to fission yeast Mto1 and Pcp1 (Flory et al 2002;Zhang and Megraw 2007;Fong et al 2008;Samejima et al 2008;Lin et al 2014). Spc29 links Spc110 to the hexagonal crystalline lattice of Spc42 that comprises the central plaque in a coupling that relies on association of Spc110 with calmodulin (Geiser et al 1993;Stirling et al 1994;Donaldson and Kilmartin 1996;Spang et al 1996;Bullit et al 1997;Sundberg and Davis 1997;Elliott et al 1999). On the cytoplasmic side of the central plaque, Spc42 anchors the Cnm67 linker protein that recruits Nud1 to the base of the outer plaque (Adams and Kilmartin 1999;Elliott et al 1999;Schaerer et al 2001).…”

High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the YeastSaccharomyces cerevisiae

“…Indeed, biochemical and genetic studies have shown that the carboxyl terminus of Spc110p along with calmodulin is present at the central plaque (Sundberg et al, 1996) in a complex with Spc42p and a 35-kDa protein (Knopp and Schiebel, 1997). The amino terminus of Spc110p links the central plaque to the inner plaque through an interaction with Spc98p, a component of the yeast ␥-tubulin complex Sundberg and Davis, 1997;Nguyen et al, 1998). Because of the physical interactions of Spc110p with both the central and inner plaques, the absence of the inner plaque in the heparin-stripped cores could have led to a disruption of the organization of proteins in this region.…”

“…A high percentage of these proteins have predicted coiled-coil domains, including Spc42p, which forms a central crystalline core (Donaldson and Kilmartin, 1996;Bullitt et al, 1997), and Spc110p, which acts as a molecular spacer between the central and inner plaques (Kilmartin et al, 1993;Kilmartin and Goh, 1996). The amino terminus of Spc110p in turn interacts with Spc98p (Geissler et al, 1996;Sundberg and Davis, 1997;Nguyen et al, 1998), which along with Spc97p (Knopp and Schiebel, 1997) and Tub4p (Sobel and Snyder, 1995;Marschall et al, 1996;Spang et al, 1996) make up the ␥-tubulin complex at the MT ends at both the inner and outer plaques. Spc72p and Cnm67p localize to the outer plaque of the SPB (Wigge et al, 1998).…”

Ppc89 Links Multiple Proteins, Including the Septation Initiation Network, to the Core of the Fission Yeast Spindle-Pole Body

“…One structural component identified is the coiled-coil protein Pcp1, the homologue of S. cerevisiae Spc110 (Flory et al, 2002). Spc110 binds calmodulin and links the gamma tubulin complex (␥-TuC) to the central plaque of the SPB (Knop and Schiebel, 1997;Sundberg and Davis, 1997). Another structural element is centrin/Cdc31.…”