Molecular architecture of the yeast monopolin complex

Abstract

The Saccharomyces cerevisiae monopolin complex directs proper chromosome segregation in meiosis I by mediating co-orientation of sister kinetochores on the meiosis I spindle. The monopolin subunits Csm1 and Lrs4 form a V-shaped complex that may directly crosslink sister kinetochores. We report here biochemical characterization of the monopolin complex subunits Mam1 and Hrr25 and of the complete four-protein monopolin complex. By purifying monopolin subcomplexes with different subunit combinations, we have determined the stoichiometry and overall architecture of the full monopolin complex. We have determined the crystal structure of Csm1 bound to a Mam1 fragment, showing how Mam1 wraps around the Csm1 dimer and alters the stoichiometry of kinetochore-protein binding by Csm1. We further show that the kinase activity of Hrr25 is altered by Mam1 binding, and we identify Hrr25 phosphorylation sites on Mam1 that may affect monopolin complex stability and/or kinetochore binding in meiosis.

Figure 1. Monopolin Complex Interactions

(A) Subunit interactions. Lrs4 is shown in green (Csm1-interacting region) and white (C-terminal uncharacterized region), Csm1 is shown in yellow (coiled coil) and blue (globular domain), Mam conserved regions are shown in magenta, and Hrr25 is shown in light blue (kinase domain), cyan (conserved extension in point-centromere fungi), and white (P/Q-rich domain). Interacting regions (from Rabitsch et al., 2003; Petronczki et al., 2006; Corbett et al., 2010) and the present work) are indicated by dashed lines; see Figures S2A–S2D for details. (B) Pulldown showing interactions between wild-type (WT), kinase-dead (K38R), and H25R/E34K mutant Hrr25^1–394 and Mam1 (CIP, calf intestinal phosphatase). (C) Hrr25^1–394 autophosphorylation. His₆-Hrr^251–394 wild-type (WT) or kinase-dead (K38R) was expressed in E. coli, alone or with Mam1^87–191, and purified by nickel-affinity pulldown. In agreement with mass spectrometry (see Figure S1A), Hrr25 WT appears less phosphorylated when coexpressed with Mam1. See also Figures S1 and S2 and Tables S1 and S2.

Figure 2. Structure of the Csm1:Mam1 Complex

(A) Overall structure of the 2 Csm1: 1 Mam1 complex, with Csm1 colored yellow and blue, and Mam1 indicated in purple. A symmetry-related copy of Csm1 that the Mam1 C-terminal region contacts is in gray. The N- and C-terminal residues of the ordered segment of Mam1 (residues 223 and 263) are labeled. (B) A 180° rotated view of the Mam1 C-terminal segment conformation as crystallized (left) and translocated based on crystallographic and Csm1-dimer symmetry onto Csm1 chain B to generate a fully wrapped model (right). See also Figure S3 and Table S3.

Figure 3. Mam1 Affects Csm1-Kinetochore Interactions

(A) Four views of the Mam1 C-terminal segment (yellow sticks) binding Csm1 (colored cyan-to-purple, based on conservation within point-centromere fungi). Close-up panel: The interaction with Csm1’s kinetochore protein-binding patch (including residues Y156 and L161; Corbett et al., 2010) is anchored by Mam1 Phe262. (B) Pulldown of in vitro-expressed ⁽³⁵⁾S-methione-labeled Dsn1 or Mif2 with Csm1 and the Csm1-Mam1 complex. (C) Pulldown of Dsn1 using equimolar amounts of Csm1 (left four lanes: 10, 5, 2, and 1 μg Csm1) and Csm1:Mam1^221–290 (right four lanes: matched molar amounts of Csm1:Mam1^221–290), showing that Csm1:Mam1^221–290 binds roughly half as much Dsn1 as does Csm1 alone.

Figure 4. Model of the Intact Monopolin Complex, with Proteins Colored as in Figure 1

Hrr25 is represented by the structure of S. pombe casein kinase I (Xu et al., 1995), with residues H25 and E34 shown in yellow and bound ATP shown as sticks. The central domain of Mam1 (magenta outline) is shown bound to the N-terminal lobe of Hrr25 (Petronczki et al., 2006), with the linker region (residues 192–220) shown as a dotted magenta line. The copy number of each protein in the complex is indicated in parentheses, and the two available Dsn1-binding sites are indicated by arrows.