Monopolin Subunit Csm1 Associates with MIND Complex to Establish Monopolar Attachment of Sister Kinetochores at Meiosis I
Figure 2
Csm1 interacts with the N-terminal domain of Dsn1.
A) Multiple alignment of the amino acid sequences of the N-terminus of Dsn1 from several members of Saccharomycetes class of yeasts (Sc- Saccharomyces cerevisiae; Zr-Zygosaccharomyces rouxii; Vp-Vanderwaltozyma polyspora; Nc- Naumovozyma Castelli; Tp- Tetrapisispora phaffia; Nd- Naumovozyma dairenensi; Ec- Eremothecium cymbala; Td- Torulaspora delbrueckii; Ag- Ashbya gossypii; Lt- Lachancea thermotolerans; Ka- Kazachatania africana). Three conserved elements are highlighted (Boxes 1–3). Note that the N-terminal domain is absent in Dsn1 homologs of S. pombe and humans. B) Dsn1 interacts with Csm1 in the 2-hybrid assay (Top panel). Transformants of AH109 strain containing pGBKT7-Dsn1 + pGAD-Csm1 or pGBKT7 + pGAD-Csm1 or pGBKT7-Dsn1 + pGAD or pGBKT7 + pGAD were spotted at different dilutions on non-selective SD-LEU-TRP plates and selective SD-LEU-TRP-ADE-HIS plates. Growth was monitored after incubation at 30°C for 2–3 days. Boxes 2 and 3 but not Box 1 in Dsn1 is required for interaction with Csm1 (Bottom panel). Transformants of AH109 strain containing pGBKT7-Dsn1 + pGAD-Csm1 or pGBKT7-Dsn1 (1-220) + pGAD-Csm1 or pGBKT7-Dsn1 (1-220B1A –Box1 alanine substituted) + pGAD-Csm1 or pGBKT7-Dsn1 (1-220B2A) + pGAD-Csm1 or pGBKT7-Dsn1 (1-220B3A) + pGAD-Csm1 were analysed by spot assay as described above. C) Csm1 interacts with N-terminal domain of Dsn1 in vitro. Csm1-MBP or MBP was incubated with extracts of yeast cells expressing Dsn1-pk6 or Dsn1-Δ110-pk6, respectively. The proteins bound to the Csm1-MBP beads and MBP beads were subjected to SDS-PAGE followed by either Western analysis using anti-PK antibodies or Coomassie staining. D) Dsn1 interacts with the C-terminal domain of Csm1 in vitro. A binding assay was performed by incubating different variants of Csm1 coupled to beads with yeast extracts from cells expressing Dsn1-pk6 and the samples were analysed as described in 2C. FL denotes the full length version of Csm1 (1–190). Data shown (panels B–D) are of single representative experiment of at least three independent repeats.