Background Duplicated chromosomes are equally segregated to daughter cells by a

Background Duplicated chromosomes are equally segregated to daughter cells by a bipolar mitotic spindle during cell division. kinetochores, Dam1-765 kinetochores do not colocalize with an equal number of plus ends. Despite the uncoupling of the kinetochores from the plus ends of MTs, the cells are viable, complete the cell cycle with the same kinetics as wild-type cells, and biorient their 28166-41-8 manufacture chromosomes as efficiently as wild-type cells. Conclusions We determine that phosphorylation of Dam1 residues S218 and S221 by Mps1 is usually required for efficient coupling of kinetochores to MT plus ends. We find that efficient plus-end coupling is usually not required for (1) maintenance of chromosome biorientation, (2) maintenance of tension between sister kinetochores, or (3) chromosome segregation. Introduction At the start of mitosis in vertebrate cells, sister kinetochores are initially captured on the sides of kinetochore microtubules (kMTs) and transported poleward along the lateral surface of the kMTs [1C3]. By metaphase, the lateral attachments have been replaced by end-on attachments, and each pair of sister kinetochores is usually bioriented on the plus ends of kMTs from opposite poles [4]. Recently, Tanaka and coworkers suggested that the same progression from lateral attachments to end-on attachments occurs during assembly of the metaphase spindle in yeast [5]. An unattached chromosome can be captured and transported along the side of a microtubule (MT) to the spindle pole body (SPB). The sister kinetochores subsequently become attached to the plus ends of kMTs from opposite SPBs by a process that has not been defined. Once kinetochores are associated with the plus end, their movements are tightly coupled to microtubule plus-end assembly and disassembly. As a result, sister kinetochores are under sufficient tension to be separated [6C10]. How the kinetochores maintain attachment to the dynamic MT plus ends is usually not known. The yeast kinetochore is usually made up of at least 65 protein arranged in six multimeric complexes [11]. The Dam1 complex is usually part of the outer kinetochore, proximal to the microtubule tip. In vitro, the Dam1 complex forms rings that hole and stabilize microtubules [12, 13]. Dam1 is usually phosphorylated by the Ipl1 Aurora kinase, but the role of the phosphorylation is usually unclear [14C16]. Previous genetic evidence also suggests a link between Dam1 and the Mps1 kinase, which regulates SPB duplication and the spindle checkpoint [17]. Inhibition of Mps1 causes multiple kinetochore defects [18]. Here we provide evidence that phosphorylation of the kinetochore component Dam1 by Mps1 kinase is usually required for efficient association of the kinetochore 28166-41-8 manufacture with the plus end of a microtubule but, amazingly, is usually not required for biorientation or chromosome segregation. Results Synthetic Lethal Screen to Identify Mutations that Alter Metaphase Spindle Architecture The temperature-sensitive allele weakens SPBs such that at the restrictive heat, the tension established during biorientation of sister chromatids causes SPBs to delaminate [19]. We reasoned that mutations that alter metaphase spindle architecture, and thereby increase stress on the SPB, would delaminate the weakened SPB even under permissive conditions and therefore be lethal or toxic to cells. We performed a synthetic lethal screen with as described in the Experimental Procedures and identified the allele is usually dominating because a diploid cell with the genotype is usually not 28166-41-8 manufacture viable at 30C unless provided with a wild-type copy of on a plasmid. contains a single mutation C662T, producing in the corresponding change in Dam1 of S221F. S221 Is usually Phosphorylated In Vitro by Mps1, Not Ipl1 Dam1 is usually a substrate for the Ipl1 kinase, and displays genetic interactions with [14, 17, 20]. To test whether S221 is usually a site of CD14 phosphorylation, we phosphorylated recombinant glutathione S-transferase (GST)-Dam1 with either recombinant GST-Ipl1 or GST-Mps1 purified from yeast and identified the sites of phosphorylation by mass spectrometric analysis. In agreement with published results [14], Ipl1 phosphorylated Dam1 residues S20, S265, and S292 (Table H1 in the Supplemental Data available with this article online). We did not detect phosphorylation by Ipl1 on S221, and S221 does not fall in an Ipl1 consensus site. In contrast, Mps1 phosphorylated S221 and five other residues (S13, S49, S217, S218, and S232) (Table H1). When Dam1-765 was phosphorylated by Ipl1 or Mps1 and then analyzed by mass spectrometric analysis, the 28166-41-8 manufacture phosphorylation pattern was.