456 The Journal of Cell Biology | Volume 156, Number 3, 2002 Figure 3. mps1-8 cells arrest in mitosis through activation of the spindle checkpoint. Asynchronously growing mps1-8 (ACY54-9b), mps2-1 (SMY-1b), and mps1-1 (WX241-10c) strains were arrested in G1 using -factor and then released at both permissive (25 C) and restrictive (36 C) temperatures. Samples for flow cytometry were collected at T 0 (G1 arrest), 2, and 3 h after release from the G1 arrest (only the 3-h time point shown for cells at 25 C). (A) The mitotic arrest observed in mps1-8 cells at the restrictive temperature is seen as the accumulation of cells with a G2 DNA content and large budded (LB) cell morphology (61%). At the permissive temper- ature, mps1-8 cells return to cycling asynchronously. (B) For comparison, mps2-1 cells also arrest in mitosis after they fail in SPB duplication at the restrictive temperature. (C) A mps1-1 strain serves as a negative control for a mutant that fails to arrest in mitosis after SPB duplication fails at the restrictive temperature. In these histo- grams, the x-axis is the relative DNA content determined by propidium iodide fluorescence, and the y-axis is the number of cells with the given DNA content (as described in Materials and methods). Peaks corresponding to normal haploid G1 and G2 DNA content are indicated on the x-axis. Each sample represents 5,000 cells. late hyperphosphorylated forms of the spindle checkpoint protein Mad1p, a molecular marker for spindle checkpoint activation (unpublished data; Hardwick et al., 1996). In contrast, the mps1-1 strain fails to arrest in mitosis when grown at the restrictive temperature and instead accumulates cells that appear aploid or aneuploid by flow cytometry (Fig. 3 C). We conclude that mps1-8 is a novel mutant allele lead- ing to defects in SPB duplication but maintaining a func- tional spindle checkpoint. Mps1-8p exhibits wild-type levels of kinase activity We were interested in assessing the level of kinase activity as- sociated with Mps1-8p. To determine this, we compared ki- nase activity in vitro at the mps1-8 permissive (25 C) and re- strictive (36 C) temperatures using protein generated from galactose-inducible glutathione S-transferase (GST) fusion constructs, containing GST alone, GST-MPS1, GST-mps1-1, GST-mps1-KD (kinase dead), and GST-mps1-8 (see Ma- terials and methods). The relative specific activity associated with GST–mps1-8p is similar to that observed for the wild- type protein at either the mps1-8 permissive or restrictive temperature (Fig. 4 A). GST–mps1-1p and GST–mps1-KD were used as controls for MPS1 mutant proteins that have negligible catalytic activity at either assay temperature (Fig. 4 B) (Lauze et al., 1995; Schutz and Winey, 1998). By West- ern analysis, both GST-Mps1p and GST–Mps1-8p migrate as broad bands slower than the predicted molecular weight of 112 kD (Fig. 4 B) (Lauze et al., 1995; Schutz and Winey, 1998). The band shift observed was shown previously for Figure 4. Autophosphorylation by GST-tagged mps1-8p is similar to wild-type Mps1p. Plasmids carrying the GST-tagged MPS1 alleles, mps1-8, mps1-1, mps1-KD (kinase dead), and MPS1 (and GST alone), were transformed into the wild-type W303 strain, and expression of the fusion proteins was induced as described in Materials and methods. The fusion proteins were isolated and used to do kinase assays in vitro at the mps1-8 permissive (25 C) and restrictive (35 C) temperatures (as described in Materials and methods). (A) Proteins were resolved on an SDS-PAGE gel and transferred to nitrocellulose. Autophosphorylation by the GST-tagged proteins and their ability to phosphorylate an exogenous substrate, myelin basic protein (MBP), were quantitated on a phosphorimager (as described in Materials and methods). (B) The amount of GST-tagged protein in each lane was quantitated using a fluorescence- based imaging system (as described in Materials and methods). Relative specific activity (S.A.) of GST–mps1-8p at both 25 C (lane 4, 1.5) and 35 C (lane 5, 1.1) is similar to that observed for GST-Mps1p at either temperature (lane 2, 1.0, and lane 3, 1.2). For comparison, the kinase-dead version of Mps1p (GST-mps1-KDp) has undetectable levels of autophosphorylation at either assay temperature (lanes 6 and 7). As reported previously (Schutz and Winey, 1998), GST–mps1-1p has minimal kinase activity at temperatures permissive (lane 8, 25 C, 0.01) for mps1-1 mutant strain growth and no kinase activity at temperatures restrictive (lane 9, 35 C, 0.03) for growth. Phosphorylation of MBP mirrors the autophosphorylation observed for the GST-tagged proteins in these kinase assays. wild-type protein to be due to autophosphorylation (Fig. 4, A and B). The ability of GST–mps1-8p to phosphorylate an exogenous substrate, myelin basic protein, was also compa- rable to that observed for the wild-type protein (Fig. 4 B). Therefore, our in vitro results suggest that the SPB duplica- tion defect associated with mps1-8 is not due to a loss of ki- nase activity itself, but may reflect inappropriate localization or other regulation of Mps1p. A dosage suppressor of mps1-8 A dosage suppressor screen was performed in a mps1-8 strain to identify genes whose products might interact with Mps1p during SPB duplication. We transformed a mps1-8 strain with a 2- URA-based yeast genomic library and screened for transformants that were able to restore growth at 36 C (see Materials and methods). In addition to isolating two