Figure 5. SPC42 is an allele-specific dosage suppressor of the mps1-8 conditional growth defect. mps1-8 (BD8WX257–5c) (A) and mps1-1 (B) strains (WX241-10c) were transformed with p2 -URA-MPS1, p2 -URA-SPC42, or vector alone. These transfor- mants were grown to OD600 3.0, plated in fivefold serial dilution on URA media, and the plates grown at either permissive (25 C) or restrictive temperatures for mps1-8 (A, 36 C) and mps1-1 (B, 30 C). (A) Increased dosage of SPC42 confers intermediate growth to the mps1-8 strain at a temperature (36 C) normally restrictive for growth. (B) This suppression is not observed for the mps1-1 strain containing p2 -URA-SPC42. As expected, both strains containing p2 -URA-MPS1 grow well at the restrictive temperature but fail to grow at the restrictive temperature when they contain the vector alone. different MPS1-containing clones (multiple times), five unique clones were identified as suppressors of the mps1-8 conditional growth defect. In one of these clones (S81), we identified SPC42 as the ORF responsible for mps1-8 sup- pression (Fig. 5 A). SPC42 mutants exhibit defects in SPB duplication, and Spc42p is a phosphoprotein that localizes to the central plaque of the SPB and duplication intermedi- ates in the satellite and duplication plaque (Donaldson and Kilmartin, 1996; Adams and Kilmartin, 1999). Increased dosage of SPC42 only suppressed the mps1-8 phenotype and not mps1-1 (Fig. 5 B), mps1-737, mps1-412, mps1-1237, or mps1-3796 (unpublished data). This suggests that the mps1-8 defect is distinct from the previously characterized condi- tional MPS1 mutants. Two well-characterized SPC42 ts mutants, spc42-10 and spc42-11 (Donaldson and Kilmartin, 1996), exhibit a mo- nopolar phenotype similar to MPS1 mutants when grown at their restrictive temperatures. We generated the double mu- tant strains, mps1-8 spc42-10 and mps1-8 spc42-11 to look for additional genetic interactions between SPC42 and MPS1 (see Materials and methods). Both double mutant strains show enhanced growth defects when compared with either single mutant strain (unpublished data). The genetic interactions between MPS1 and SPC42 suggest that the gene products may interact in vivo. Mps1p localizes to the SPB and kinetochores The genetic interaction of MPS1 with a bona fide integral SPB component suggested that Mps1p might localize to SPBs, which we initially tested using immunofluorescence microscopy on whole cells. Asynchronously growing cells containing myc epitope-tagged MPS1 at the MPS1 locus Mps1p regulates assembly of Spc42p | Castillo et al. 457 (Mps1p-myc) and SPC42 tagged with green fluorescent pro- tein (GFP) (Spc42-GFP) were analyzed by immunofluores- cense (see Materials and methods). We observe a strong sig- nal of Mps1p-myc (red) that partially overlaps Spc42p-GFP (green), primarily in unbudded cells, and diffuse nuclear staining (Fig. 6 A). Similar localization was observed with a ProA-tagged Mps1 protein (Schutz et al., 1997; Steiner, 1998). This Mps1p signal is suggestive of SPB and/or kinet- ochore localization, since yeast kinetochores are adjacent to the SPB during G1 of the cell cycle (Wigge et al., 1998; Wigge and Kilmartin, 2001). To better characterize Mps1p localization, we determined if Mps1p-myc colocalizes with the kinetochore protein Ndc10p using chromosome spread analysis. A strain con- taining Mps1p-myc and Ndc10p-HA was grown to midlog phase and samples were harvested for chromosome spread analysis. The diffuse nuclear Mps1p-myc signal we observed by whole cell immunofluorescence appears to overlap with signal from Ndc10p-HA (Fig. 6 B). We also observed Mps1p-myc colocalization with Spc42p-GFP using chro- mosome spread analysis (Fig. 6 B). In examining chromo- some spreads where a single SPB was labeled by Spc42-GFP, and the criterion of 90% overlap of Mps1p-myc with Spc42-GFP was considered colocalization, we found that 43% of the spreads (n 54) exhibited colocalization of these proteins. As a control, we did the same analysis in spreads of strains expressing Spc42-GFP and Ndc10p-HA, and we found only 20% of these spreads showed 90% overlap of the two proteins (n 54), indicating that chro- mosome spreads can be used to resolve SPBs and kineto- chores. Using this technique, Mps1p is found at both or- ganelles. Finally, we performed immuno-EM on asynchronously growing strains containing Mps1p-myc. Colloidal gold sig- nal overlapping the SPB in the plane of the nuclear envelope suggests that Mps1p localizes to the Spc42p central plaque region (5 examples in 24 cells examined) (Fig. 6 C). Mps1p signal is also detected at the end of microtubules by im- muno-EM as is seen for other kinetochore proteins (Wigge et al., 1998; Wigge and Kilmartin, 2001) (18 examples in 24 cells examined) (Fig. 6 D). This dual localization is consis- tent with a role for Mps1p in SPB duplication and the spin- dle checkpoint. Mps1p and Spc42p physically interact The genetic interactions between MPS1 and SPC42 and their colocalization at the SPB prompted us to investigate their physical interaction in the cell. Extracts were prepared from cells containing (a) Mps1p-myc, (b) Ndc1p-myc, as a control for nonspecific interaction with the myc epitope, and (c) no tag. These proteins were immunoprecipitated with anti-myc antibody conjugated to agarose beads and re- solved using SDS-PAGE. The presence of Mps1p-myc and Ndc1p-myc was detected using an anti-myc antibody (Fig. 7 B, lanes 5 and 6). Polyclonal anti-Spc42p antibody was used in a duplicate Western analysis and detected a band migrat- ing at the expected molecular weight (46–51 kD) for Spc42p in the Mps1p immunoprecipitate but not the Ndc1p immunoprecipitate (Fig. 7 A, lanes 1 and 2). Indeed, this band migrates at approximately the same position as