bly assay was performed in MPS1 and mps1-1 strains at per- missive (25 C) and restrictive temperatures (30 and 34 C) for mps1-1. These strains also contained a myc epitope- tagged version of SPC42 under the control of a galactose- inducible promoter (GAL-SPC42-myc). Proper formation of the “super plaque” was first analyzed using immunofluorescence. The MPS1 GAL-SPC42-myc and mps1-1 GAL-SPC42-myc strains were grown in nonin- ducing media (no galactose), arrested in G1 using -factor, and then released into galactose-containing media at both 25 and 30 C for 3 h to induce expression of GAL-SPC42- myc. At the permissive temperature, the mps1-1 (Fig. 9 A) and MPS1 (unpublished data) strains form Spc42p-myc– dependent dome-like structures (super plaque in green) that are associated with DNA (blue) and microtubule (red) sig- nal. The “super plaque” formed in the wild-type strain at 30 C is identical to what we observe for this strain at 25 C (Fig. 9 C). By contrast, in the mps1-1 strain at 30 C we ob- serve a decrease in Spc42p-myc signal at the SPB, and it has a much less organized appearance (Fig. 9 E). To characterize these structures at higher resolution, we analyzed them using EM (see Materials and methods). Overexpression of SPC42-myc in the wild-type and mps1-1 strain at the permissive temperature results in the forma- tion of the organized dome-shaped “super plaque.” These structures extend laterally from the central plaque of the SPB but appear to be constrained vertically to 23 nm as reported previously for the “super plaque” (Fig. 9, B and D) (Bullitt et al., 1997). In contrast, it is clear that Spc42p-myc signal in the mps1-1 strain at the restrictive temperature is an unorganized mass of electron-dense material associated with the half-bridge of the unduplicated SPB (Fig. 9 F). Based on the EM section width (80 nm), we estimate that the struc- ture shown is 300 nm 480 nm 250 nm (Fig. 9 F). This structure is similar to one formed when spc42-S:A, a mutant allele in which 34 serines have been mutated to ala- nines, is overexpressed at similar levels (Adams and Kilmar- tin, 1999). This data suggests that Mps1p function, via phosphorylation of Spc42p, is required for the proper as- sembly of the central layer of the new SPB during SPB du- plication. Discussion We have identified a physical interaction between the inte- gral SPB component Spc42p and Mps1p, a regulator of Spc42p assembly during SPB duplication. We show that Mps1p is capable of phosphorylating Spc42p in vitro and its phosphorylation is dependent on Mps1p in vivo. Addition- ally, in vivo assembly of Spc42p is compromised in a MPS1 mutant (mps1-1). Mps1-1p has essentially no kinase activity in vitro; therefore, we believe that phosphorylation of Spc42p by Mps1p is important for the assembly of Spc42p into the SPB. We investigated interactions between Mps1p and Spc42p based on our isolation of SPC42 as a dosage suppressor of a unique MPS1 mutant, mps1-8. This condi- tional mutant was identified in a screen designed to separate the role of MPS1 in SPB duplication from its role in the spindle checkpoint. Using immunofluorescence and EM, we show that mps1-8 mutants fail to duplicate their SPBs at re- Mps1p regulates assembly of Spc42p | Castillo et al. 461 strictive temperatures. Unlike previously characterized con- ditional MPS1 mutants, mps1-8 cells are competent to arrest in mitosis through activation of the spindle checkpoint after SPB duplication fails (Schutz and Winey, 1998). Therefore, mps1-8 is an important genetic tool for our further investiga- tion of the role of Mps1p in SPB duplication. mps1-8 is a unique allele Mutations in the noncatalytic region of MPS1 generated a conditional mutant, mps1-8, that is specifically defective in SPB duplication. We targeted the noncatalytic region of MPS1 for mutagenesis, believing that this region might con- fer spatial or temporal regulation of Mps1p for each of its roles. In fact, our screen also identified a MPS1 allele that appears to be specifically defective in the spindle checkpoint (to be described elsewhere). In this paper, we show that the molecular defect associated with mps1-8 is likely distinct from our previously characterized conditional MPS1 mu- tants. Mutations are in the noncatalytic region of mps1-8 and do not affect kinase activity of the mutant protein. By contrast, other conditional MPS1 mutants previously char- acterized in our lab have mutations in the catalytic region of the gene that affect kinase activity measured in vitro (Schutz and Winey, 1998). Consistent with the notion that the mps1-8 defect is dis- tinct from other conditional MPS1 mutants, mps1-8 shows unique genetic interactions. We show that mps1-8 but not the other conditional MPS1 mutants is suppressed by an in- creased dosage of the Mps1p in vitro substrate, Spc42p. By contrast, several of the MPS1 kinase mutants but not mps1-8 are suppressed by an increased dosage of the molecular chap- erone CDC37 (Schutz et al., 1997). Although it is likely that CDC37 suppresses through stabilization of the jeopardized Mps1p kinase, it is less clear how SPC42 suppression occurs. We propose that suppression by SPC42 may be through sta- bilizing an interaction between Spc42p and Mps1-8p. Alter- natively, extra Spc42p may help localize Mps1-8p to the SPB or another site of action required for SPB duplication. Future localization studies and coimmunoprecipitation ex- periments using Mps1-8p should address these questions. Multiple requirements for Mps1p in SPB duplication This and previous analyses suggest that Mps1p is required for multiple steps in SPB duplication (Winey et al., 1991; Schutz and Winey, 1998). Here, we show that mps1-8 mu- tant cells fail very early in SPB duplication when grown at their restrictive temperature. By EM analysis, the undupli- cated SPB in mps1-8 cells is similar to those observed in cdc31 and kar1 mutants (Baum et al., 1986; Rose et al., 1986). The unduplicated SPB of mps1-8 does not have an extended half-bridge that is characteristic of several other MPS1 mutants (Winey et al., 1991; Schutz and Winey, 1998). This suggests that there are two distinct requirements for Mps1p early in SPB duplication. Alternatively, the func- tions defined by mps1-8 and the other conditional MPS1 mutants are not mutually exclusive; proper spatial regulation of Mps1p and Mps1p kinase activity may both be required for its early SPB function. Presumably, the MPS1 kinase do- main mutants proceed further in SPB duplication because the mutant proteins localize properly or make appropriate