454 The Journal of Cell Biology | Volume 156, Number 3, 2002 called the duplication plaque (Adams and Kilmartin, 1999; O’Toole et al., 1999). Immuno-EM analysis of the satellite and duplication plaque show both structures are composed of the core SPB components, Spc29p, Spc94p/Nud1p, Spc42p, and Cnm67p (Adams and Kilmartin, 1999). Assembly of the new SPB is completed when the duplication plaque is in- serted into the nuclear envelope and associates with addi- tional SPB components that will make up the inner (nuclear) plaque layers (Adams and Kilmartin, 1999). The terminal phenotype of various SPB duplication mu- tants has suggested when the gene products might be re- quired in the process (Byers and Goetsch, 1975; Rose and Fink, 1987; Winey et al., 1991; Schutz et al., 1997; Schutz and Winey, 1998). For example, yeast containing a mutant SPC42 gene fail in SPB duplication after satellite formation (Donaldson and Kilmartin, 1996). The SPC42 gene prod- uct forms the electron-dense (two-dimensional crystalline) central layer of the SPB, and Spc42p is found in the duplica- tion intermediates, the satellite, and duplication plaque (Donaldson and Kilmartin, 1996; Bullitt et al., 1997; Ad- ams and Kilmartin, 1999; O’Toole et al., 1999). Unlike mu- tant alleles of SPC42, different mutant alleles of MPS1, which encodes a dual specificity protein kinase required for SPB duplication, fail at two distinct points in SPB duplica- tion (Winey et al., 1991; Lauze et al., 1995; Schutz and Winey, 1998). This suggests that Mps1p is required for multiple events in SPB duplication. Mps1p is unusual in that it has a role in the spindle check- point and in SPB duplication (Hardwick and Murray, 1995; Weiss and Winey, 1996). Kinetochores that are not attached to microtubules activate the spindle checkpoint (Wang and Burke, 1995; Pangilinan and Spencer, 1996). Failed SPB du- plication also triggers this checkpoint, possibly because a mo- nopolar spindle does not nucleate a sufficient number of mi- crotubules to capture all of the kinetochores and cannot produce tension via bipolar spindle attachment (Winey and O’Toole, 2001). The conditional MPS1 mutants isolated thus far are defective in both pathways; therefore, under restrictive conditions MPS1 mutant cells proceed through mitosis with a monopolar spindle, aberrantly segregate their DNA, and rap- idly lose viability (Winey et al., 1991; Schutz and Winey, 1998). Although the role of Mps1p in this checkpoint is not yet clearly defined, it requires kinase activity and is probably accomplished through phosphorylation of another checkpoint component, Mad1p (Hardwick et al., 1996). Previously, we used a mps1-1 strain in genetic screens to identify interactions that would enhance our understanding of the role of Mps1p in SPB duplication and the spindle checkpoint (Schutz et al., 1997; Jones et al., 1999). We identified genes involved in the spindle checkpoint, spindle function, and those involved in stabilizing the Mps1p kinase (Hofmann et al., 1998; Jones et al., 1999). However, we did not identify SPB components. Here, we characterize a novel conditional allele, mps1-8, that is specifically defective in SPB duplication and use this allele in a dosage suppressor screen. We identified SPC42, a gene that encodes an integral SPB component, as a dosage suppressor of the mps1-8 con- ditional growth defect. We use genetic and biochemical techniques to investigate the interaction between Mps1p and Spc42p, taking advantage of an Spc42p in vivo assem- Figure 1. A new MPS1 allele, mps1-8, contains mutations in the noncatalytic region that result in conditional growth at 36 C. (A) A wild-type (WX257-14c) and mps1-8 (ACY54-9b) strain was grown to saturation at 25 C and plated in fivefold serial dilution on rich media. These plates were incubated at 25 or 36 C for 4 d. The mps1-8 strains fail to grow at the restrictive temperature of 36 C. (B) Sequencing of the noncatalytic region (amino acids 1-450) of mps1-8 revealed 10 point mutations that resulted in amino acid changes (K13E, R74G, D143V, I158M, N235I, I244V, E254G, R319G, V415A, N429D). bly assay (Donaldson and Kilmartin, 1996) to show that Mps1p is required for Spc42p assembly. Results A novel mps1 temperature-sensitive for growth allele We have investigated further the essential role of MPS1 by generating and characterizing a novel conditional MPS1 mutation, defective only in SPB duplication. This MPS1 al- lele was isolated from a library of mutagenized MPS1 clones prepared by amplifying the noncatalytic (NH2-terminal) re- gion of MPS1 using error-prone PCR. We targeted this re- gion of MPS1 for mutagenesis because (a) existing condi- tional MPS1 alleles have mutations in the catalytic domain and are defective in both Mps1p functions and (b) deletion analysis showed the NH2 terminus is required for function (unpublished data; Schutz and Winey, 1998). We used a strain that allowed us to screen simultaneously for different MPS1-associated phenotypes (mps1 ::KanMX cin8 ::HIS3, supported by counter-selectable plasmids) (see Materials and methods). Counter selection of the plasmid containing CIN8 allowed us to identify MPS1 alleles that might be de- fective in the spindle checkpoint function of MPS1 (see Ma-