462 The Journal of Cell Biology | Volume 156, Number 3, 2002 physical interactions, though they are unable to function; the extended half-bridge might be a result of failed attempts to initiate SPB duplication. MPS1 also functions late in SPB duplication. mps1-737 mutants assemble the central and outer layers of the new SPB but like ndc1, mps2, and bbp1 mutants fail to insert this structure into the nuclear envelope (Winey et al., 1991, 1993; Schramm et al., 2000). Whereas these proteins may function to insert the duplication plaque into the nuclear envelope by providing an opening in the nuclear envelope, it is unclear how Mps1p is involved in this process (Chial et al., 1998; Munoz-Centeno et al., 1999; Schramm et al., 2000). One possibility is that Mps1p facilitates insertion of the duplication plaque into the nuclear envelope by control- ling assembly of the inner plaque, a structure not formed in the mps1-737 mutant. Two of the six proteins that make up the inner plaque, Spc110p and Spc98p, are substrates for Mps1p in vitro, and their phosphorylation is dependent on Mps1p in vivo (Pereira et al., 1998; Friedman et al., 2001). The mps1-737 mutant phenotype might reflect a disrupted interaction with Spc110p and Spc98p or the inability of Mps1-737p to phosphorylate Spc110p and Spc98p. The multiple MPS1 mutant phenotypes are consistent with Mps1p, playing a regulatory role in SPB duplication. Mps1p is required for Spc42p assembly We investigated the role of Mps1p as a regulator of SPB as- sembly using a Spc42p in vivo assembly assay. Overexpres- sion of Spc42p at very high levels causes the central plaque of the SPB (normal site of Spc42p localization) to extend laterally and form a structure we call the “super plaque.” We believe that Mps1p might function in assembly of the cen- tral plaque during SPB duplication based on MPS1 mutant phenotypes and because Mps1p physically interacts with and colocalizes with Spc42p in vivo and regulates Spc42p phosphorylation. When Spc42p was overexpressed in a mps1-1 mutant at restrictive temperatures, we observed a de- fect in assembly of the “super plaque.” This structure is not vertically confined to the two-dimensional crystalline lattice typically seen in MPS1 cells overexpressing SPC42 (Donald- son and Kilmartin, 1996; Adams and Kilmartin, 1999). A similar structure is detected when spc42-S:A, a mutant allele in which 34 serines have been mutated to alanines, is overex- pressed at similar levels (Adams and Kilmartin, 1999). Since spc42-S:A is functional at endogenous levels, we believe this common overexpression phenotype reveals the importance of phosphorylation for proper plaque assembly that appar- ently results in different phenotypes when some phosphory- lation is lost (the spc42-S:A allele with threonines intact) ver- sus when most phosphorylation is lost (mutations in the kinase) (Adams and Kilmartin, 1999). In fact, Mps1-1p is severely compromised for kinase activity, and the mps1-1 mutant accumulates fewer phosphorylated forms of Spc42p. We suspect that the phenotype we observe results from the inability of Mps1-1p to properly phosphorylate Spc42p. Failure of Mps1-1p to phosphorylate Spc42p during SPB duplication should then prevent assembly of the central plaque of the new SPB; consistent with this prediction, mps1-1 cells do not show accumulation of satellite material at the half-bridge (Winey et al., 1991). Is Mps1p function required for assembly of other satellite components? We suspect that Mps1p is required indirectly for assembly of Nud1p, Cnm67p, and Spc29p; Mps1p is re- quired for assembly of Spc42p that may serve as a scaffold upon which the other satellite components assemble. This is supported by the unique suppression of mps1-8 by increased dosage of SPC42. NUD1, CNM67, and SPC29 were not identified in our screen as dosage suppressors of mps1-8 or earlier as dosage suppressors of the mps1-1 (Schutz et al., 1997). We tested SPC29 directly and verified that increased dosage of SPC29 does not suppress mps1-8 (unpublished data). This does not preclude that the function of Mps1p is required for assembly of the other satellite components; the development of assembly assays for Nud1p, Cnm67p, and Spc29p will allow for investigation of the requirement for Mps1p in assembling these components. Regardless of other requirements for Mps1p in SPB duplication, the specific in- teraction established between Mps1p and Spc42p in this analysis indicates that their association is essential for assem- bly of the central plaque, an event that may be critical for launching SPB duplication. Materials and methods Yeast and Escherichia coli culture and genetic techniques Yeast strains used in this study are listed in Table I and are derived from S288c and W303 backgrounds. Yeast and E. coli genetic techniques were as described by Ausubel et al. (1997). Yeast synchronization and 5-fluoro- orotic acid screening were done as in Jones et al. (1999). Galactose (GAL) induction was done as in Hardwick et al. (1996). Protein techniques and kinase assays GST fusion proteins were induced and isolated and kinase assays were performed as in Schutz and Winey (1998). Mps1p-13Xmyc (Ndc1p-3Xmyc isolated similarly, HC12–2b) was isolated from an asynchronously grow- ing strain (SBY650) using techniques similar to those described in Schutz and Winey (1998). Mps1p-13Xmyc and recombinant Spc42p expressed in baculovirus-infected insect cells (a gift from Danni Vinh, Trisha Davis Lab, University of Washington, WA) were used in kinase assays as described in Lauze et al. (1995). Samples for all kinase assays were resolved on 15% Anderson SDS-PAGE gels (Anderson et al., 1973). Gels were subjected to electrophoretic transfer onto nitrocellulose membrane (Schleicher and Schuell Inc.) and membranes were used to expose a phosphorimager screen and for Western analysis to quantitate protein levels. 32P incorpora- tion was quantitated using a Storm 860 phosphorImager with the Im- ageQuaNT analysis package (Molecular Dynamics). For Western analysis, nitrocellulose membranes were blocked using Odyssey blocker. Detection was performed using secondary antibodies labeled with the near-infrared fluorescent dyes IRDye800 and Cy5.5, and blots were scanned with an Odyssey Infrared Imager (Odyssey reagents, instruments, and software from LI-COR Biosciences). GST-Mps1 proteins were detected with goat an- tiglutathione primary antibody (1:1,000; Amersham Pharmacia Biotech) and an anti–goat antibody labeled with the IRDye800 fluorophore (1:10,000). Mps1p-13Xmyc was detected using an anti-myc primary anti- body (1:1,000; Santa Cruz Biotechnology, Inc.) and an anti–mouse anti- body labeled with IRDye800 (1:10,000). Spc42p was detected using a polyclonal anti-Spc42p primary antibody (a gift from John Kilmartin, Med- ical Research Council Laboratory of Molecular Biology, London, UK) and an anti–rabbit antibody labeled with Cy5.5 (1:10,000). Protein was visual- ized and quantitated with the Odyssey Imaging System. We determined relative specific activity for each fusion protein by using the Storm860 phosphorImager and ImageQuaNT analysis package to mea- sure 32P incorporation and by using the Odyssey Infrared Imager and anal- ysis software to measure protein amounts. The number representing 32P in- corporation was divided by the number representing protein amount to yield relative specific activity. In the coimmunoprecipitate experiment, the immunoprecipitated mate- rial was resolved on an 8.5% laemmli SDS-PAGE gel (Ausubel et al., 1997). Gels were subjected to electrophoretic transfer onto a polyvi- nylidene difluoride membrane (Millipore). Polyvinylidene difluoride mem-