458 The Journal of Cell Biology | Volume 156, Number 3, 2002 Figure 6. Mps1p-myc localizes to the SPB and the kinetochores. (A) Cells containing MPS1-myc and SPC42-GFP (JM7) were grown to mid-log phase, harvested, and then fixed and stained for indirect immunofluorescence. DAPI was used to visualize DNA (blue), an affinity purified anti-myc polyclonal antibody was used to identify Mps1p-myc (red), and a polyclonal anti-GFP antibody was used against Spc42-GFP (green). (A) Mps1p-myc signal is observed as faint dots coincident with DAPI staining and a more intense dot that colocalizes with Spc42-GFP signal. (B) Chromosome spreads prepared from a strain containing Mps1p-myc (red) and Ndc10p-HA (green) (JM16) or Mps1p-myc (red) and Spc42p-GFP (green) (JM43) show localization of Mps1p-myc to the kinetochores and SPB. (C and D) Immuno-EM of strains containing Mps1p-myc shows colloidal gold signal coincident with the central plaque of the SPB (C, arrows) and at the plus end of microtubules nucleated from the SPB (D, arrows). Arrowheads in C and D indicate microtubules. Bars: (A and B) 1.0 m; (C and D) 0.1 m. Figure 7. Mps1p and Spc42p physically interact. Clarified extracts (as described in Materials and methods) made from asynchronously growing cultures of a MPS1-myc strain (SBY650), a NDC1-myc strain (HC12–2b), and an untagged strain were incubated with anti-myc monoclonal antibody conjugated to agarose beads to immunoprecipitate the myc-tagged proteins. Immunoprecipitated Spc42p-myc, isolated from a strain overexpressing Spc42p- myc (Fig. 7 A, lane 4). Thus, Mps1p and Spc42p both local- ize at the SPB and physically interact with each other. Spc42p is a substrate in vitro for Mps1p, and Spc42p phosphorylation is dependent on Mps1p Spc42p is a phosphoprotein whose phosphorylation state varies throughout the cell cycle (Donaldson and Kilmartin, 1996). To determine if Spc42p is a substrate for Mps1p, we used Mps1p-myc and recombinant Spc42p in a kinase assay protein samples were divided and resolved by SDS-PAGE to produce duplicate gels. One blot (A, lanes 1–4) was probed using polyclonal anti-Spc42p, and a second blot (B, lanes 5–7) was probed with an anti-myc antibody. Spc42p specifically coimmunoprecipitates with Mps1p-myc (lane 1), since Spc42p is not detected in either the Ndc1p-myc or untagged control lanes (lanes 2 and 3). A whole cell lysate from a strain overexpressing Spc42p-myc (ACY122-1c) was used to indicate where Spc42p would migrate. Lanes 5 and 6 show that the Mps1-myc and NDC1-myc proteins are present. Lane 7 contains an immunoprecipitate from the untagged strain and shows where the IgG band migrates (IgG band also seen in lanes 5 and 6).