JCB Article The yeast protein kinase Mps1p is required for assembly of the integral spindle pole body component Spc42p Andrea R. Castillo,1 Janet B. Meehl,1 Garry Morgan,1 Amy Schutz-Geschwender,2 and Mark Winey1 1MCD Biology, UCB 347, University of Colorado, Boulder, CO 80309 2LI-COR Inc., Lincoln, NE 68504 Sprotein kinase that has roles in spindle pole body accharomyces cerevisiae MPS1 encodes an essential (SPB) duplication and the spindle checkpoint. Previously characterized MPS1 mutants fail in both functions, leading to aberrant DNA segregation with lethal consequences. Here, we report the identiﬁcation of a unique conditional allele, mps1–8, that is defective in SPB duplication but not the spindle checkpoint. The mutations in mps1-8 are in the noncatalytic region of MPS1, and analysis of the mutant protein indicates that Mps1-8p has wild-type kinase activity in vitro. A screen for dosage suppressors of the mps1-8 conditional growth phenotype identiﬁed the gene encoding the integral SPB component SPC42. Additional analysis Introduction In Saccharomyces cerevisiae, the spindle pole body (SPB)* serves as the centrosome equivalent organelle. SPBs are duplicated once each cell cycle and function to nucleate microtubules that will form the mitotic spindle. Proper SPB duplication is required to form a bipolar spindle, which in turn is essential for cells to accurately segregate their DNA. SPB morphology is distinct from the typical cen- trosome of animal cells, having a trilaminar disc-like structure instead of centrioles surrounded by pericentri- olar material (Byers and Goetsch, 1974; Kochanski and Borisy, 1990). The SPB remains embedded in the nuclear envelope throughout the cell cycle, allowing it to simulta- neously nucleate nuclear and cytoplasmic microtubules Address correspondence to Mark Winey, MCD Biology/UCB 347, University of Colorado, Boulder, CO 80309-0347. Tel.: (303) 492- 3409. Fax: (303) 492-7744. E-mail: mark.winey@colorado.edu *Abbreviations used in this paper: GFP, green fluorescent protein; GST, glutathione S-transferase; SPB, spindle pole body. A.R. Castillo’s present address is Div. of Basic Sciences, Fred Hutchison Cancer Research Laboratory, 1100 Fairview Ave. N. A2-168, Seattle, WA 98109. Key words: budding yeast; spindle pole body; MPS1; SPC42; protein kinase  The Rockefeller University Press, 0021-9525/2002/2/453/13 $5.00 The Journal of Cell Biology, Volume 156, Number 3, February 4, 2002 453–465 http://www.jcb.org/cgi/doi/10.1083/jcb.200111025 revealed that mps1-8 exhibits synthetic growth defects when combined with certain mutant alleles of SPC42. An epitope-tagged version of Mps1p (Mps1p-myc) localizes to SPBs and kinetochores by immunoﬂuorescence microscopy and immuno-EM analysis. This is consistent with the physical interaction we detect between Mps1p and Spc42p by coimmunoprecipitation. Spc42p is a substrate for Mps1p phosphorylation in vitro, and Spc42p phosphorylation is dependent on Mps1p in vivo. Finally, Spc42p assembly is abnormal in a mps1-1 mutant strain. We conclude that Mps1p regulates assembly of the integral SPB component Spc42p during SPB duplication. (Byers and Goetsch, 1974). Despite morphological differ- ences between the SPB and centrosome, there are several conserved components (Adams and Kilmartin, 2000). These include proteins that form the -tubulin complex in S. cere- visiae (Spc98p, Spc97p, and Tub4p), the centrin homo- logue, Cdc31p, and the molecular spacer protein, Spc110p (Kendrin) (Byers, 1981; Kilmartin et al., 1993; Geissler et al., 1996; Spang et al., 1996; Knop et al., 1997; Middendorp et al., 1997; Flory et al., 2000). Although the -tubulin complex clearly serves to nucleate microtubules in both the SPB and the centrosome, the conserved function performed by Cdc31p and Spc110p is still unclear (Moritz et al., 1995; Zheng et al., 1995; Knop and Schiebel, 1997; Marshall and Wilson, 1997; Adams and Kilmartin, 2000). The SPB duplication pathway has been described through EM analysis of wild-type cells and mutant cells that fail at dif- ferent stages of SPB duplication (Byers and Goetsch, 1975; Adams and Kilmartin, 1999). Duplication of the SPB occurs in G1 of the cell cycle, beginning with the accumulation of SPB components (the satellite) onto the cytoplasmic face of the half-bridge, a modification of the nuclear envelope dis- tal to the SPB (Byers and Goetsch, 1975). The amorphous satellite appears to develop into a larger ordered structure 453